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American Journal of Pathology, Vol. 151, No.

6, December 1997
Copynight ) American Society for Investigative Pathology

Human Cytotrophoblast Differentiation/Invasion Is


Abnormal in Pre-Eclampsia

Kee-Hak Lim,* Yan Zhou,t Mary Janatpour,t


Michael McMaster,t Kathryn Bass,t
Sun-Hee Chun,* and Susan J. Fisher*tt
From the Departments of Obstetrics, Gynecology and
Reproductive Sciences,* Stomatologyt Anatomy,' and
Pharmaceutical Chemistry,5 University of California San
Francisco, San Francisco, California

During human placental development, cytotrophoblast stem cells differentiate and invade the uterus.
Simultaneously, the cells modulate their expression
of several classes of stage-specific antigens that mark
transitions in the differentiation process and play a
role in either uterine invasion (integrin cell-extracellular matrix receptors and matrix metailoproteinase-9) or immune interactions (HLA-G). The pregnancy disease pre-eclampsia is associated with
shallow cytotrophoblast invasion. Previously we
showed, by immunofluorescence localization on placental tissue, that in pre-eclampsia invasive cytotrophoblasts fail to properly modulate their integrin repertoire. This finding suggests possible abnormalities
in the differentiation pathway that leads to uterine
invasion. Here we used a culture system that supports
this differentiation process to compare the differentiative and invasive potential of cytotrophoblasts obtained from control (n = 8, 22 to 38 weeks) and
pre-eclamptic (n = 9, 24 to 38 weeks) placentas. In
culture, the cells from pre-eclamptic placentas failed
to properly modulate al integrin and matrix metalloproteinase-9 expression at the protein and mRNA
levels. Their invasive potential was also greatly reduced. Likewise, the cells failed to up-regulate HLA-G
protein and mRNA expression. These results suggest
that defective cytotrophoblast differentiation/invasion can have significant consequences to the outcome of human pregnancy (ie, development of preeclampsia) and that, by the time delivery becomes
necessary, the defect is not reversed by removing the
cells from the maternal environment. (Am J Pathol
1997, 151:1809-1818)

Formation of the maternal-fetal interface, a consequence


of cytotrophoblast differentiation, is critical to pregnancy
outcome. In humans, two mutually exclusive differentiation pathways give rise to morphologically and functionally distinct trophoblast populations that compose the

fetal portion of this interface.1' 2 Cytotrophoblast stem


cells are attached to the basement membrane that surrounds the stromal core of chorionic villi. Many of these
cells differentiate by fusing to form a syncytial layer that
covers the villus and transports nutrients, wastes, and
gases between fetal and maternal blood. At selected
sites, cytotrophoblasts break through the syncytium and
form multilayered columns of cells that attach to and
invade the uterus. As a result of interstitial invasion, a
subpopulation of cytotrophoblasts (termed extravillous,
invasive, or intermediate)3 are normally found throughout
the entire decidua and the first third of the myometrium.
As a result of endovascular invasion, these cells also
invade uterine arterioles, replacing the endothelial lining
and most of the musculoelastic tissue in the vessel
wall.,45 Consequently, uterine arterioles become highflow, low-resistance vessels that are capable of responding to increases in fetal requirements for maternal blood
as pregnancy progresses.6
We hypothesized that fetal cytotrophoblast invasion of
the uterus requires these cells to up-regulate expression
of molecules that function during this process. The morphology of the maternal-fetal interface suggests that the
cells' ability to acquire an invasive phenotype, while
avoiding maternal immune recognition, is particularly crucial. In studying invasiveness, we showed that cytotrophoblasts normally modulate their expression of several
integrin cell-extracellular matrix (ECM) receptors7 and
MMP-9.6-10 We used an in vitro model of this differentiation process to analyze the role these molecules play.9'11
The results showed that, during invasion, the cells upregulated their expression, first of molecules that inhibit
invasion (eg, the integrin a5 fibronectin receptor) and
then of molecules that promote it (eg, the integrin al
laminin and collagen (types and IV) receptor; MMP-9).
Therefore, correctly balancing these mechanisms is likely
to be critical to normal placental development. In studying how these cells evade maternal immune recognition,
we showed that the subpopulation of cells that invade the
uterus express HLA-G, a very unusual class lb major
histocompatibility antigen that may protect them from
natural killer cell lysis.12-14

Supported by National Institutes of Health grant HD 30367.


Accepted for publication September 11, 1997.
Address reprint requests to Dr. Susan Fisher, Department of Stomatology, HSW 604, 513 Parnassus Avenue, University of California San Francisco, San Francisco, CA 94143-0512.

