AJRI 2001; 46:124131

Printed in Ireland - all rights reser6ed.

Thrombophilic Gene Mutations and Recurrent

Spontaneous Abortion: Prothrombin Mutation
Increases the Risk in the First Trimester
RUDOLF PIHUSCH, TINA BUCHHOLZ, PETER LOHSE, HEIKE RUBSAMEN, NINA ROGENHOFER, UWE
HASBARGEN, ERHARD HILLER, AND CHRISTIAN J. THALER

Pihusch R, Buchholz T, Lohse P, Rubsamen H, Rogenhofer N, Hasbargen U, Hiller E,

Thaler CJ. Thrombophilic gene mutations and recurrent spontaneous abortion:
Prothrombin mutation increases the risk in the first trimester. AJRI 2001; 46:124131
Munksgaard, 2001
PROBLEM: Thrombophilic predisposition may be one of the underlying causes of
recurrent spontaneous abortions (RSA). We studied the prevalence of five thrombophilic gene mutations in patients with RSA.
METHOD OF STUDY: 102 patients with two or more consecutive abortions and 128
women without miscarriage were analyzed for factor V Leiden mutation (FVL),
prothrombin G20210A mutation (PTM), C677T mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, glycoprotein IIIa (GPIIIa) C1565T polymorphism, and b-fibrinogen G-455A polymorphism by polymerase chain reaction
(PCR) techniques.
RESULTS: No differences in the prevalence of FVL, MTHFR T/T, GPIIIa and
b-fibrinogen polymorphism were detected. Heterozygous PTM occurred more often in
patients with RSA. This effect was significant in a subgroup with abortions exclusively
in the first trimester (6.7% vs. 0.8%, P= 0.027, OR 8.5).
CONCLUSIONS: In contrast to the other mutations and polymorphisms, heterozygous PTM is more common in patients with abortions in the first trimester. This
might reflect an influence of PTM on pathogenesis of early pregnancy loss.

Key words:
b-Fibrinogen, factor V Leiden,
genetic thrombophilia, GPIIIa,
habitual abortion, MTHFR
RUDOLF PIHUSCH
ERHARD HILLER
Haemostaseology Research
Laboratory, Department of
Haematology and Oncology,
Klinikum der Universitat
Munchen-Grohadern, Munich,
Germany
TINA BUCHHOLZ
NINA ROGENHOFER
UWE HASBARGEN
CHRISTIAN J. THALER
Department of Obstetrics and
Gynecology, Klinikum der
Universitat
Munchen-Grohadern, Munich,
Germany
PETER LOHSE
HEIKE RU8 BSAMEN
Molecular Biology Laboratory,
Department of Clinical
Chemistry, Klinikum der
Universitat
Munchen-Grohadern, Munich,
Germany
Address reprint requests to
Christian J. Thaler, Department
of Obstetrics and Gynecology,
Klinikum der Universitat
Munchen-Grohadern, 81377
Munich, Germany.
E-mail:
thaler@gyn.med.uni-muenchen.de
Submitted September 25, 2000;
revised December 5, 2000;
accepted December 6, 2000.

MUNKSGAARD, 2001

GENETIC THROMBOPHILIA AND RECURRENT SPONTANEOUS ABORTIONS

INTRODUCTION
Recurrent spontaneous abortions (RSA) are a major
concern in gynecology, affecting about 1 5% of couples1,2 and frequently accompanied by maternal morbidity as well as a considerable psychological burden.
Despite intense anatomic, endocrinologic, and immunologic screening efforts, up to 30 50% of RSA
remain unexplained.3,4
Common findings in placentae from recurrent
aborti are fibrin deposition and thrombi in intervillous
spaces and fetal stem vessels, associated with fetal
hypoperfusion, hypoxia, and fetal demise.5 The underlying pathophysiological mechanisms remain unclear,
although it has been speculated that increased coagulation or decreased fibrinolytic activities may be potential causes of RSA. The precedence of this disorder
is the antiphospholid syndrome, where activation of

TABLE I.

