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Human Reproduction Vol.19, No.1 pp.

191195, 2004

DOI: 10.1093/humrep/deg512

Prevalence of circulating procoagulant microparticles in


women with recurrent miscarriage: a case-controlled study
Howard Carp1,2,4, Rima Dardik3, Aharon Lubetsky3, Ophira Salomon3, Regina Eskaraev3,
Esther Rosenthal3 and Aida Inbal3
1

Department of Obstetrics & Gynecology, 2Department of Embryology and 3Institute of Thrombosis and Hemostasis, Sheba Medical
Center, Tel Hashomer, and Sackler Faculty of Medicine, Tel Aviv University, Israel
4

To whom correspondence should be addressed at: Department of Obstetrics & Gynecology, Sheba Medical Center, Tel Hashomer,
52621, Israel. E-mail: carp@netvision.net.il

Key words: circulating endothelial microparticles/habitual abortion/recurrent pregnancy loss

Introduction
Pregnancy itself is a hypercoaguable state associated with
increased levels of coagulant factors (Stirling et al., 1984) and
decreased levels of naturally occurring anticoagulants such as
protein S (Comp et al., 1986) and brinolysis due to increased
plaminogen activator inhibitor-1 (PAI1) (Sattar et al., 1999).
Thrombosis in decidual vessels is believed to be one possible
cause of recurrent miscarriage. Antiphospholipid antibodies
(aPL) can cause pregnancy loss (Blank et al., 1991; Bakimer
et al., 1992), and thrombosis has been described as one of the
mechanisms whereby aPL may cause pregnancy loss (De Wolf
et al., 1982). More recently, hereditary thrombophilias have
been described as risk factors for pregnancy loss (Preston et al.,
1996; Rai et al., 1996; Brenner et al., 1997; Grandone et al.,
1997; Tal et al., 1999; Martinelli et al., 2000). Greer (2001) has
suggested that various complications of pregnancy, including
pregnancy loss, may be associated with procoagulant changes
in general, rather than congenital or acquired thrombophilias in
particular.
Circulating microparticles can be produced from the cell
membrane after apoptotic cell death. They are released

following cell activation when there is remodelling of the


membrane leading to externalization of phospholipids, such as
phosphotidylserine. Microparticles in turn lead to increased
expression of adhesion molecules (Barry et al., 1997; Mesri
and Altieri, 1998), so amplifying the procoagulant and/or
inammatory response on the endothelial cell surface.
Microparticles have been found in increased numbers in
normal pregnancy (Bretelle et al., 2003), and have been
associated with several prothrombotic conditions such as
thrombotic thrombocytopenic purpura (Galli et al., 1996),
myocardial infarction (Mallat et al., 2000), sepsis (Joop et al.,
2001), heparin-induced thrombocytopenia (Hughes et al.,
2000; Walenga et al., 2000) and even pre-eclampsia (Greer,
1999; Bretelle et al., 2003).
Laude et al. (2001) have measured total microparticles in
women with a history of recurrent pregnancy loss compared
with controls, and found microparticle levels to be above the
upper limit of normal in 59% of patients. The purpose of this
study was to assess the prevalence of circulating endothelial
microparticles in a large group of recurrently miscarrying
women, compared with that of parous control women.

Human Reproduction vol. 19 no. 1 European Society of Human Reproduction and Embryology 2004; all rights reserved