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Lim et al

AJP December 1997, Vol. 151, No. 6

We reasoned that pregnancy diseases in which cytotrophoblast invasion is abnormal at a microscopic level
could be associated with disruption of invasion at a molecular level, ie, result from an imbalance of the regulatory
mechanisms that govern invasion. Previous work suggested that pre-eclampsia could be an interesting example of such a disease. Histological studies of the maternal-fetal interface (eg, placental bed biopsies) show that,
in pre-eclampsia, interstitial cytotrophoblast invasion is
often shallow and endovascular invasion nearly absent.15-17 Pre-eclampsia is important because it occurs
frequently (7% of nulliparous patients) and has serious
consequences for both the mother (eg, high blood pres-.
sure, proteinuria, and edema) and the fetus (intrauterine
growth retardation). As a result, pre-eclampsia and hypertensive diseases of pregnancy are a leading cause of
maternal death18 and contribute significantly to premature deliveries in the United States.19
We began studying cytotrophoblast differentiation in
this syndrome by using immunolocalization techniques to
determine whether differentiating/invading cytotrophoblasts in tissue sections of the maternal-fetal interface
correctly switch their integrin repertoire. In normal pregnancies, cytotrophoblasts that are differentiating along
the invasive pathway first up-regulate the expression of
integrin a5/01, then al/f1.7 In pre-eclampsia, invasive
cytotrophoblasts do not completely switch their integrin
repertoire. The cells express integrin a5/f31 but not al/
11,17 thus displaying an adhesion phenotype our function-perturbation studies suggest could inhibit invasion.11
In addition, Redline20 recently showed that, in pregnancies complicated by pre-eclampsia, intermediate/invasive cytotrophoblasts have an increased proliferative index, as evidenced by increased staining for proliferating
cell nuclear antigen (PCNA) and reduced expression of
human placental lactogen, another antigen that is produced as a consequence of the differentiation process.
Together, these results suggest that, in pre-eclampsia,
cytotrophoblasts start to differentiate along the invasive
pathway but cannot complete this process. However,
studies performed on tissue sections do not allow us to
examine the differentiative potential of cytotrophoblasts
and to determine whether the observed defect is reversible.
In experiments described here, we used a culture
system that promotes cytotrophoblast stem cell differentiation along the invasive pathway to compare the differentiative potential of cells isolated from placentas of gestation-matched control and pre-eclamptic patients. The
results showed that, in pre-eclampsia, cytotrophoblast
integrin switching in vitro was altered in a pattern that
replicated the changes we previously described in vivo,
ie, in tissue sections of placental bed biopsies. Cytotrophoblast expression of al integrin protein and mRNA
was all but absent. Their ability to up-regulate MMP-9
production, as well as to invade ECM in vitro, was also
impaired. Likewise, their expression of HLA-G, a stagespecific antigen that probably plays an important role in
maternal-fetal immune interactions, dramatically decreased. Together, these data suggest that, in preeclampsia, cytotrophoblasts start to differentiate along

the invasive pathway, fail to complete the process, and


consequently are less able to invade. As neither stagespecific antigen expression nor invasiveness could be
restored to control levels by removing the cells from the
maternal environment, these changes are irreversible by
the time the symptoms necessitate delivery.

Materials and Methods


Placental Sources
Cytotrophoblasts were isolated from the placentas of patients with pre-eclampsia diagnosed according to the
following criteria, recommended by Chesley21: nulliparity, no history of hypertension before pregnancy, increase
in diastolic pressure of 15 mm Hg or systolic pressure of
30 mm Hg compared with blood pressure obtained before 20 weeks of gestation, proteinuria of .0.5 g/24 hours
or .300 mg/dl (or 1 + on urine dipstick) in a catheterized
specimen, hyperuricemia of >5.5 mg/dl (or one SD
greater than the normal mean value before term), and
return to normal blood pressure and resolution of proteinuria by 12 weeks postpartum. The American College of
Obstetricians and Gynecologists' definitions for severe
pre-eclampsia19 and published criteria for the syndrome
of hemolysis, elevated liver enzymes, and low platelets
(HELLP)22 were used: systolic blood pressure of .160
mm Hg and/or diastolic pressure of .110 mm Hg, proteinuria of .5 gm in a 24-hour period or 3+ on urine
dipstick, presence of cerebral or visual disturbances,
epigastric pain, oliguria (<500 ml in 24 hours), pulmonary
edema, evidence of hemolysis on peripheral smear, increased bilirubin of .1.2 mg/dl, increased lactic dehydrogenase of >600 IU/L, platelet counts of <100,000/
mm3, and serum glutamic-oxaloacetic transaminase level
of .70 lU/ml. Eclampsia was defined as an onset of
seizure activity in the setting of pre-eclampsia in a patient
without a history of seizure disorder. Of the nine patients
whose placentas we obtained, one was diagnosed with
pre-eclampsia at 38 weeks of gestation, five with severe
pre-eclampsia at 24, 27, 28, 29, and 30 weeks of gestation, two with HELLP syndrome at 28 and 32 weeks, and
one with eclampsia/severe pre-eclampsia at 29 weeks.
Of the eight total control patients, seven patients delivered at or before 33 weeks. Two delivered at 27 and 32
weeks due to cervical incompetence. Three underwent
pregnancy terminations at 22 to 24 weeks, and the sixth
patient delivered at 33 weeks because of microinvasive
carcinoma of the cervix diagnosed during pregnancy.
The seventh patient underwent elective delivery at 26
weeks of gestation for nonoperable conjoined twins.
None of the patients had evidence of pre-eclampsia,
gestational hypertension, chorioamnionitis, chronic hypertension, or a medical history that suggested they were
at an increased risk for developing pre-eclampsia. None
of the placentas had abnormalities that could be detected either grossly or histologically. The eighth patient
delivered at 38 weeks after a normal, uncomplicated
pregnancy.