/ 125

coagulation generates arterial and venous clot formation and secondary placental insufficiency.6
During the last years, several genetic risk factors of
thrombophilia have been identified.7 14 The known
thrombophilic mutations, which were considered in
this study, are recapitulated in Table I. Besides causing hereditary thrombophilia, several studies demonstrated that factor V Leiden mutation (FVL),
prothrombin mutation (PTM), and 5,10-methylenetetrahydrofolate reductase (MTHFR) mutations may
also increase a womans risk of recurrent pregnancy
loss in the second and third trimester,15 25 possibly by
affecting the maternal fetal circulation.
We studied these mutations in our collective consisting of abortions mainly in the first trimenon and
furthermore included polymorphisms in the glycoprotein IIIa (GPIIIa) and b-fibrinogen gene loci that
have so far not been investigated in this collective,

Genetic Risk Factors for Thrombosis7 14,26 30

Name

Mutation

Pathomechanism

FVL

1691 GA substitution
in the gene of
coagulation factor V

PTM

20210 G A substitution
in the 3%-untranslated
region of the
prothrombin gene

TL-MTHFR

Homozygosity for the

677 C T substitution
in the MTHFR gene

Blocked inactivation
Heterozygosity
Heterozygosity increases
of factor Va by
approximately 510% the risk for thrombosis
activated protein C,
by approximately
resulting in reduced
seven-fold,
clearance of factor
homozygosity
Va
increases it by
approximately 80-fold
Elevated prothrombin Heterozygosity
Heterozygosity increases
levels in plasma
approximately 12%
risk for thrombosis by
approximately two-fold;
increased risk of
myocardial infarction
and cerebral vein
thrombosis
Thermolabile variant of TL-MTHFR
Possible increased risk
the enzyme with
approximately 10%
for venous thrombosis
reduced catalytic
and arterial infarction
activity and elevated
plasma levels of
homocysteine
Platelet GPIIIa is
PIA2 allele
PIA2 allele potentially
essential for
approximately 15%
more often in subjects
with unstable angina
aggregation and
thrombus formation
and myocardial
infarction
A/A genotype causes A allele
Possibly associated with
higher plasma
approximately 20%
arterial complications
fibrinogen levels

GPIIIa PIA1/A2
1565 C T substitution
polymorphism
in the gene for platelet
GPIIIa (PIA2 allele)

b-Fibrinogen
455 G A substitution
polymorphism
in the promotor of the
gene for the b-chain

Prevalence (Caucasians) Clinical effect

FVL, factor V Leiden mutation; PTM, prothrombin mutation; TL-MTHFR, thermolabile 5,10 -methylenetetrahydrofolate reductase, GPIIIa,
glycoprotein IIIa.
AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY VOL. 46, 2001

126 /

PIHUSCH ET AL.

although they may be of importance in the pathogenesis of arterial occlusions.14,26 30

MATERIALS AND METHODS

RSA Patients and Controls
We analyzed 102 Caucasian women with two or more
unexplained consecutive abortions at 5 25 weeks of
gestation. The routine work up of these patients included vaginal ultrasound, hormonal evaluation (follicle-stimulating hormone, luteinizing hormone,
prolactin, dehydroepiandosterone [DHEA], testosterone, thyroid-stimulating hormone), determination of
auto-antibodies (antinuclear antibodies [ANA], antimitochondrial antibodies [AMA], IgG and IgM anticardiolipin antibodies), dilute Russell viper venom time
(DRVVT) and lupus-sensitive activated partial thromboplastin time (aPTT).
If anticardiolipin antibodies (ACA) IgG was greater
than 20 IU/mL or ACA IgM was greater than 12
IU/mL in two consecutive measurements (more than 4
weeks apart) or if the DRVVT test and the lupus-sensitive aPTT were positive, patients were considered as
having an antiphospholipid syndrome and were excluded from the study. All functional tests were performed at least 2 months after the last pregnancy.
Patients with chromosomal aberrations were also excluded. None of the patients had deficiencies of antithrombin, protein C, or protein S as determined by
functional assays.
One hundred and twenty-eight women with at least
one healthy term delivery and no history of abortions
or pregnancy-associated complications served as controls. They were recruited from a series of consecutive
pregnant women that delivered healthy term infants at
our institution between November 1998 and February
1999. Patients after artificial reproduction techniques
were excluded.
Each RSA patient and each control patient gave a
written informed consent allowing polymerase chain
reaction (PCR) testing for the mutations and polymorphisms mentioned in this paper.