191

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BACKGROUND: Circulating microparticles are markers of cell activation associated with various prothrombotic
states. As hypoxia due to uteroplacental thrombosis is considered to be one of the causes of pregnancy loss, microparticles may be associated with pregnancy loss, in addition to, or as part of, other procoagulant states such as antiphospholipid syndrome or hereditary thrombophilias. The objective of this study was to examine the prevalence of
circulating microparticles in women with recurrent pregnancy loss. METHODS: A total of 96 women with recurrent pregnancy losses were enrolled in a casecontrol study and compared with 90 parous women. Microparticles
were measured by ow cytometry using uorescent anti-CD51/CD31 antibodies. RESULTS: Microparticle levels >2
SD above the mean of controls (57 700/ml) were detected in 12 out of 96 women with recurrent miscarriages
(12.5%), compared with two of the 90 control women (2.2%), P < 0.008. The titre of microparticles did not correlate
with age, number of pregnancy losses, primary secondary or tertiary aborters status, or with pregnancy losses in
the 1st or 2nd trimesters. CONCLUSIONS: A proportion of women with pregnancy loss have elevated endothelial
microparticles suggesting that endothelial damage or activation might be associated with the pathogenesis of pregnancy loss.

H.Carp et al.

Materials and methods

Laboratory testing
Circulating endothelial microparticles were measured in platelet-poor
plasma (PPP), 34 months after the previous miscarriage, according to
the method of Combes et al. (1999). Briey, PPP was obtained from
nine parts of whole blood drawn into one part of 3.8% sodium citrate,
centrifuged for 10 min at 1500 g, followed by centrifugation of PPP
for 5 min at 13 000 g, and stored at 35C. Aliquots of 100 ml of
plasma were incubated for 30 min at 4C with 10 ml of PhycoErythrinconjugated PECAM monoclonal antibody to CD31 (Immunotech,
France) and 10 ml of FITC-conjugated av monoclonal antibody to
CD51 (Immunotech). 105 beads of 3 mm (Sigma, USA) were added
and the number of uorescence-positive microparticles was quantied
by ow cytometry on a Coulter Epics (Coulter, UK). Thrombophilia
markers, Factor V Leiden, Factor II G20210A and MTHFR C677T
were measured as previously described (Carp et al., 2002).
An increased level of endothelial microparticles was dened as a
level >2 SD from the mean of control parous women.

Statistical analysis
The data were entered into a computerized database (SPSS, Chicago,
USA). The unpaired Student's t-test was used to compare continuous
variables between patients and controls, and c2 with Yates' correction
for categorical variables. Fisher's exact test was used where the
numbers were small. P < 0.05 was taken to be statistically signicant.

192

Age (years),
median 6 SD (range)
Factor V Leiden (%)
FII G20210A (%)
MTHFR 677Ta (%)
Total (%)

Recurrent
pregnancy loss
(n = 96)

Controls

(n = 90)

31.5 6 5 (1942)

35.2 6 6.9 (2552)

0.001

4 (4.2)
4 (4.2)
12 (12.5)
20 (20.8)

5 (5.56)
5 (5.56)
8 (8.89)
18 (20)

NS
NS
NS
NS

aRefers to the homozygous state.


NS = non-signicant.

Figure 1. Distribution of microparticles in patients and controls.


EMP = endothelial microparticles.
Linear regression analysis was used to assess whether age had an
inuence on microparticle levels.

Results
The 96 cases were slightly older than the controls (P = 0.001)
(Table I). There was no difference in the prevalence of the
measured thrombophilia markers between patients and controls
(Table I).
The distribution of microparticles in the peripheral blood of
cases and parous control women was parametric, ranging from
0 to 120 000/ml in cases and 0 to 71 000/ml in controls. Levels
>2 SD from the mean of control women were considered to be
abnormal. The cut-off value was 57 700/ml. Twelve out of 96
patients with recurrent miscarriages had an increased number
of circulating endothelial microparticles (12.5%), compared
with only two of 90 controls (2.2%) (P < 0.008). Figure 1
shows the distribution of microparticle levels for each patient
and control tested. It can be seen that a signicantly higher
proportion of women with recurrent miscarriage had raised
microparticle levels. Linear regression analysis showed no
association between age and endothelial microparticles. The
odds ratio for recurrent miscarriage in the presence of elevated