Abnormal Cytotrophoblast Differentiation/Invasion in Pre-Eclampsia

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AJP December 1997, Vol. 151, No. 6

Isolation and Culture of Cytotrophoblasts


Cytotrophoblasts were isolated from second-trimester
placentas according to the methods described previously.8'9 Briefly, chorionic villi were subjected to enzymatic
dissociation using trypsin, DNAse 1, and collagenase.
The resulting cells were enriched using Percoll gradient
centrifugation. The cytotrophoblast-enriched fraction
from the Percoll gradient was purified further by removing
bone-marrow-derived cells using magnetic beads (PerSeptive Diagnostics, Cambridge, MA) coated with antiCD45 antibody, which recognizes all bone-marrow-derived cells (purified from ascites fluid of the GAP 8.3
hybridoma, American Type Culture Collection, Rockville,
MD). Cytotrophoblasts from third-trimester placentas
were isolated by similar methods except that the chorionic villi were subjected to three cycles of enzymatic
digestion using trypsin and DNAse 1. All isolated cytotrophoblasts were cultured in DME H21 minimal essential
medium containing 2% Nutridoma (Boehringer Mannheim, Indianapolis, IN) and 50 ,ug/ml gentamicin. Viability
after 72 hours in culture was 90 to 95% as determined by
trypan blue exclusion.

Immunohistochemistry
Mouse monoclonal antibody to the human integrin al
subunit (IgG) was obtained from T-Cell Laboratory, Boston, MA. Rat monoclonal antibodies to human integrin
subunit a5 (BIIG2) and cytokeratin (7D3) were made in
this laboratory and described previously.7 Mouse monoclonal antibody to HLA-G (IgM) was also produced in this
laboratory.23 Species-specific monoclonal antibodies to
IgG and IgM were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA).
Highly purified cytotrophoblasts were cultured at a
concentration of 2.5 x 105 cells/ml on Matrigel-coated
(Collaborative Research, Bedford, MA) 12-mm coverslips
in serum-free medium supplemented with 2% Nutridoma.
For time zero analysis, cytotrophoblasts were allowed to
adhere to the coverslips for 10 minutes at 370C. After 0,
12, 24, 36, and 48 hours of culture, the cells were washed
with phosphate-buffered saline (PBS) and fixed in 2.5%
paraformaldehyde at room temperature for 10 minutes.
Next, cells were permeabilized with 100% methanol at
-200C, and nonspecific reactivity was blocked by incubating them with 0.2% bovine serum albumin for 20 minutes. Primary antibody was added at 40C for 10 to 12
hours. Then the cells were washed in PBS and incubated
with species-specific secondary antibodies at room temperature for 1 hour. In all cases, the cells were double
stained with antibodies that detect cytokeratin as described previously.8 Cytokeratin-positive cells were
counted on an Axiophot microscope (Zeiss), and the
percentage of these cells that expressed integrin al or
a5 and HLA-G was calculated.