units Taq DNA Polymerase (Sigma-Aldrich, Deisenhofen, Germany). After denaturation at 95C for 3 min,
DNA was amplified for 40 cycles at 95C for 30 s, 59C
(GPIIIa), 60C (FVL and PTM), 62C (b-fibrinogen),
65C (MTHFR), and finally annealed at 72C for 2 min.
The positive assay control was DNA from a normal
(FVL and b-fibrinogen) or homozygous mutant subject
(GPIIIa, MTHFR, and PTM), while a water blank
served as negative control for each analysis.
Following PCR, the newly generated products were
digested with the appropriate restriction enzyme (New
England BioLabs) and electrophoresed in 2 3% low
melting point agarose gels (Gibco BRL Life Technologies, Karlsruhe, Germany). MnlI digestion of the 288bp PCR product of the factor V gene generated
fragments of 158, 93, and 37 bp for the normal allele.
Digestion products of the mutant allele were 158 and
130 bp in size. The prothrombin fragment of 230 bp was
cleaved by HindIII in case the G A mutation was
present and yielded two smaller ones of 190 and 40 bp.
The 219-bp PCR product of the normal MTHFR allele
was also not digested by HinfI, whereas the mutant
allele was characterized by two fragments, 176 and 43
bp in length.
For the PIA1/A2 polymorphism, the resultant PCR
product was digested with MspI, generating fragments
of 275, 69, and 6 bp in case of the A1 allele and of 173,
102, 69, and 6 bp in case of the A2 allele. The 5%-flanking
region and part of the first exon of the b-fibrinogen gene
was enzymatically amplified by PCR as described by
Thomas et al.,14 using slightly modified oligonucleotides.

Statistical Analysis
Results of the two groups were compared with the
MannWhitney U-test, Fishers exact test, and Pearsons chi-square test for categorial variables. Odds ratio
(OR) and its 95% confidence intervals (CI) were calculated. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS for
Windows 9.0, SPSS Inc., Chicago, IL).

RESULTS
Genetic Studies

The FVL, PTM, MTHFR, GPIIIa, and b-fibrinogen

genotype analyses were performed in all patients and
controls. Genomic DNA was extracted from white
blood cells using the QIAmp DNA blood mini kit
(QIAGEN, Hilden, Germany) and amplified by the
PCR with gene-specific primer pairs. Each 50-mL reaction contained 10 mM Tris HCl, pH 8.3, 50 mM KCl,
1.5 mM MgCl2, 0,01% gelatin, 200 mM of each dNTP,
20 mM of each forward and reverse primer, approximately 200 ng of high molecular weight DNA, and 1.25
MUNKSGAARD, 2001

Patient Demographics and Pregnancy Data

The demographic characteristics and pregnancy data of
the RSA patients and the controls are shown in Table
II. Women with recurrent abortions were significantly
older (PB 0.001), had a significant higher number of
pregnancies (P B0.001), and less healthy children (PB
0.001).
We documented 351 abortions in the RSA group. Of
the RSA patients, 65.7% had two or three abortions and
34.3% had four or more abortions. The median per

GENETIC THROMBOPHILIA AND RECURRENT SPONTANEOUS ABORTIONS

TABLE II.

/ 127

Demographic and Pregnancy Data of the Study Populations

All patients

P value

First-trimester patients

P value

75

Controls

Number

102

128

Age

35 (2248)

B0.001

35 (2248)

B0.001

32 (1844)

Pregnancies

4 (29)

B0.001

4 (29)

B0.001

1 (14)

Pregnancy Losses
First trimester
Second trimester
Primary
Secondary

3 (29)
3 (08)
0 (03)
61.8% (63)
38.2% (39)

B0.001

3 (28)
3 (28)

61.3%(46)
38.7%(29)

B0.001

0 (00)

Deliveries

0 (04)

B0.001

0 (04)

B0.001

1 (14)

All 6alues are medians (range) and percentage (numbers), respecti6ely.

