Placenta (2006), 27, 56e61doi:10.1016/j.placenta.2004.11.007

Excess Syncytiotrophoblast Microparticle Shedding is a

Feature of Early-onset Pre-eclampsia, but not Normotensive

Intrauterine Growth Restriction

D. Goswamia, D. S. Tannetta

b, L. A. Magee

c,d, A. Fuchisawa

a, C. W. G. Redman

b,

I. L. Sargentband P. von Dadelszen

a,d,*

a Department of Obstetrics and Gynaecology, University of British Columbia, 4500 Oak Street, Vancouver BC V6H 3N1,Canada; b Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women’s Centre, John Radcliffe Hospital,Headley Way, Oxford, Oxon OX3 9DU, UK; c Department of Medicine, University of British Columbia, 4500 Oak Street,Vancouver BC V6H 3N1, Canada; d Centre for Healthcare Innovation and Improvement, University of British Columbia, 4500Oak Street, Vancouver BC V6H 3N1, Canada

Paper accepted 8 November 2004

Rationale: Syncytiotrophoblast microparticles (STBM) are shed into the maternal circulation in higher amounts in pre-eclampsia

compared to normal pregnancy and are believed to be the stimulus for the systemic inflammatory response and endothelial cell

damage which characterises the maternal syndrome. The excess shedding of STBM may be caused by hypoxia as a result of poor

placentation, which is often a feature of pre-eclampsia. Similar placental pathology occurs in some cases of normotensive

intrauterine growth restriction (nIUGR), but in the absence of maternal disease.

Objective: To examine whether the shedding of STBM in nIUGR occurs to the same extent as in pre-eclampsia.

Methods: A prospective caseecontrol study in a tertiary referral centre of: 1) women with early-onset pre-eclampsia (EOPET

!34 week), 2) women with late-onset pre-eclampsia (LOPET R 34 week), 3) women with nIUGR), 4) matched normal pregnant

women (NPC), and 5) non-pregnant women. An ELISA using the antitrophoblast antibody NDOG2 was used to measure STBM

levels in peripheral venous plasma. Non-parametric analyses were conducted with statistical significance set at p! 0.05.

Results: STBM levels rise during normal pregnancy. EOPET was associated with increased STBM levels (EOPET (median):

41 ng/ml, nZ 15) compared with matched normal pregnancy (16 ng/ml, nZ 15; Wilcoxon pZ 0.005). LOPET (50 ng/ml,

nZ 10) and nIUGR (18 ng/ml, nZ 8) STBM levels did not differ from matched normal pregnancy (36 ng/ml, nZ 15, and

36 ng/ml, nZ 8, respectively). Background levels in non-pregnant plasma were 0.49 ng/ml, nZ 10.

Conclusions: Increased STBM levels were found in EOPET but not in nIUGR providing further evidence for their role in the

pathogenesis of the maternal syndrome.

Placenta (2006), 27, 56e61 � 2004 Elsevier Ltd. All rights reserved.

Keywords: Pre-eclampsia; Pregnancy; Fetal growth retardation; Placenta; Apoptosis; Syncytiotrophoblast

INTRODUCTION

Pre-eclampsia remains one of the most common causes of

maternal mortality in the developed world [1,2]. The maternal

syndrome of pre-eclampsia, characterised by hypertension and

proteinuria, defines the disease. When pre-eclampsia presents

remote from term, the fetus is at increased risk of intrauterine

growth restriction (IUGR). IUGR can also occur in the

absence of maternal hypertension, a state that we have termed

* Corresponding author. 2H30 e 4500 Oak Street, Vancouver BCV6H 3N1, Canada. Tel.: C1 604 875 3108; fax: C1 604 875 2725.E-mail address: pvd@cw.bc.ca (P. von Dadelszen).

0143e4004/$esee front matter

normotensive IUGR. Given that incomplete placentation is

shared by pre-eclampsia and normotensive IUGR [3], the

latter may represent the fetal consequences of a shared

placental pathology occurring in isolation.

The cogent model for the pathogenesis of the maternal

syndrome of pre-eclampsia describes a process by which

a placental factor is released into the maternal circulation,

which damages the maternal endothelium, causing a syndrome

of systemic endothelial dysfunction [8]. It is now apparent that

this endothelial dysfunction is part of a wider maternal

systemic inflammatory response which occurs in normal

pregnancy but is far more intense in pre-eclampsia [4e6].

