Introduction
ecurrent pregnancy loss or
miscarriage can be defined as the
loss of three or more successive
pregnancies before viability and includes
all pregnancy losses from the time of
conception until 24 weeks of gestation
[1, 2]. Pregnancy loss is divided into
biochemical and clinical loss. The
biochemical loss is a transient positive
pregnancy test without ultrasonic
visualization of the pregnancy [3].The
term clinical miscarriage is used when
ultrasound examination or histological
evidence has confirmed that an
intrauterine pregnancy has existed.
Clinical miscarriages can be subdivided
into early clinical pregnancy losses
(which is most common) that occur
before the twelfth week of gestation, and
late clinical pregnancy losses (which
constitute small proportion of pregnancy
losses) that occur in the twelfth week to
twenty first week of gestation [4,5].
Despite a wide range of investigations,
the cause of recurrent miscarriage
remains unknown (idiopathic) in more
than 50% of cases [6, 7], but several
hypotheses have been proposed [8]
including the following: genetic disorder,
uterine anatomic malformation,
immunological risk factors, infection,
endocrine disorders and thrombophilia.
Thrombophilia is a term which describes
the increased tendency of excessive
blood clotting due to inherited or
acquired causes. It may occur during
pregnancy, where there is an increase in
most clotting factors and decreased
levels of anticoagulant factors with
reduced fibrinolytic activity. This can
result in placental insufficiency and
abortion [9,10].Factors associated with
thrombophilia include Factor V Leiden
(FVL) mutation associated activated
Protein C Resistance (APCR),
prothrombin G20210A gene mutation,
anti-thrombin III deficiency, protein S
deficiency, protein C deficiency and
hyperhomocysteinaemia (methylenetetrahydrofolate
reductase mutation,
MTHFR) stasis and endothelial cell
dysfunction[11].
Protein C (PC) is a vitamin Kdependent
glycoprotein activated
bythrombin-thrombomodulin complex on
the surface of endothelial cells [12].
Protein C is a precursor of the serine
protease, activated protein C (APC) [13].
In the presence of protein S which is a
cofactor for APC, phospholipidsand
calcium, APC inactivates membrane
boundFVa and FVIIIa so results in
attenuation of thrombin generation which
leads to inhibit clot formation [14].
Protein C deficiency is generally
subdivided into two types: type I
(quantitative deficiency) decreased levels
of protein C. and type II (qualitative
deficiency) decreased functional activity
of protein C. Most patients with PC
deficiency have type I deficiency, while
Type II deficiency is observed in 10-
15% of the cases [15, 16]. Protein C
deficiency is inherited as autosomal
dominant disorders and, in most cases,
derived from heterozygous mutations
[17]. Acquired PC deficiency can
develop with vitamin K deficiency, liver
disease, treatment with vitamin K
antagonists, severe and chronic
inflammation, autoimmune syndromes,
nephritic syndrome, ordisseminated
intravascular coagulation (DIC) [18]. A
recent study stated an observation of a
higher rate of late fetal loss in patients
with protein C deficiency compared to
non-deficient patients [19].There are two
main types of assays: activity assay
(qualitative)which is either clotting timebased
assay or chromogenic by
spectrophotometer [20]. Quantitative
assay for protein C antigen which are
immunoassays generally done by using
ELISA; it is considered to measure the
quantity of protein C irrespective of its
function [18].Genetic testing by DNA
sequencing is indicated if the results of
functional and antigenic assays do not
approve the diagnosis clearly[21].
Protein S (PS) is a vitamin Kdependent
glycoprotein which acts as
natural anticoagulant [22].Protein S
exists in plasma both free (40%) and
bound to the complement C4b binding
protein (60%) [23].Protein S has both
APC-dependent and independent
anticoagulant properties and
consequently is an important protector in
controlling thrombin generation and
fibrinolysis [24, 25]. The PS deficiency
is identified more than PC deficiency and
its prevalence has been assessed with
about 0.5% in the healthy population and
2% to 12% of thrombophilic patients
[25,17]. Regarding pregnancy
complications, a meta-analysis
designated that protein S deficiency
consulted an overall 15-fold increased
risk of recurrent pregnancy loss and a 7-
fold higher risk of late fetal loss [26].
Hereditary PS deficiency is an autosomal
dominant disorder. Acquired deficiency
of protein S is detected in several
pathological conditions and could be
associated with an increased risk of
thrombosis. These include nephrotic
syndrome, disseminated intravascular
coagulation (DIC), liver disease, and the
use of oral anticoagulants drugs [27, 28].
There are two types of PS assays:
Immunoassays for the determination of
total and free PS levels and clotting
assays to measure APC cofactor activity.