Immunoblotting
Conditioned medium was collected from control and preeclamptic cytotrophoblasts at 12-hour intervals for 3 days

and stored at -800C. MMP-9 protein in these samples


was detected by immunoblotting as previously described.10 Briefly, conditioned medium (10 ,l) was solubilized in loading buffer, boiled for 5 minutes, subjected
to sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (10% gels) and transferred by blotting to
nitrocellulose. Nonspecific reactivity was blocked by incubating the transfers for 1 hour in Tris-buffered saline
containing Tween-20 (TBST; 0.01 mol/L Tris/HCI, 0.15
mol/L NaCI, and 0.05% Tween-20, pH 8.0) and 5% nonfat
dried milk (Carnation). The transfers were then incubated
for 2 hours at 25C with anti-MMP-9 antibody (7-1 1C24),
which was suspended (5 ,ug/ml) in TBST containing 5%
nonfat dried milk. The blots were washed three times (10
minutes each time) in TBST. Primary antibody was detected by incubating the blot for 2 hours at 250C with
horseradish-peroxidase-conjugated rabbit anti-mouse
IgG (Jackson ImmunoResearch Laboratories) diluted 1:
5000 in TBST containing 5% nonfat dried milk. The membranes were washed in TBST, rinsed in PBS, and developed for enhanced chemiluminescence with a
commercially available kit (Amersham, Arlington Heights,
IL).

RNA Extraction
RNA was extracted from cytotrophoblasts cultured at a
concentration of 1.0 x 106 cells/ml in serum-free medium
on Matrigel as described above. Total RNA was extracted according to published methods.25 Briefly, 5 x
106 cells per sample were homogenized in 500 ,ul of
guanidine buffer (4 mol/L guanidine isothiocyanate, 25
mol/L sodium citrate, 0.5% sarcosyl), followed by the
addition of 50 ,ul of 2 mol/L sodium acetate (pH 4.0), 500
,ul of water-saturated phenol and 100 ,ul of chloroform.
After centrifugation, the RNA was pelleted from the aqueous phase by the addition of 500 Al of isopropanol and
reprecipitated from a solution containing 10 mmol/L Tris
(pH 7.5), 1 mmol/L EDTA, and 0.5% SDS. The pellet was
then washed with cold (40C) 70% ethanol, vacuum dried,
and dissolved in sterile water. The concentration of RNA
was determined by measuring the absorbance at 260
nm.

Northern Blotting
Total RNA (8 ,ug) was separated by formaldehyde-agarose gel electrophoresis, transferred to Nytran membranes (Schleicher and Schuell, Keene, NH), and analyzed by Northern blot hybridization as described
previously.2627 The probe for human integrin al was
synthesized by random priming of full-length cDNA (the
gift of Dr. E. Marcantonio, Columbia University) using
[32P]CTP and the Klenow fragment of DNA polymerase 1.
The probe for MMP-9 was a 384-bp PCR product near the
5' end of the cDNA that has the least homology to MMP2,28 and the probe for HLA-G was synthesized by random priming of the 450-bp Pvull fragment from the 3'
untranslated region of HLA-G. Probes had a specific
activity of 2 x 109 dpm/,g. In all experiments, gels were

1812 Lim et al
AJP December 1997, Vol. 151, No. 6

Figure 1. Cytotrophoblast (CTB) expression of integrin al protein dramatically decreases in syndrome of hemolysis, elevated liver enzymes, and low platelets
(HELLP). Control CTBs isolated from the placenta of a patient who delivered at 27 weeks of gestation due to cervical incompetence were plated on Matrigel-coated
coverslips, cultured in serum-free medium for 48 hours and then stained with monoclonal antibodies against integrin a 1 (A) and cytokeratin (CK; B). CTBs isolated
from the placenta of a patient who delivered at 28 weeks of gestation due to HELLP syndrome were cultured under the same conditions for 48 hours, after which
their integrin al (C) and CK expression (D) was assessed. By 48 hours in culture many of the control CTBs stained with an antibody against the al integrin subunit
(A), whereas few of the CTBs isolated from the placenta of the patient with HELLP syndrome reacted with this antibody (C).

stained with acridine orange before transfer to ensure


integrity of the RNA samples and to confirm that equal
amounts of RNA had been loaded onto each lane. The
final post-hybridization wash was carried out in 0.3 x
SSC (0.45 M NaCI, 4.5 mmol/L sodium citrate) and 0.1%
SDS at 650C.

Invasion Assay
Invasion assays were performed using one of two methods previously described.29'30 In the first method, 2.5 x
105 cytotrophoblasts were cultured on Transwell inserts
(6.5 mm; Costar, Cambridge, MA) containing polycarbonate filters that had been coated with 10 ,l of Matrigel.
After 72 hours of culture, the cells were stained with
anti-cytokeratin antibody, and the number of cell projections on the bottom side of the filter was counted using a
fluorescence microscope as described.29 In the second
method, after the 72-hour culture period, cells were fixed
and processed for scanning electron microscopy.30 The
filters were removed from the inserts, critical-point dried,
sputter-coated with gold palladium, and then imaged in
an ETEC Autoscan Model U-1 scanning electron microscope. Invasion was quantified by counting the number
of cells and processes on the underside of the filter.