P 6alues were determined by the MannWhitney U-test.

RSA patient was 3 (range 2 9), whereas, by definition,

none of the controls had abortions. The abortions were
classified as primary in 63 patients (61.8%) and as
secondary in 39 patients (38.2%). This amounts to a
total of 217 primary and 134 secondary abortions. The
patients with secondary abortions had significantly
more pregnancies (median 5, range 3 9 vs. median 4,
range 29; P B0.001) and, by definition, more normal
deliveries with healthy infants (median 1, range 1 4 vs.
median 0, range 02) than the primary aborters.
Seventy-five (73.5%) of the women had abortions
exclusively in the first trimester (at 5 12 weeks of
gestation), the remaining 27 (26.5%) had additional
abortions in the second trimester (gestation week 13
24). The first-trimester RSA patients had a total of 244
abortions, 46 (61.3%) were classified as having primary
abortions, 29 (38.7%) as having secondary abortions.

Pre6alence of Thrombophilic Mutations

Eight women with RSA and 11 women of the control
group (7.9 vs. 8.6%, NS) were heterozygous for FVL,
while homozygous carriers were not detected (Table
III). The frequency of the 1691 A allele was 4.0 vs.
4.3% (NS). The PTM occurred only as the heterozygous form in five RSA patients and in one control
(4.9 vs. 0.8%, NS). The frequency of the 20210 A allele
was 2.5 vs. 0.4% (P = 0.0265). The OR among heterozygous carriers for the PTM was 6.27 (95% CI
0.7552.8). Heterozygosity for the MTHFR 677C/T
mutation was found in 47 RSA patients and in 61
controls (46.1 vs. 47.7%, NS), homozygosity (thermolabile [TL]-MTHFR) in 14 RSA patients and in 12
controls (13.7 vs. 9.4%, NS). The frequency of the
677T allele was 36.8 vs. 33.2% (NS). The 1565 C/T
(PIA1/A2) genotype of platelet GPIIIa was found in 21

RSA patients and in 31 controls (20.6 vs. 24.4%, NS),

while the 1565 T/T (PIA2/A2) genotype was present in
one RSA patient and in two controls (1.0 vs. 1.6%,
NS). The frequency of the 1565 T (PIA2) allele was 11.3
vs. 13.8% (NS). The 455 G/A genotype in the
promoter region of the b-fibrinogen gene was detected
in 33 RSA patients and in 48 controls (32.4 vs. 37.5%,
NS), the 455 A/A genotype in eight RSA patients
and in 11 controls (7.8 vs. 8.6%, NS). Accordingly, the
frequency of the 455 A allele was 24.0 vs. 27.3%
(NS).
Subgroup analysis of those 75 RSA patients with
abortions occurring exclusively in the first trimester
demonstrated no significant difference in the prevalence of the FVL and 677C/T MTHFR mutations or
of the GPIIIa and b-fibrinogen polymorphisms. However, heterozygosity for the PTM occurred significantly
more often in the group of first-trimester RSA when
compared to all RSA patients as well as to the controls
(6.7 vs. 0.8%, P=0.027, Table IV). The OR was 8.53
(95% CI 1.0271.7) for heterozygous carriers of the
G20210A PTM. Correspondingly, in the controls, the
prothrombin G/G genotype (wildtype) was significantly increased (99.2 vs. 93.3%, P= 0.027) and the
prevalence of the 20210A allele 3.3 vs. 0.4% (P=
0.009). Because there was from literature an a priori
basis for evaluating PTM, the PB0.009 value does not
require a Benferroni correction for multiple comparisons. No statistically significant difference existed between patients with primary and secondary abortions.