The placental factor responsible is not known but candidates

� 2004 Elsevier Ltd. All rights reserved.

Goswami et al.: Excess Syncytiotrophoblast Microparticle Shedding 57

include sFlt-1, peroxides, eicosanoids, cytokines, and syncy-

tiotrophoblast microparticles (STBM).

We have previously shown that STBM prepared from

normal placentae cause endothelial cell dysfunction in vitro [7]

and in isolated vessels [8], and that pre-eclampsia plasma

inhibits endothelial cell proliferation [9]. STBM are detectable

in the plasma of pregnant women by both flow cytometry and

enzyme-linked immunosorbent assay (ELISA) [10] and

significantly higher levels were found in women with pre-

eclampsia [10]. A significant correlation was found between the

plasma concentration of STBM and endothelial inhibition,

suggesting that STBM may contribute to the maternal

endothelial dysfunction [10]. There is also an excess of

circulating cellular syncytial debris in pre-eclampsia [11]. The

release of syncytiotrophoblast debris into the maternal

circulation is thought to be the result of syncytial apoptosis,

which is part of a normal process of turnover and repair [12]

and/or necrosis [13]. Syncytiotrophoblast apoptosis is in-

creased in pre-eclampsia [14] and this could explain the

increased debris in the maternal circulation. It has been

proposed that this increase in apoptosis may result from

oxidative stress in the placenta caused by a failure of spiral

artery adaptation leading to a poorly developed blood supply

[12]. The other consequence of this placental pathology is

intrauterine growth restriction (IUGR) of the fetus.

The poor placentation and fetal growth restriction seen in

some cases of pre-eclampsia, however, is not unique to this

disorder. Similar pathology is also seen in some, but not all,

cases of normotensive IUGR. Interestingly, increased syncytial

apoptosis has also been reported in these pregnancies [15]

which would be expected to result in the increased shedding of

syncytiotrophoblast debris. According to this hypothesis, this

should precipitate the maternal syndrome which it clearly does

not. This could be due either to a lack of increased shedding in

normotensive IUGR or a difference in the way that the

mother’s innate immune system and endothelial cells respond

in this condition. Therefore, the purpose of this study was to

measure STBM levels in the maternal circulation in normal

pregnancy and to compare them with those seen in pre-

eclampsia, normotensive IUGR, and non-pregnancy.

METHODS

This was a prospective caseecontrol study using clinical

plasma samples obtained from the maternity services at

a tertiary referral centre (Children’s and Women’s Health

Centre of British Columbia). These samples were frozen

at �80 (C and transported to Oxford, UK, for analysis.

Pre-eclampsia was defined by the criteria of the National

High Blood Pressure Education Program [16]. Only singletons

were investigated. IUGR was defined as either an ultrasound

estimate of fetal weight or an ultrasound measurement of the

fetal abdomen !5th centile for gestational age, confirmed at

delivery (birthweight !5th centile for age and gender) and

associated with neither aneuploidy, structural anomalies, nor

congenital infection. The histopathology diagnoses of all

women were reviewed, when available, to confirm the presence

or absence of abnormal placental findings in cases and controls,

respectively.

Following informed consent, peripheral venous blood was

drawn from the following:

1. 15 women with early-onset pre-eclampsia (!34 weeks’

gestation),

2. 10 women with late-onset pre-eclampsia (R 34 weeks’

gestation),

3. 10 women with normotensive IUGR (abdominal circum-

ference !5th centile for gestational age with birthweight

!5th centile confirmed postnatally, excluding both

aneuploidy and congenital infections),

4. 35 normal pregnant women matched for age (G5 years),

gestation (G14 days) and parity (0, 1, R 2) (one control

per case in groups 1e3), and

5. 10 non-pregnant women aged 20e40 years, not using

hormonal contraception.

The sample collection was co-ordinated by a dedicated full-

time research co-ordinator and was approved by both the

University of British Columbia Clinical Research Ethics Board

and the Children’s and Women’s Health Centre of British

Columbia Ethics Board. For women with both pre-eclampsia

and normotensive IUGR blood sampling was performed at the

time of diagnosis of the respective pregnancy complication.