Statistical Analysis
All data were analyzed using analysis of variance with
Scheffe F-test for post hoc analysis (StatView, Abacus
Concepts, Berkeley, CA). A P value of <0.05 was considered significant.

Results
We compared the ability of cytotrophoblasts isolated
from normal and pre-eclamptic placentas to differentiate
along the invasive pathway by culturing these cells under
conditions that promote this process. First, we assayed
their expression of integrins and MMP-9, stage-specific
antigens that modulate their invasiveness. The integrins
of interest were the al and a5 subunits. By 48 hours in
culture, cells isolated from a control placenta (27 weeks)
reacted with an antibody that recognizes the al subunit
(Figure 1A). Cytotrophoblasts isolated from placentas
that were obtained from a pregnancy (28 weeks) complicated by pre-eclampsia all reacted with cytokeratin antibody (Figure 1D), but almost none reacted with the anti-al antibody (Figure 1C). In contrast, cells isolated from
both the control and pre-eclamptic placentas expressed

Abnormal Cytotrophoblast Differentiation/Invasion in Pre-Eclampsia

1813

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integrin ca5 (Figure 2, A and C) and reacted with anticytokeratin antibody (Figure 2, B and D).
To quantify these results, we determined the number of
cytokeratin-positive cells (ie, cytotrophoblasts) in both
groups that expressed integrin a1 immediately after isolation and after culture for 12, 24, 36, and 48 hours. The
results are shown in Figure 3. At time 0, an average of 8%
of control cytotrophoblasts (23 to 33 weeks) reacted with
the anti-cal integrin antibody, and by 48 hours this percentage increased to 36%. However, cells isolated from
pregnancies complicated by pre-eclampsia (24 to 32
weeks) demonstrated a marked decrease in their ability
to up-regulate integrin al expression in culture. Starting
at time 0, 1% of these cytotrophoblasts expressed integrin al, and after 48 hours, only 4% of the cells reacted
with the anti-ca1 integrin antibody. In contrast, a similar
percentage of cytotrophoblasts isolated from the placentas of normal and pre-eclamptic patients expressed integrin a5 in vitro at all times analyzed (Figure 4). Before
culture, approximately 53% of cells in both groups reacted with the anti-a5 antibody, and this increased to an
average of 80% after 48 hours.
Because our previous studies showed that MMP-9 expression is also required for cytotrophoblast invasiveness
in vitro, 0 we used Western blotting to compare the ability
of cytotrophoblasts in both groups to up-regulate expression of this proteinase in vitro (Figure 5). The amount of

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1814

Lim et al

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Figure 4. Simnilar pcrcentagexs of C113s 'plre'ss
ietegililn a) protpein ill prieclampsipia ind in gestation-illtched colntrol piregnancies. 'th'le patients and
Iletliocds of anllx'iS iire decriiidil in Intlre 3, except that an antihiody to
iintc1Wi a ti toin aSXes the
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ilnteg'till nn inl vil/r is connsisient xx-tilt thilei exptession of this inte'grin i)i viltO
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MMP-9 in samples of conditioned medium from 26-week


control cytotrophoblasts increased steadily during 60
hours in culture. Cytotrophoblasts isolated from a 27week pre-eclamptic patient also up-regulated MMP-9 secretion during 60 hours in culture. However, the amount
detected in the medium, quantified by scanning densitometry, was fourfold less than amounts in control medium throughout the culture period. We obtained essentially the same results in two other experiments in which
we assayed the ability of cytotrophoblasts isolated from a
28-week HELLP patient and a 29-week severely pre-