DISCUSSION
In this casecontrol study, the prevalence of FVL and
the TL-MTHFR was not increased in women who

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PIHUSCH ET AL.

had RSA while otherwise healthy. This is in contrast

to previously published data: Ridker et al.15 found an
OR of 2.3 for FVL in 113 women with three or more
recurrent abortions before the third trimenon compared to 437 postmenopausal multipara without abortions. Meinardi et al.16 also reported an increased risk
of fetal loss (even single) and stillbirth in 228 carriers
of FVL compared to 121 healthy relatives. The OR
were 2.08 (95% CI 1.33 3.25) for loss before 20 weeks
of gestation and 1.60 (95% CI 0.58 4.43) for stillbirth,
with homozygous carriers having an even higher risk.
In contrast to these two studies, however, our
TABLE III.

cohort included mainly women with abortions in the

first trimester. It has been proposed that the pathogenesis of first-trimester abortions is different from
loss of pregnancies occurring in the second and third
trimester, as profound structural and genetic abnormalities are common at this early stage of embryonal
development.31 This might dilute the influence of FVL
on pathogenesis of abortion in our study. Indeed, the
study of Younis et al.,25 who concentrated only on
pregnancy losses after sonographic detection of a
fetal pulse, thereby excluding very early abortions
and many pregnancy losses due to failure of early

Prevalence of the Genotypes in all Recurrent Spontaneous Abortion Patients and Controls

Total

Patients

Controls

102

128

P value

FVL
1691 G/G
1691 A/G
1691 A/A
Frequency of 1691 G
Frequency of 1691 A

92.1%
7.9%
0.0%
96.0%
4.0%

(93)
(8)
(0)
(194)
(8)

91.4%
8.6%
0.0%
95.7%
4.3%

(117)
(11)
(0)
(245)
(11)

NS
NS
NS
NS
NS

MTHFR mutation
677 C/C
677 C/T
677 T/T
Frequency of 677C
Frequency of 677T

40.2%
46.1%
13.7%
63.2%
36.8%

(41)
(47)
(14)
(129)
(75)

43.0%
47.7%
9.4%
66.8%
33.2%

(55)
(61)
(12)
(171)
(85)

NS
NS
NS
NS
NS

GPIIIa mutation
1565 C/C (PIA1/A1)
1565 C/T (PIA1/A2)
1565 T/T (PIA2/A2)
Frequency of 1565 C
Frequency of 1565 T

78.4%
20.6%
1.0%
88.7%
11.3%

(80)
(21)
(1)
(181)
(23)

74.0%
24.4%
1.6%
86.2%
13.8%

(94)
(31)
(2)
(219)
(35)

NS
NS
NS
NS
NS

b-fibrinogen mutation
455 G/G
455 G/A
455 A/A
Frequency of 455 G
Frequency of 455 A

59.8%
32.4%
7.8%
76.0%
24.0%

(61)
(33)
(8)
(155)
(49)

53.9%
37.5%
8.6%
72.7%
27.3%

(69)
(48)
(11)
(186)
(70)

NS
NS
NS
NS
NS

PTM
20210 G/G
20210 A/G
20210 A/A
Frequency of 20210 G
Frequency of 20210 A

95.1%
4.9%
0.0%
97.5%
2.5%

(97)
(5)
(0)
(199)
(5)

99.2%
0.8%
0.0%
99.6%
0.4%

(127)
(1)
(0)
(255)
(1)

0.062
0.062
NS
0.027
0.027

OR (95% CI)

6.27 (0.7552.8)

OR, odds ratio; CI, confidence inter6al; FVL, factor V Leiden mutation; MTHFR, 5,10 -methylenetetrahydrofolate reductase; GPIIIa,
glycoprotein IIIa; PTM, prothrombin mutation; NS, not significant.
Percentage (number) of patients and controls with the respecti6e genotype.
MUNKSGAARD, 2001

GENETIC THROMBOPHILIA AND RECURRENT SPONTANEOUS ABORTIONS

/ 129

TABLE IV. Prevalence of Prothrombin Genotype in Patients with First-trimester Recurrent Spontaneous Abortion
and Controls
Patients

Controls

Total

75

128

PTM
20210 G/G
20210 A/G
20210 A/A
Frequency of 20210 G
Frequency of 20210 A

93.3% (70)
6.7% (5)
0.0% (0)
96.7% (145)
3.3% (5)

99.2% (127)
0.8% (1)
0.0% (0)
99.6% (255)
0.4% (1)

P value

0.027
0.027
NS
0.009
0.009

OR (95% CI)

8.53 (1.0271.7)

OR, odds ratio; CI, confidence inter6al; PTM, prothrombin mutation.

