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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References
Bakimer R, Fishman P, Blank M, Sredni B, Djaldetti M and Shoenfeld Y
(1992) Induction of primary antiphospholipid syndrome in mice by
immunization with a human monoclonal anticardiolipin antibody (H-3). J
Clin Invest 89,15581563.
Barry OP, Practico D, Lawson JA and Fitzgerald GA (1997) Transcellular
activation of platelets and endothelial cells by bioactive lipids in platelet
microparticles. J Clin Invest 99,21182127.
Blank M, Cohen J, Toder V and Shoenfeld Y (1991) Induction of antiphospholipid syndrome in naive mice with mouse lupus monoclonal and
human polyclonal anti-cardiolipin antibodies. Proc Natl Acad Sci USA
88,30693073.
Brenner B, Mandel H, Lanir N, Younis J, Rothbart H, Ohel G and Blumenfeld
Z (1997) Activated protein C resistance can be associated with recurrent
fetal loss. Br J Haematol 97,551554.
Bretelle F, Sabatier F, Desprez D, Camoin L, Grunebaum L, Combes V,
D'Ercole C and Dignat-George F (2003) Circulating microparticles: a
marker of procoagulant state in normal pregnancy and pregnancy
complicated by preeclampsia or intrauterine growth restriction. Thromb
Haemost 89,486492.
Carp HJA, Menashe Y, Frenke Y, Many A, Nebel L, Toder V, Serr DM and
Mashiach S (1993) Lupus anticoagulant: signicance in rst trimester
habitual abortion. J Reprod Med 38,549552.
Carp HJA, Toder V, Orgad S, Aviram A, Danieli M, Mashiach S and Barkai G
(2001) Karyotype of the abortus in recurrent miscarriage. Fertil Steril
5,678682.
Carp HJA, Salomon O, Seidman D, Dardik R, Rosenberg N and Inbal A
(2002) Prevalence of genetic markers for thrombophilia in recurrent
pregnancy loss. Hum Reprod 17,16331637.
Carp HJA, Dolitzky M and Inbal A (2003) Thromboprophylaxis improves the
live birth rate in women with consecutive recurrent miscarriages and
hereditary thrombophilia. J Thromb Hemost 1,433438.
Combes V, Simon AC, Grau GE, Arnoux D, Camoin L, Sabatier F, Mutin M,
Sanmarco M, Sampol J and Dignat-George F (1999) In vitro generation of
endothelial microparticles and possible prothrombotic activity in patients
with lupus anticoagulant. J Clin Invest 104,93102.
Comp PC, Thurnau G.R, Welsh J, Esmon CT (1986) Functional and
immunologic protein S levels are decreased during pregnancy. Blood
68,881885.
De Wolf F, Carreras LO, Moerman P, Vermylen J, Van Assche A and Renaer
M (1982) Decidual vasculopathy and extensive placental infarction in a
patient with repeated thromboembolic accidents, recurrent fetal loss, and a
lupus anticoagulant. Am J Obstet Gynecol 142,829834.
Galli M, Grassi A and Barbui T (1996) Platelet-derived microvesicles in
thrombotic thrombocytopenic purpitra and hemolytic uremic syndrome.
Thromb Haemost 75,427431.
Grandone E, Margaglione M, Colaizzo D, D'Addedda M, Cappucci G,
Vecchione G, Scianname N, Pavone G and Di Minno G (1997) Factor V
Leiden is associated with repeated and recurrent unexplained fetal losses.
Thromb Haemost 77,822824.