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As a first step toward understanding why cytotrophoblasts from placentas of pre-eclamptic patients fail to
correctly modulate integrin al and MMP-9 protein expression in vitro, we performed Northern hybridization to
compare levels of the corresponding mRNAs in these
cells with levels in gestation-matched control cytotrophoblasts. RNA was extracted from the cells at 0 hour, ie,
immediately after isolation, or after culture for 12 and 36
hours. Northern hybridization was performed using cDNA
probes that detect either integrin al mRNA (4.1 kb and
5.2 to 6.5 kb) or MMP-9 mRNA (2.8 kb). At 0 hour, integrin
l1 mRNA was detected in control 26-week cytotrophoblasts, and there was a small increase in message level
after the cells had been cultured for 12 hours (Figure 6A).
In contrast, cytotrophoblasts isolated from the 27-week
nl,qr-ant_
Pnrl_qmntirinvnracc2arl
d yuiL.. nntiiont
IILLI a-1
iUZ,3t U co
tiU littla
P dUl IcI ofam a-qyIdnra-(a.ii
LIui IILA
mRNA at either time point that the band is not visible in
the photograph. After 36 hours in culture, there was a
very slight increase in ca1 mRNA expression by both
pre-eclamptic and control cells (data not shown). Analysis of cytotrophoblast integrin a1 mRNA levels in another
sample pair (24-week control/29-week pre-eclamptic)
gave essentially the same results.
A different blot of the same RNA samples shown in
Figure 6A was probed to detect MMP-9 mRNA (Figure
6B). In accord with our protein data, no message was
detected in control cells at 0 hour. By 12 hours MMP-9
mRNA was found, and levels continued to increase during the 36-hour culture period. In contrast, no MMP-9
mRNA was detected at either 0 or 12 hours in preeclamptic cytotrophoblasts. By 36 hours message was
present, but the levels, quantified by scanning densitometry, were approximately 15-fold less than those found in
control cells. These data suggest that the impaired ability
of cytotrophoblasts isolated from the placentas of preeclamptic patients to switch on expression of integrin al
and MMP-9 in culture is due, at least in part, to a dramatic
decrease in the levels of the corresponding mRNAs.
As these molecules mediate cytotrophoblast invasion
in vitro, we examined the ability of pre-eclamptic cytotrophoblasts to invade Matrigel substrates. We compared
the invasiveness of cells isolated from the placentas of
patients with pre-eclampsia who delivered at 29, 30, 27,
and 28 weeks to cells isolated from control patients who
delivered at 22, 24, 26, and 38 weeks. A graph summarizing these data is shown in Figure 7. In all cases, the
cells isolated from patients with pre-eclampsia were severalfold less invasive than the control cells. Overall, these
observations suggest that, in pre-eclampsia, the invasive
ability of cytotrophoblasts is dramatically decreased.
Finally, we also compared the ability of cytotrophoblasts in the two groups to modulate their expression of
stage-specific antigens that are likely to play a role in
immune interactions with maternal cells. In this case, the
antigen of interest was HLA-G, an unusual major histocompatibility complex class lb molecule expressed by
fully differentiated cytotrophoblasts that have invaded the
uterus."a Immediately after isolation, an average of 21%
of control preterm cytotrophoblasts expressed HLA-G,

Abnormal Cytotrophoblast Differentiation/Invasion in Pre-Eclampsia 1815


AJP December 1997, Vol. 151, No. 6

A.

CON
Oh 12h

PE
Oh 12h
.., .

__ n_

,-.

.......-7-77"-

.I

........T

I;

- 28S

-28S

alto

MMP
-18S

-18S

Figure 6. CTB expression of integrin al and MMP-9 mRNA is reduced in pre-eclampsia. Total RNA was prepared from CTBs isolated from the placenta of a patient
with pre-eclampsia (PE; 27 weeks) and a gestationally matched control patient (CON; 26 weeks) and analyzed by Northern blotting as described in Materials and
Methods. Control CTBs expressed al integrin mRNA immediately after isolation (0 h), and the level increased only slightly after 12 hours of culture (A). In contrast,
CTBs isolated from the placenta of a pre-eclamptic patient expressed so little al mRNA at either 0 or 12 hours that the bands are not visible in the photograph.
CTB expression of mRNA for MMP-9 was also affected by pre-eclampsia (B). As compared with control cells, the onset of expression was delayed and the amount
detected was decreased.

and at 48 hours, the number had increased to 47%


(Figure 8). In contrast, an average of 3% of cytotrophoblasts isolated from pre-eclamptic patients expressed
HLA-G immediately after isolation, rising only to 23% after
48 hours.
We further investigated whether HLA-G mRNA levels
were also reduced in pre-eclamptic cytotrophoblasts.
The blot shown in Figure 4A was stripped and rehybridized with a cDNA probe that corresponds to a 450-bp
region of the 3' untranslated region of HLA-G (Figure 9).
Consistent with our previously published data,23 relatively high levels of HLA-G mRNA were detected in control cytotrophoblasts immediately after isolation (0 hour),
and levels did not change significantly after the cells
were cultured. In contrast, HLA-G mRNA was not detected in pre-eclamptic cytotrophoblasts at any time. We
also compared levels of this message in two other pairs
of pre-eclamptic and control cytotrophoblast samples: 1)
28-week pre-eclamptic/40-week control and 2) 29-week

pre-eclamptic/26-week control. In both cases, HLA-G


mRNA expression by pre-eclamptic cytotrophoblasts
was less than 15% of the levels detected in control cells.

Discussion
Our results suggest that cytotrophoblast differentiation
along the invasive pathway is abnormal in pre-eclampsia.
Specifically, the cells start to differentiate, as shown by
their up-regulation of integrin a5 expression. But their
ability to up-regulate expression of integrin al, MMP-9,
and HLA-G, stage-specific antigens that are associated
with later stages of the differentiation process, is impaired. We also show that this defect has functional consequences; cytotrophoblasts isolated from the placentas
of pre-eclamptic patients were much less invasive in vitro

0
Cytotrophoblast

Invasion

._A
._
en
m
100 -

0
0

80

7;
za
C)

0.