Immunoassays for free and total PS are
preferred for screening [29]. If the
results of functional and antigenic assays
do not confirm the diagnosis, genetic
testing is indicated [21].
Materials and Methods
90 women were involved in the
present study, 45 of them have three or
more miscarriages in the first trimester
(32 women) and second trimester
(13women) considered as patient group,
45 healthy women at time of full term
delivery with at least one alive child as
the control group. The study is carried
out through a period from November
2013 to August 2014 at two hospitals;
Babylon Teaching Hospital for
gynecology and Pediatric and Al-Hilla
Teaching Hospital. Complete history was
taken including medical history, surgical
history, drug history and family history
of thrombosis for patient and control
groups with exclusion criteria for other
causes of pregnancy loss. The blood
samples that obtained from the patient
and control groups consist of 2ml of
blood drawn in a tube contain ethylene
diamine tetra acetate (EDTA) as anticoagulants
to prevent clotting of blood to
be used for platelets count by auto
analyzer (CELL-DYN Ruby,Abbots).
1.8mL of blood drawn in tube contain
0.2mL of 3.8% trisodium citrate solution,
minutes at 1500-2000 g for plasma
preparation and then take the plasma into
plane tube and preserved in -20°C for
testing the levels of protein C and protein
S by ELISA reader (BioTek, U.S.A)
using kit of Aeskulisa Diagnostics.
Statistical analysis was carried out using
SPSS version 20. Categorical variables
were presented as frequencies and
percentages. Pearson’s chi square (X2)
test and fisher exact test were used to
find the association between the
categorical variables. A p-value of ? 0.05
was considered as significant.
Results
The results showed a significant
relation of low protein S with recurrent
miscarriages (p=0.002) OR=2.250
(95%C.I. 1.764-2.870) as shown in
table1. Patients with low protein S were 2
times more likely to have recurrent abortion.
The relation of protein C and platelets were
not significant with recurrent miscarriage
(P>0.05).
There were 71.1% (32/45) of
patients had abortions occurred in the
first trimester (<13 weeks of gestation),
and 13 patients (28.9%) had abortions
occurred in the second trimester (?13
weeks of gestation). There was
significant association of low protein S
andlow protein C with the abortion
occurred in the second trimester (P<0.05,
P=0.016 respectively), however, no
significant association was seen in
combined proteins(C and S) deficiencies
with gestational age. (Table 2)
This study demonstrated that 17.8% of the
patients had positive family history of
thrombosis while in the control group the
percentage of positive family history of
thrombosis was 6.7 %. So there is significant
relationship (p < 0.01) between positive
family history of thrombosis and recurrent
abortions (Figure 1).
Discussion
In this study a significant
association was found between RPL and
protein S deficiency (p=0.002), this is in
agreement with many previous studies
[30, 31, 32, 33, 34, 35, 36]. Rey et al.,
(2003) in meta-analysis consisting of 31
studies, established the association of PS
deficiency with RPL (OR 14.72, 95% CI
0.99-218.01)[32].
Protein C had no significant
difference between patients and controls,
this is in agreement with Vora et al.
(2008) [33] and Parandet al. (2013) [36].
Rey et al. (2003) [32] excluded PC and
antithrombin (AT) deficiencies as
thrombophilia risk factors for pregnancy
loss. Yamada et al. (2001) [37] could not
find an increased incidence of protein C
deficiency in patient with RPL. While
Jyotsna et al. (2011) [34] found a
significant association in the mean value
of protein C in patient group comparing
with control group.
In the combined defect of both
protein C and S which was documented
in (4.4%) of the patients, there was no
statistical significant comparing with
control group. While the study of Cosmi
B. et al. (2013) [38] and Yildizet al.
(2012) [39] stated that the combination
of more than one inherited thrombophilic
gene defect has been recognized as a
cause of early and late RPL. In spite of
that, the incidence of combined
thrombophilia is not clear [9].
Protein C and protein S deficiency
had a significant association with second
trimester pregnancy loss. This is
consistent with the meta-analysis of 31
retrospective study [32] which had
shown that the relationship of
thrombophilia with late pregnancy loss is
stronger than early miscarriages. Alonso
et al. (2002)[40] also stated that the
prevalence of thrombophilia was more
prominent in second trimester, also in
agreement with studies done by (Rey and
Regan, 2000)[41] which established that
protein C deficiency has been associated
with an increased risk of secondtrimester
miscarriage and stillbirth.
However, a systemic review of 25 studies
by Robertson et al. (2005) [42] revealed
a positive association between early
pregnancy loss and thrombophilia, which
disagree with our study.
Conclusions
There is a significant association
of protein S deficiency with recurrent
miscarriage and the incidence is more in
the miscarriage occurring in the second
trimester. Positive family history of
thrombosis is considering a risk factor
for recurrent miscarriage.