Following informed consent, 5 ml of antecubital vein blood

was taken antenatally. The plasma was prepared from this

lithium heparin anticoagulated peripheral venous blood by

high speed centrifugation, and stored at �80 (C for transport

from Vancouver to Oxford. The tube containing the plasma

was thawed to room temperature and 2 ml of plasma was used

per sample assay. The sample was topped up with endotoxin

free phosphate buffered saline (PBS-E, Sigma, St Louis, MO),

ensuring the sample was diluted at least 1:2. The plasma/

PBS-E mixture was then transferred to an ultracentrifuge tube

(14! 89 mm Ultra-Clear tube, Beckman Coulter, High

Wycombe, Bucks, UK). To pellet any STBM, the samples

were spun on a Beckman L8-80M ultracentrifuge at 150,000gfor 45 min at 4 (C. This was based on a protocol known to

pellet ribosomes. The supernatant was discarded and the final

pellet was resuspended in 350 ml 0.1% bovine serum albumin

(BSA, Research Diagnostics Inc, Flanders, NJ) in PBS-E. The

samples were then transferred to 0.7 ml screw-top tubes and

kept at �80 (C until use.

Standards for the STBM enzyme-linked

immunosorbent assay (ELISA)

Syncytiotrophoblast microparticles (STBM) were prepared

from normal placentae by a modification of the method of

Smith et al. [7] and used as standards for the STBM ELISA.

The protein content of the STBM suspension was 9.9 mg/ml.

Fifty microlitres of this was added to 1 ml of diluting buffer

58 Placenta (2006), Vol. 27

(1% BSA, 0.05% Tween 20 (Sigma) in PBS-E) to make

a stock solution of 495 mg/ml. Eight quadruple dilutions from

4000 ng/ml down to 1 ng/ml were then prepared, using the

diluting buffer.

Measurement of free STBM in plasma

samples by ELISA

The STBM ELISA was developed ‘in house’ by Dr S Kumar

(DPhil thesis, University of Oxford). A 96-well Maxi Sorp

plate (Nunc plasticware, Life Technologies, Paisley, UK) was

coated with NDOG2 antibody, at a concentration of 10 mg/ml

(in PBS-E), using 100 ml per well. NDOG2 is an antitropho-

blast antibody which recognises placental alkaline phosphatase

[10]. The plate was then incubated overnight at room

temperature under moist conditions in a covered box. The

following day, the plate was washed five times, by hand, with

wash buffer (0.05% Tween 20 in TBS). To prevent any non-

specific binding, 300 ml of blocking buffer (5% BSA in PBS-E)

was added per well and left for at least 3 h at room

temperature. Following this blocking period, the plate was

then washed a further five times with wash buffer.

The plasma samples and standards were then added in

triplicate to the appropriate wells (100 ml per well) and

incubated overnight at room temperature in moist conditions

in a covered box.

The following day, the plate was washed 10 times with wash

buffer. The final step was an ELISA Amplification System

(Gibco BRL, Life Technologies, Paisley, Scotland, UK). This

utilized endogenous alkaline phosphatase on the STBM

microparticles as the enzyme for the colorimetric reaction.

Fifty microliters of neat substrate per well was added and left

for 1 h at room temperature. Without any further wash, 50 mlof neat amplifier per well was added. The colour started to

develop immediately. The plates were read at 2 time points,

after 5 min, on a MRX Microplate reader (Dynex Technol-

ogies, Billinghurst, W Sussex, UK) at 490 nm. The best

standard curve was obtained only after 5 min. The standard

curve was used to determine the STBM concentration in each

350 ml sample in ng/ml. As the samples had been concentrated

by ultracentrifugation, this figure had to be divided by

a concentration factor of x/0.35 (where xZ volume of plasma

in ml) in order to calculate the STBM level in the original

plasma sample.

A detectable relationship between gestational age and

STBM level prompted an evaluation of the ‘observed/

expected’ values, where the expected values were derived

from the linear regression equation for normal pregnancy

samples in this study and the gestational age at sampling. This

approach corrected for the influence of gestational age on the

data (and any influence of imperfect matching on the results)

and made the differences between groups more explicit.

Non-parametric (ManneWhitney U and Wilcoxon, as

appropriate) analyses were used for continuous variables, and

c2 for categorical variables. Statistical significance was set at

p! 0.05. Statistical and linear regression calculations were

made using Prism 3.0 (GraphPad Software Inc, SanDiego, CA).