60*

control

PE

40

20 -

Hours in Culture
0

Figure 8. CTB expression of HLA-G protein decreases in pre-eclampsia.

72 hrs

Hours

In

culture

Figure 7. Cytotrophoblast invasiveness is markedly reduced in pre-eclampsia. Invasion was quantified using the first assay method described in Materials and Methods. The data are expressed as percentage of control invasion.
The mean cell count (n = 4 separate experiments) of 639.3 + 191.6 (SEM)
was used as the 100% value. The absolute value for pre-eclamptic cells was
45.7 + 20.2. The difference between the two groups was statistically significant (P

0.037).

CTBs isolated from placentas of patients with pre-eclampsia (n = 4, 24 to 32


weeks) and control patients (n = 4, 23 to 33 weeks) were cultured on
Matrigel-coated coverslips in serum-free medium for 0, 12, 24, 36, and 48
hours. The cells were fixed and stained with a monoclonal antibody to
HLA-G as described in Materials and Methods. The percentage of CK-positive
cells that also expressed HLA-G was '500s of control levels when the
pregnancy was complicated by pre-eclampsia. Except for time 0 (P = 0.054),
the differences between the two groups were statistically significant (P value
of 0.01, 0.03, 0.002, and 0.03 at 12, 24, 36, and 48 hours, respectively).

1816

Lim et al

AJP December 1997, Vol. 151, No. 6

-266

-168

HLA-GO

Figure 9. CTB expression of HLA-G mRNA is markedly reduced in preeclampsia. The blot shown in Figure 6A was stripped and rehybridized with
a cDNA probe that corresponds to a 450-bp region of the 3' untranslated
region of HLA-G. PE, pre-eclampsia; CON, control.

than gestation-matched control cells. These data (diagrammed in Figure 10) suggest that, in pre-eclampsia,
cytotrophoblasts differentiating along the invasive pathway cannot complete this process. As a result, they do
not express normal levels of molecules that are required
for invasion (eg, integrin ca1 and MMP-9), a factor that
could restrict invasion to the superficial portions of uterine
arterioles, the placental pathology most frequently associated with pre-eclampsia. Our finding that HLA-G expression is also impaired raises the interesting possibility
that interactions between the invasive subpopulation of
cytotrophoblasts and maternal immune cells change in
pre-eclampsia.
Understanding the end result of pre-eclampsia's effect
on cytotrophoblast differentiation/invasion is helping us to
identify the factors that are critical to normal cytotrophoblast invasion. To date we have identified two circum-

stances in which normal cytotrophoblasts exhibit elements of the pre-eclampsia phenotype. The first is when
first-trimester cytotrophoblasts are cultured in an environment of reduced oxygen tension. We hypothesized that
the lack of endovascular invasion could lead to hypoxia
at the maternal-fetal interface, initiating a feedback
mechanism in which a reduction in oxygen tension further
impairs cytotrophoblast differentiation/invasion. To test
this idea experimentally, we compared the differentiative
and invasive capacity of normal first-trimester cytotrophoblasts cultured either under standard tissue culture conditions (20% 02) or in a hypoxic environment (2% 02). We
previously showed that cytotrophoblasts cultured in 20%
02 undergo normal differentiation along the invasive
pathway, ie, switch their repertoire of stage-specific antigens, and rapidly degrade ECMs on which they are
plated. In hypoxia, the cells' integrin expression is altered
to the pattern observed in pre-eclampsia; ie, they express integrin a5 but not cr1. In addition, their ability to
invade ECM is markedly reduced.29 These results suggest that a reduction in oxygen tension at the maternalfetal interface can lead to abnormalities in cytotrophoblast differentiation/invasion that are similar to those
observed in pre-eclampsia.
The second circumstance in which normal cytotrophoblasts exhibit a pre-eclampsia phenotype is at term.
Starting with the earliest-gestation samples that are routinely available to us (6 weeks), we have noted a progressive decline in the cytotrophoblasts' differentiative and
invasive capacity as gestation progresses. With regard to
differentiation, cells isolated from term placentas have a
greatly reduced ability to up-regulate expression of integrins cr5 and al,11 MMP-9,8 and HLA-G23 in vitro. For
example, the percentage of normal 12- to 16-week cytotrophoblasts that react with antibody against integrin ar1

Normal Pregnancy

Preecfampsla

A. Morphology
FETUS

iAIL

fhnloww
iviovp
ysvv
FE'TUS
(plcenta

,~~~~~abM

M H
MO~~~~~~(tHerus

(place

CTS bMW~ ~ ~ ~ U6

04,

MOTHmER
bod

8w~~

Zoel

Zone/im

zne iv

B. Differendtiation
cra Smm CeliS _0 a" 1/HLAG -_w- azPI/MMpm9

I~

ZoneI

zon I/m

ZoneIV

zo I

B.

zoua/m

zoe IV

Dlfferentiadon

cm SoC s u.asIptIl&A-G@.zpllMMp.,
N g
Z !