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Patients
Between 1998 and 2001, 96 consecutive patients with three or more
consecutive miscarriages (cases) were assessed for microparticles.
The control group consisted of 90 healthy women, with at least two
living children and no miscarriages, no concurrent disease, on no
medication, with no previous history of thromboses, and no
antinuclear factor, or aPL. Most of the patients and controls were
obtained from the cohort included in our previous publication (Carp
et al., 2002).
The clinical features of each patient and her miscarriages were
recorded, paying particular attention to whether the patients were
primary, secondary or tertiary aborters (primary aborters were women
with no previous live births, secondary if there was a live birth
followed by miscarriages, and tertiary if there were miscarriages
followed by a live birth and at least three subsequent miscarriages).
The following additional features were also noted: whether previous
miscarriages occurred in the rst or second trimesters, whether they
presented as blighted ova, fetal deaths etc., and the presence of
hereditary thrombophilias, e.g. Factor V Leiden and prothrombin gene
(G20210A) mutations and methyltetrahydrofolate reductase
(MTHFR) homozygosity determined as previously described (Carp
et al., 2002).
Cases or controls were excluded from this study if there was a
previous history of thrombosis, pregnancy at the time of investigation,
or if using oral contraceptives. Cases were only included after other
presumptive aetiological factors were found to be normal: karyotype
of both parents, glucose tolerance test, toxoplasmosis serology,
hysterosalpingogram, thereby excluding anatomical abnormalities,
intrauterine adhesions and cervical incompetence, thyroid function,
serum prolactin levels, normal luteal phase of >12 days and plasma
progesterone >24.8 ng/ml, absence of antinuclear factor, or aPL.
The Human Investigation Review Board of the Sheba Medical
Center approved the study. Each patient signed an informed consent
form.

Table I. Prevalence of thrombophilia polymorphisms in women with


recurrent pregnancy loss and controls

Microparticles and recurrent miscarriage


Table II. Details of cases with raised microparticles compared with normal microparticle levels

Age (years), mean 6 SD


Mean no. of miscarriages 6 SD (range)
1st trimester miscarriages only (%)
1st and 2nd trimester miscarriages (%)
Prevalence of thrombophiliasa (%)

Elevated microparticle levels


(n = 12)

Normal microparticle levels


(n = 84)

33.4 6 6.3
4.25 6 1.6 (38)
9 (75)
3 (25)
5 (41.6)

31.8 6 5.4
4.23 6 1.99 (316)
61 (72.6)
23 (27.4)
38 (45.2)

NS
NS
NS
NS
NS

aThrombophilias refers to the presence of any one of the following: Factor V Leiden, prothrombin gene (G20210A) mutation, MTHFR homozygosity, proteins
C, S or antithrombin deciencies.

Discussion
The results showed that a signicantly higher number of
recurrently miscarrying women had increased levels of circulating endothelial microparticles (12.5% as opposed to 2.2% of
parous control women), indicating that endothelial microparticles are associated with pregnancy loss. The odds ratio for
recurrent miscarriage was 6.29 (95% CI = 1.3728.93) in the
presence of endothelial microparticles. The presence of
endothelial microparticles was independent of maternal age,
number of prior miscarriages or hereditary thrombophilias.
None of the patients in this series had any apparent
thrombotic phenomena. It is conceivable that other procoagulant features of pregnancy, such as those listed in the
introduction, or possibly the activation of Th-1 cytokines
such as tumour necrosis factor (TNF)-a and interleukin-6
(which have been reported to initiate thrombosis, by causing
release of tissue factor (Ten Cate et al., 1999), may be
necessary to effect thrombosis. In other conditions of
prothrombotic states leading to pregnancy loss, such as aPL
or congenital thrombophilias, the association with second
trimester loss has been much greater than the association with
rst trimester loss (Lockshin, 1992; Carp et al., 1993; Preston
et al., 1996; Martinelli et al., 2000). However, in this series,
there was no signicant association between the prevalence of
microparticles and the clinical presentation of the pregnancy
loss (Table II). In fact, 75% of the women with raised
microparticle levels had only experienced rst trimester
miscarriages.
Microparticles have been shown to be elevated in normal
pregnancy (Bretelle et al., 2003), indicating an ongoing process
of cell activation in pregnancy. Constant deportation of

trophoblast also occurs into the maternal circulation.