RESULTS

The patient details are summarized in Table 1. Four of the 15

women with early-onset pre-eclampsia and one of the 10

women with late-onset pre-eclampsia delivered infants below

the 5th centile for sex and gestational age. When using the

10th centile of birthweight for gestational age as the definition

of ‘IUGR’ then 12 of early-onset pre-eclampsia infants

fulfilled that definition. Two cases of women identified

antenatally as normotensive IUGR were not confirmed

postnatally, and their data were removed from the analyses.

When subdivided by case type, the gestational ages at

sampling (median [range]) were 29.7 [25.3, 34.9], 35.9 [32.7,

40.3], and 33.8 [30.1, 37.6] weeks, for ‘‘early-onset pre-

eclampsia controls’’, ‘‘late-onset pre-eclampsia controls’’, and

‘‘normotensive IUGR controls’’, respectively.

Histopathology results were available for 12, eight, and

seven women with pregnancies complicated by early-onset

pre-eclampsia, late-onset pre-eclampsia, and normotensive

IUGR, respectively. All the subset of cases whose placentae

were examined had confirmed placental abnormalities (e.g.

Table 1. Clinical characteristics (n (%), median [range])

VariableEOPET(nZ 15)

LOPET(nZ 10)

nIUGR(nZ 10)

Normal pregnancy(nZ 35)

Non-pregnancy(nZ 10)

Age 33 [18, 42] 35 [30, 42] 30 [16, 37] 31.5 [23, 41] 31.5 [23, 40]Primigravid (%) 72.7 71.4 72.7 68.2GA at sampling (weeks) 30.9 [24.1, 33.4] 35.5 [34.0, 39.1] 33.3 [28.1, 39.0] 32.7 [25.3, 40.3]MAP (mmHg) 117 [100, 167] 117 [100, 143] 85 [75, 98] 89 [68, 101]Platelets (!1012/L) 229 [136, 380] 165 [114, 249]AST (mM) 28 [19, 193] 26 [16, 46]Uric acid (mM) 364 [221, 572] 407 [325, 518]Absent or reversed EDF 2 (13) 0 (0) 3 (30) 0 (0)GA at delivery (weeks) 31.6 [26.1, 38.0] 37.2 [34.3, 39.1] 37.7 [30.1, 41.7] 40.0 [37.7, 42.1]Birthweight (g) 1505 [530, 3440] 2360 [1565, 4445] 1815 [915, 2780] 3523 [2805, 4275]Placental abnormality 12 (100) (nZ 12) 8 (100) (nZ 8) 7 (100) (nZ 7) 0 (0) (nZ 1)

Goswami et al.: Excess Syncytiotrophoblast Microparticle Shedding 59

acute atherosis, syncytial knots, infarction, perivillus throm-

bosis, villitis of unknown aetiology, and advanced villus

maturation).

The plasma concentration of STBM for each group is shown

in Figure 1. The background levels for the assay are shown in

the non-pregnancy samples. Only early-onset pre-eclampsia

was associated with increased levels of STBM when compared

with matched normal pregnancy controls. STBM levels in both

late-onset pre-eclampsia and normotensive IUGR were similar

to those seen in normal pregnancy. However, there was

a tendency for late-onset pre-eclampsia to have higher levels of

STBM than normal pregnancy, and, when analysed as a single

group, all pre-eclampsia cases (45.4 ng/ml, nZ 25) were

associated with increased levels of STBM than matched

controls (20.4 ng/ml, nZ 25, Wilcoxon pZ 0.007).

The influence of gestational age on STBM concentration is

shown in Figure 2. There was a linear relationship between

STBM concentrations and gestational age for normal preg-

nancy. This linear relationship was consistent with that

determined previously by us (Germain S, DPhil thesis, Oxford,

submitted). Fifteen percent of the variation in STBM

concentration could be explained by gestational age alone.

There was no relationship between STBM concentrations

and parameters of clinical disease severity (mean arterial

pressure, total leukocyte count, uric acid, platelet count, mean

platelet volume, fibrinogen, aspartate transaminase, alanine

transaminase, and plasma albumin) among the pre-eclampsia

cases (data not shown).