EwzarV

Figure 10. Schema illustrating how pre-eclampsia changes cytotrophoblast invasion at the morphological and molecular levels. Comparison of the morphology
of the matemal-fetal interface in normal pregnancy (left, A) and in pre-eclampsia (right, A) shows that this syndrome is associated with abnormally shallow uterine
invasion and, in particular, with failure of CTB to penetrate the uterine arterioles. We theorize that these morphological abnormalities are the result of a defect
in CTB differentiation (B). Our data suggest that in pre-eclampsia, the cells start to differentiate along the invasive pathway (as shown by their ability to express
integrin a5) but do not complete this process (as shown by their reduced ability to express HLA-G, integrin arl, and MMP-9).

Abnormal Cytotrophoblast Differentiation/Invasion in Pre-Eclampsia

1817

AJP December 1997, Vol. 151, No. 6

increases from 15% immediately after isolation to 60%


after 48 hours in culture.11 In contrast, only 2% of term
cytotrophoblasts initially react with the antibody, and this
percentage does not change during the culture period.11
The percentage of integrin-al-positive cells noted in the
gestation-matched controls for the experiments reported
here (23 to 33 weeks; Figure 3) was intermediate between the values for early second-trimester and term
cytotrophoblasts. With regard to invasion, the cells' ability
to penetrate ECM substrates is dramatically reduced at
term.9 Thus, the placental defects that occur in preeclampsia could also be attributed to an acceleration of
the normal, gestation-related changes that result in cytotrophoblast loss of differentiative/invasive capacity at
term.
The data presented in this paper do not allow us to
differentiate unequivocally between these two possible
explanations for why cytotrophoblast invasion is abnormal and placentation shallow in pre-eclampsia. Nevertheless, taken together, our results to date tend to favor
the hypothesis that disease-related alterations in the differentiative/invasive capacity of cytotrophoblasts are the
result of placental hypoxia rather than an acceleration of
changes that normally occur as pregnancy advances.
The strongest evidence we have to support this theory
comes from analysis of the cells' integrin al expression in
a variety of circumstances. Although term cells cannot
up-regulate expression of this adhesion molecule in vitro
(eg, differentiate1 1), cytotrophoblasts in tissue sections of
the uterine wall express al at the end of normal pregnancy but not in pre-eclampsia.17 The discordance in
integrin al expression in these two situations suggests
that the effects of gestation-related changes and preeclampsia on cytotrophoblast differentiation may be separable phenomena. If this is the case, then it is likely that
at least some of the phenotypic alterations that are associated with invasive cytotrophoblasts in pre-eclampsia
can be attributed to a reduction in oxygen tension at the
maternal-fetal interface. Currently, we are continuing to
test this hypothesis by determining whether hypoxia and
pre-eclampsia have similar or different effects on a number of other parameters that allow assessment of the
cells' functional and differentiative capacity.
Our data also suggest that, by the time the symptoms
of pre-eclampsia necessitate delivery, the deleterious effects of the disease on cytotrophoblasts cannot be reversed. The interesting question remains as to whether
the inability of these cells to differentiate/invade is a primary cause of the disease or is secondary to the pathogenesis. With regard to primary defects, transcriptional
mechanisms that regulate trophoblast differentiation in
the mouse have recently been described,3132 and it is
likely that similar mechanisms play an important role in
human placental development. Thus, it is possible that
alterations in the expression of these genes, or the regulatory pathways in which they operate, could be associated with some cases of pre-eclampsia. However, the
critical role that these genes play in development suggests that such mutations might be relatively rare, and
their effects might lead to abortion, rather than faulty
placentation. Thus, it seems more likely that the placental

defects that occur in pre-eclampsia are the downstream


consequences of the pathogenesis of the disease. If this
is the case, then understanding the association between
pre-eclampsia and the reduced capacity of cytotrophoblasts to differentiate/invade necessitates a better understanding of the events that lie upstream of the placental
pathology. In either case, knowing the phenotype of the
cytotrophoblast defect that is associated with preeclampsia is critical for determining whether the inability
of the cells to differentiate/invade is the cause or the
result of this disease.

Acknowledgments
We thank Ms. Evangeline Leash for editing the manuscript.

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