Percentage (number) of patients and controls with the respecti6e genotype.
NS, not significant.

embryonic differentiation, was able to show an increased prevalence of FVL in women who recurrently
aborted within the first trimester. However, as these
data reflect ethnic specificities and analyze a relatively
small number of highly selected patients (n = 37), they
might overestimate the true role of FVL.
Perhaps more relevant, impaired placental perfusion
might not be critical for embryonic development during very early gestation, due to very favorable placental/embryonic ratio at this stage. In agreement with
this explanation, several cohort studies17 23 found an
increased prevalence of FVL and TL-MTHFR only in
patients loosing their pregnancies in the second and
third trimester. Even more persuasive is the prospective study of Preston et al.24 In a large European
cohort, FVL and other coagulation inhibitor deficiencies (antithrombin, protein C, or protein S) increased
the risk of fetal death only after 28 weeks of gestation.
The fact, that ]2 consecutive abortions were used as
inclusion criterion in our study, does not seem to
explain our findings, as even those reports, which
concentrate on patients with ] 3 abortions within the
first trimester,15,17,18 failed to show an increased risk
with FVL or TL-MTHFR. In summary, we conclude
that, in unselected populations, FVL and TLMTHFR do not appear to play a relevant role for
first-trimester abortions.
In our study, we were unable to detect an influence
of the GPIIIa PIA2 allele on RSA. The GPIIIa PIA2/A2
has been associated with unstable angina, myocardial
infarction,26 and stent occlusion27; however, these data
have been contradicted by other studies.28,29 At
present, our data do not suggest an unfavorable influence of GPIIIa PIA2/A2 polymorphism on pregnancies.
Furthermore, our study did not reveal any effects of
the 455 G/A polymorphism in the promotor of the
b-fibrinogen gene on recurrent fetal loss in the first or
second trimester. This gene polymorphism has been

associated with elevated fibrinogen levels30 and consecutive risk of ischemic heart disease. However, our
data do not indicate an increased risk of placental
perfusion or functional problems caused by the discrete rise in fibrinogen concentrations that has been
shown for the b-fibrinogen 455 G/A polymorphism.
Our study demonstrates that the prevalence of the
prothrombin 20210GA genotype is significantly increased in patients with first-trimester abortions with
an OR of 8.4 and, therefore, has to be considered as
an important risk factor for RSA. In earlier studies,
Kutteh et al.,17 Gris et al.,21 and Brenner et al.22 have
identified this genotype as a risk factor for secondand third-trimester abortions. Our significant findings
for the first trimester might be due to the composition
of the study group, as previous studies had relatively
small numbers of patients with early abortions that, in
contrast, represents 73% of the patients in our collective. This may also explain the observed differences in
risk: our study revealed a much higher OR in the first
trimester than that reported for second- and thirdtrimester abortions. Our findings implicate that PCR
testing for PTM might be valuable in all patients with
a history of abortions, as clinical clues for the presence of the mutation (history of thrombosis) are not
reliable and screening tests (PTM, aPTT) are normal
in these patients.
As PTM causes only a moderately thrombophilic
state,9,10 it is presently not known how it might cause
failure of first-trimester pregnancies. In contrast to
FVL, however, the PTM is associated with problems
in both venous9 and arterial system.10 In this respect,
PTM might resemble the antiphospholipid syndrome
that has been well documented to cause abortions6 as
well as arterial and venous thromboses. In addition,
increased prothrombin levels in heterozygous mothers
might not only affect plasmatic coagulation. Besides
fibrin generation, thrombin also activates platelets,

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PIHUSCH ET AL.

smooth muscle cells, fibroblasts, mesangial cells, and

macrophages,32 all of which are represented within
placental tissues. Thrombin is able to induce cellular
responses, such as proliferation, chemotaxis, and inhibition of neuronal outgrowth. Thus, increased
prothrombin levels might affect placental function by
influencing pivotal mechanisms, such as cell adhesion,
smooth muscle cell proliferation, and vasculogenesis.33
It might be rewarding to study these aspects in
miscarriages of mothers carrying the heterocygous
prothrombin mutation.

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