DISCUSSION

These data show that the increased concentration of STBM

previously noted in pre-eclampsia [10] seems specific to

EO

P

ET

EO

P

ET

c

on

tro

ls

LO

P

ET

LO

P

ET

c

on

tro

ls

nIU

GR

nIU

GR

c

on

tro

ls

no

np

re

g

0

25

50

75

100

125

150

175

W p=0.005

MWu p<0.001

[N

DO

G2] (n

g/m

l)

Figure 1. Peripheral venous blood syncytiotrophoblast microparticle(NDOG2) concentrations in women with early-onset pre-eclampsia (EOPET;open triangles: birthweight !5th centile for gestational age), late-onset pre-eclampsia (LOPET; open triangles: birthweight !5th centile for gestationalage), normotensive intrauterine growth restriction (nIUGR), normal pre-gnancy, and non-pregnancy (nonpreg). Horizontal bars represent medianvalues. Non-pregnancy values were significantly lower than all pregnancygroups. MWu: ManneWhitney U test; W: Wilcoxon test.

pre-eclampsia, and that in cases of normotensive IUGR defined

solely by birthweight for gestational age there is no increase in

circulating STBM. This is despite the reported increase in

placental apoptosis and/or necrosis (as determined by infarc-

tion) in both pre-eclampsia and normotensive IUGR pregnan-

cies [15,16]. This suggests a central role for increased STBM

shedding in either the pathogenesis and/or the maintenance of

the maternal syndrome of pre-eclampsia [7,9e11,17].

The characteristic endothelial cell [18] and innate immune

cell [4,6] activation of pre-eclampsia may well be secondary to

this excess circulating trophoblast debris. We have previously

described the adverse effects of STBM on cultured endothelial

cell function [7] and isolated small arterial function [8].

Perturbation of in vitro endothelial cell function is plasma-

specific [9], and the effect of pre-eclampsia plasma on

endothelial cell function is directly related to STBM

concentration [9]. Disruption of normal endothelial cell

function in vitro may be mediated by adhesion molecules

expressed on the surface of STBM [19] and not by intrinsic

proteases [20]. Also, the conditioned medium from endothelial

cells co-cultured with STBM fragments activates peripheral

blood leukocytes in vitro [21].

The results from this study may reflect the differential rate

of syncytiotrophoblast apoptosis and/or necrosis noted

between pre-eclampsia and normotensive IUGR placentae

[15,16]. However, Ishihara and colleagues [15] also found

increased syncytiotrophoblast apoptosis in normotensive

IUGR compared to normal pregnancy placentae that we could

not confirm by examination of circulating trophoblast debris.

This may be explained, in part, by reduced villous area in

normotensive IUGR placentae, whereas the villous area of pre-

eclampsia placentae is preserved [22]. Therefore, a modest

increase in syncytiotrophoblast apoptosis and/or necrosis in

normotensive IUGR might be masked by the smaller placental

volume from which the STBM would be derived. While

peripherally sampled STBM may result from placental

apoptosis and/or necrosis [23], they are, at best, an indirect

measure of this process.

20 25 30 35 40 45

0

10

20

30

40

50

60

70

Gestational age (weeks)

[N

DO

G2

] (n

g/m

l)

Figure 2. Concentration of circulating syncytiotrophoblast microparticles(NDOG2) is associated with increasing gestational age for normal pregnancycontrols. SlopeZ 1.77; r2 Z 0.15; p Z 0.018.

60 Placenta (2006), Vol. 27

Another explanation might be that increased syncytiotro-

phoblast necrosis predominates in pre-eclampsia, while

apoptosis might be similar between IUGR and pre-eclampsia

placentae [24]. In this model, Huppertz and Kingdom [24]