Extrusion of trophoblast may lead to the formation of
microparticles. The question arises whether endothelial
microparticles may be causative of pregnancy loss or may be
a byproduct of embryonic demise. In the case of rst trimester
miscarriage, 2960% of recurrent pregnancy losses are due to
chromosomal aberrations (Stern et al., 1996; Ogasawara et al.,
2000; Carp et al., 2001), which invariably cause abortion,
irrespective of the presence of microparticles, or other
associations or causes of pregnancy loss. Structural anomalies
have been found in 10 of 19 missed abortions examined by
transcervical embryoscopy (prior to curettage) (Philipp and
Kalousek, 2001). Embryonic structural anomalies have been
associated with increased cytokines such as transforming
growth factor b (Jaskoll et al., 1996), TNFa (Ivnitsky et al.,
1998), and apoptosis (Torchinsky et al., 1995). The above
suggest that embryonic death may occur due to genetic or
structural anomalies, proinammatory cytokines or inappropriate apoptosis. All of these mechanisms may trigger subsequent microparticle formation and thrombosis. However, in
certain patients, circulating endothelial microparticles may
themselves act as a trigger for thrombosis and subsequent loss
of a normal pregnancy.
The only other paper to examine microparticles in women
with pregnancy losses was that of Laude et al. (2001). In their
paper, microparticles were found in 59% of patients with
recurrent pregnancy losses. The lower prevalence in the current
study may be due to assessing endothelial microparticles rather
than total microparticles. Endothelial microparticles are only a
fraction of the total microparticles. This study concentrated on
endothelial microparticles as endothelial activation and
pertubation may contribute to placental dysfunction and
subsequent miscarriage. The presence of endothelial microparticles in the interval between pregnancies may be a chronic
state of blood vessel activation which only becomes apparent in
pregnancy.
The obstetric complications in which microparticles have
been studied include pre-eclampsia and intrauterine growth
restriction. Microparticles have been reported to have a
causative role in pre-eclampsia by impairing endotheliumdependent relaxation of isolated myometrial arteries in vitro
(Van Wijk et al., 2002a), and increasing thrombin generation
(Van Wijk et al., 2002b). In pre-eclampsia, the number of
platelet microparticles has been reported to be decreased
(Bretelle et al., 2003) which may reect their consumption in
thrombus generation.
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endothelial microparticles is 6.29 [95% condence interval


(CI) = 1.3728.93].
Table II shows the clinical features of the patients with
normal and increased levels of microparticles. Both groups of
patients were similar with regard to the mean number and
trimester of previous miscarriages. Although the recurrently
miscarrying women were younger than the control women,
there was no signicant age difference between recurrently
miscarrying women with normal or raised microparticle levels.
Due to the small number of patients with raised microparticle
levels (n = 12), no inferences can be drawn about different
prevalences of hereditary thrombophilias in the patients with
raised and normal endothelial microparticle levels.

H.Carp et al.

If microparticles do cause thrombosis in certain patients with


recurrent pregnancy loss, the question arises whether anticoagulants are indicated to prevent subsequent thrombosis, and
subsequent pregnancy losses. Although anticoagulants have
been reported to have a benecial effect in antiphospholipid
syndrome (Kutteh, 1996; Rai et al., 1997) and hereditary
thrombophilia (Carp et al., 2003) they may also have an effect
in patients with increased levels of procoagulant microparticles. However, before this question can be answered, it is
necessary to have a comparative cohort study comparing the
prognosis for the subsequent pregnancy in patients with and
without increased circulating microparticles. The results of
such a study should be corrected for patients losing genetically
or otherwise aberrant embryos, as anticoagulants are administered for maternal conditions which cause the loss of normal
embryos.

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Submitted on May 9, 2003; resubmitted on June 24, 2003; accepted on
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