Greer IA (1999) Thrombosis in pregnancy: maternal and fetal issues. Lancet

353,12581265.
Greer IA (2001) Procoagulant microparticles: new insights and opportunities
in pregnancy loss? Thromb Haemost 85,34.
Hughes M, Hayward CP, Warkentin TE, Horsewood P, Chorneyko KA and
Kelton JG (2000) Morphological analysis of microparticle generation in
heparin-induced thrombocytopenia. Blood 96,188194.
Ivnitsky I, Torchinsky A, Gorivodsky M, Zemlyak I, Orenstein H, Savion S,
Shepshelovich J, Carp HJA, Fein A and Toder V (1998) Tnf-a expression in
embryos exposed to a teratogen. Am J Reprod Immunol 40,431440.
Jaskoll TH, Choy HA, Chen H and Melnick M (1996) Developmental
expression and CORT-regulation of TGF-b and EGE receptor mRNA
during mouse palatal morphogenesis: correlation between CORT-induced
cleft palate and TGF-b2 mRNA expression. Teratology 54,3444.
Joop K, Berckmans RJ, Nieuwland R, Berkhout J, Romijn FP, Hack CE and
Sturk A (2001) Microparticles from patients with multiple organ
dysfunction syndrome and sepsis support coagulation through multiple
mechanisms. Thromb Haemost 85,810820.
Kutteh WH (1996) Antiphospholipid antibody-associated recurrent pregnancy
loss: treatment with heparin and low-dose aspirin is superior to low-dose
aspirin alone. Am J Obstet Gynecol 174,15841589.
Laude I, Rongieres-Bertrand C, Boyer-Neumann C, Wolf M, Mairovitz V,
Hugel B, Freysinnet JM, Frydman R, Meyer D and Eschwege V (2001)
Circulating procoagulant microparticles in women with unexplained
pregnancy loss: a new insight. Thromb Haemost 85,1821.
Lockshin MD (1992) Antiphospholipid antibody syndrome. J Am Med Assoc,
268,14511453.
Mallat Z, Benamer H, Hugel B and Benessiano J (2000) Elevated levels of
shed membrane microparticles with procoagulant potential in the peripheral
circulating blood of patients with acute coronary syndromes. Circulation
101,841848.
Martinelli L, Taioli E, Cetin I, Marinoni A, Gerosa S, Villa MV, Bozzo M and
Mannucci PM (2000) Mutations in coagulation factors in women with
unexplained late fetal loss. N Engl J Med 343,10151018.
Mesri M and Altieri DC (1998) Endothelial cell activation by leukocyte
microparticles. J Immunol 161,43824387.
Ogasawara M, Aoki K, Okada S and Suzumori K (2000) Embryonic karyotype
of abortuses in relation to the number of previous miscarriages. Fertil Steril
73,300304.
Philipp T and Kalousek, DK (2001) Transcervical embryoscopy in missed
abortion. J Assist Reprod Genet 18,285290.
Preston FE, Rosendaal FR, Walker ID, Briot E, Berntorp E, Conard J,
Fontcuberta J, Makris M, Mariani G et al. (1996) Increased fetal loss in
women with heritable thrombophilia. Lancet 348,913916.
Rai R, Regan L, Hadley E, Dave M and Cohen H (1996) Second-trimester
pregnancy loss is associated with activated C resistance. Br J Haematol
92,489490.
Rai R, Cohen H, Dave M and Regan L (1997) Randomised controlled trial of
aspirin and aspirin plus heparin in pregnant women with recurrent
miscarriage associated with phospholipid antibodies (or antiphospholipid
antibodies). Br Med J 314,253257.
Sarig G, Younis JS, Hoffman R, Lanir N, Blumenfeld Z and Brenner B (2002)
Thrombophilia is common in women with idiopathic pregnancy loss and is
associated with late pregnancy wastage. Fertil Steril 77,342347.
Sattar N, Greer IA, Rumley A, Stewart G, Shepherd J, Packard CJ and Lowe
GD (1999) A longitudinal study of the relationships between haemostatic,
lipid, and oestradiol changes during normal human pregnancy. Thromb
Haemost 81,7175.
Stern JJ, Dorfman AD and Gutierez-Najar MD (1996) Frequency of abnormal
karyotype among abortuses from women with and without a history of
recurrent spontaneous abortion. Fertil Steril 65,250253.
Stirling Y, Woolf L, North WR, Seghatchian MJ and Meade TW (1984)
Haemostasis in normal pregnancy. Thromb Haemost 52,176182.
Tal J, Schliamser LM, Leibovitz Z, Ohel G and Attias D (1999) A possible
role for activated protein C resistance in patients with rst and second
trimester pregnancy failure. Hum Reprod 14,16241627.
Ten Cate H, Timmerman JJ and Levi M (1999) The pathophysiology of
disseminated intravascular coagulation. Thromb Haemost 82,713717.
Torchinsky A, Savion S, Gorivodsky M, Shepshelovich J, Zaslavsky Z, Fein A
and Toder V (1995) Cyclophosphamide-induced teratogenesis in ICR mice:
the role of apoptosis. Teratogen Mutagen Carcinogen 15,179190.
Van Wijk MJ, Svedas E, Boer K, Nieuwland R, Vanbavel E and Kublickiene
KR (2002a) Isolated microparticles, but not whole plasma, from women
with preeclampsia impair endothelium-dependent relaxation in isolated

Microparticles and recurrent miscarriage

myometrial arteries from healthy pregnant women. Am J Obstet Gynecol
187,16861693.
Van Wijk MJ, Boer K, Berckmans RJ, Meijers JC, Van der Post JA, Sturk A,
VanBavel E and Nieuwland R (2002b) Enhanced coagulation activation in
preeclampsia: the role of APC resistance, microparticles and other plasma
constituents. Thromb Haemost 88,415420.

Walenga JM, Jeske WP and Messmore HL (2000) Mechanisms of venous and

arterial thrombosis in heparin-induced thrombocytopenia. J Thromb
Thrombolysis 10 (Suppl. 1), 1320.
Submitted on May 9, 2003; resubmitted on June 24, 2003; accepted on
September 16, 2003

Downloaded from http://humrep.oxfordjournals.org/ by guest on October 6, 2015

195