state that it may be that apoptotic material released from the

placenta will be predominately corpuscular in nature, will be

mostly trapped in pulmonary capillaries lung and, therefore,

will not reach the systemic circulation. In contrast, it is

possible that increased amounts of subcellular necrotic

trophoblast material in pre-eclampsia will not be cleared by

the lungs and will be detected in peripheral blood. However,

we have determined that much of the STBM (and subcellular

debris from other sources) found in the maternal circulation

may be apoptotic in origin, in both normal and pre-eclampsia

pregnancies [23]. The concentrations of both STBM and

Annexin V-binding microparticles change in parallel in normal

pregnancy and pre-eclampsia [23]. The levels of Annexin V-

binding microparticles are raised in pre-eclampsia compared

with normal pregnancy and these could be both placental and

maternal in origin [23]. We have postulated that this debris is

capable of immune system modification in normal pregnancy

(Th1 downregulation [25] and monocyte activation [23,25]),

and that excessive placental debris of apoptotic and/or

necrotic origin stimulates the exaggerated maternal inflamma-

tory response characteristic of pre-eclampsia [4e6,25,26]. The

degree, duration, and reversibility of the ischaemia-reperfusion

insult endured by a placenta may control the proportion of

apoptotic versus necrotic debris found in the circulation

[27].

It is recognised that the large corpuscular syncytiotropho-

blast debris noted in uterine venous, but not in peripheral

venous, blood will be cleared by the pulmonary circulation.

This debris is seen in excess in women with pre-eclampsia [11].

Therefore, we feel that our findings reflect true differences

between the amounts of circulating placental debris in pre-

eclampsia, normotensive IUGR, and normal pregnancies.

It is important to note that the cases of normotensive IUGR

included pregnancies with abnormal uterine arterial (one case)

and umbilical arterial (two cases: absent end diastolic flow, and

one case: reversed end diastolic flow) Doppler velocimetry

waveforms. We accept that some of the normotensive IUGR

cases may have been constitutionally small fetuses, as uterine

arterial Doppler studies and placental pathology were not

performed on all cases.

These data support the view that there are changes specific to

the maternal syndrome of pre-eclampsia and not shared by

normotensive IUGR.We have previously found that abnormally

delayed neutrophil apoptosis [26] and levels of antibodies against

atherogenic organisms [28] were specific to pre-eclampsia, and

not found in normotensive IUGR. The Poston group has found

biomarkers (leptin, placenta growth factor, the plasminogen

activator inhibitor (PAI-1)/PAI-2 ratio, and uric acid) that

selectively predict the development of later pre-eclampsia,

whereas reduced ascorbate levels predicted the later develop-

ment of both pre-eclampsia and normotensive IUGR [29].

We have demonstrated the gestational age effect on STBM

shedding into the maternal circulation in normal pregnancy,

previously noted by us (Germain S, DPhil thesis, Oxford,

submitted). Therefore, although the absolute concentrations of

STBM were similar in both early- and late-onset pre-

eclampsia, when corrected for the effect of gestational age by

matching, the increase in circulating placental debris was

relatively greater in women with early-onset disease. What

remains unknown is whether or not increased placental debris

can be detected in the circulation prior to the onset of clinical

disease. This might help to determine whether the excess

shedding of STBM into the maternal circulation plays a central

role in the development of the maternal syndrome, or whether

the excess shedding occurs in response to other pathologies

(e.g., acute atherosis or ischaemia-reperfusion injury) and

plays a central role in the maintenance of the condition.

In addition, the pattern of the excessive STBM shedding

needs to be determined, as that might reflect recurrent

ischaemia-reperfusion injuries during the evolution of acute

atherosis. We speculate that placental ischaemia-reperfusion

events may underlie the episodic spikes in maternal blood

pressure and transient fluctuations in platelet counts and liver

enzyme abnormalities that can be observed in women

expectantly managed remote from term. Timing phlebotomy

to coincide with these transient events in women, and during

intervals between them, might help to determine the

mechanisms that underlie the deteriorating clinical syndrome

that compels clinicians to deliver women remote from term.

These findings contribute to our understanding of the role

played by trophoblast debris in the maternal syndrome of pre-

eclampsia. They may help to differentiate between the

mechanisms of disease that are specific to pre-eclampsia and

those shared with normotensive IUGR.

ACKNOWLEDGEMENTS

This study was funded, in Canada, by the Canadian Foundation for Women’s Health, the BC Research Institute for Children’s and Women’s Health, and the BC

Women’s Hospital and Health Centre Foundation. In the UK, Dionne Tannetta was supported by Action Research.

We gratefully acknowledge the help and support of Terry Viczko, Shelley Soanes, and Vesna Popovska for recruiting both cases and controls for this study, and

our medical, nursing, and clinical laboratory colleagues for their support. Finally, we thank Thurl Wilkins for her scientific and technical expertise brought to this

and many other projects over decades.

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