Download Acquired and inherited thrombophilia: implication

Human Reproduction Vol.21, No.10 pp. 2694–2698, 2006
doi:10.1093/humrep/del203
Advance Access publication July 11, 2006.
Acquired and inherited thrombophilia: implication
in recurrent IVF and embryo transfer failure
Hussein S.Qublan1,7, Suhair S.Eid2, Hani A.Ababneh3, Zouhair O.Amarin4, Aiman Z.Smadi1,
Farakaid F.Al-Khafaji5 and Yousef S.Khader6
1
Department of Obstetrics and Gynecology, 2Department of Coagulation and Hematology, 3Department of Immunology, Royal Medical
Services, Jordan Armed Forces, 4Department of Obstetrics and Gynecology, Jordan University of Science and Technology, 5Department
of Clinical Chemistry, Royal Medical Services, Jordan Armed Forces and 6Department of Public Health and Community Medicine,
Jordan University of Science and Technology, Irbid, Jordan
7
To whom correspondence should be addressed at: Irbid-Aidoun, P.O. Box 97, Irbid 21166, Jordan. E-mail: [email protected]
BACKGROUND: The objective of this study was to determine the incidence of undiagnosed thrombophilic factors
and its relation to IVF and embryo transfer failure in women who have had three or more previous IVF–embryo
transfer cycles. METHODS: The study group comprised of 90 consecutive women with three or more previously
failed IVF–embryo transfer cycles (group A). Two control groups were enrolled: group B (n = 90) included women
who have had successful pregnancy after their first IVF–embryo transfer cycle, and group C (n = 100) included
women who conceived spontaneously with at least one uneventful pregnancy and no previous history of miscarriage.
All women were tested for the presence of inherited [factor V Leiden (FVL) mutation, prothrombin mutation, methylenetetrahydrofolate reductase (MTHFR) mutation and deficiencies in proteins S and C and antithrombin III] or
acquired (lupus anticoagulant and anticardiolipin) thrombophilic factors. RESULTS: An increase in the incidences
of FVL, MTHFR and antiphospholipid antibodies was found in the study group compared with the two control
groups. At least one inherited or acquired thrombophilic factor was detected in 68.9% of women with repeated IVF
failure compared with 25.6 and 25% in the groups B and C, respectively (P < 0.01). Combined thrombophilia (two or
more thrombophilic factors) was significantly higher in women who have had repeated IVF failure as compared with
the two control groups (35.6 versus 4.4 and 3%) (P < 0.0001). CONCLUSION: Thrombophilia has a significant role
in IVF–embryo transfer implantation failure. Women with repeated IVF–embryo transfer failure should be screened
for thrombophilia.
Key words: implantation failure/IVF/thrombophilia
Introduction
About one-third of women undergoing IVF and embryo transfer
will achieve an ongoing pregnancy. Thus, failure to achieve
pregnancy implies failure of the pregnancy at implantation or
at a time shortly thereafter. Several factors have been recognized to affect either success or failure rate of IVF–embryo
transfer. Such factors include age, parity, previously successful
pregnancy, basal hormonal levels, number of antral follicles
before stimulation, endometrial thickness, embryo grading,
position and length of uterus and technique of embryo transfer
(Templeton et al., 1996; Egbase et al., 2000; Ng et al., 2000;
Surrey and Schoolcraft, 2000; Schoolcraft et al., 2001;
Alkande et al., 2002; Kovacs et al., 2003; Qublan et al., 2005).
Inherited [factor V Leiden (FVL) mutation, methylenetetrahydrofolate reductase mutation (MTHFR) (C677T), prothrombin
gene mutation (G20210A), protein C deficiency, protein S deficiency and antithrombin III (ATIII) deficiency] or acquired [lupus
anticoagulant (LA) and anticardiolipin (ACL)] thrombophilia
have been recently implicated in early pregnancy loss and IVF
implantation failure, by impairing the initial vascularization
process occurring at implantation, which is necessary for a successful pregnancy (Geva et al., 1995; Grandone et al., 2001;
Azem et al., 2004; Kujovich, 2004). There are limited data in
the literature associating thrombophilia and repeated IVF–embryo
transfer failure (Grandone et al., 2001; Martinelli et al., 2003;
Azem et al., 2004; Coulam et al., 2006). We conducted this
study to determine the incidence of undiagnosed thrombophilic
factors and its relation to IVF–embryo transfer failure in women
who have had three or more failed previous IVF–embryo transfer
cycles.
Materials and methods
Ninety consecutive women with a history of at least three previously
failed IVF–embryo transfer treatments, presenting to the infertility
clinic between January 2001 and August 2005, were included in this
2694 © The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Thrombophilia and IVF failure
study (group A). Women’s age ranged from 23 to 44 years (mean ±
SD, 31 ± 4.2). Two control groups were enrolled: group B (n = 90)
included women who have had successful pregnancy after their first
IVF–embryo transfer cycle. Their ages ranged from 22 to 40 years
(mean ± SD, 30 ± 3.1). The second group (group C) consisted of 100
women who had conceived spontaneously with at least one uneventful
pregnancy and no previous history of miscarriages. Women’s age in
this group ranged from 17 to 41 years (mean ± SD, 30 ± 2.8). Women
with endometriosis, hydrosalpinx, abnormal uterine cavity on the hysterosalpingogram and history of thromboembolic disease and those
who were receiving hormonal treatment were not included in the
study group. All women were investigated for the presence of inherited (FVL mutation, prothrombin mutation, MTHFR mutation and
deficiencies in proteins S and C and ATIII) or acquired (LA and ACL)
thrombophilic factors. In the IVF–embryo transfer cycles, only cycles
in which grade 1 and 2 embryos were transferred were included in the
study group. Indications for IVF treatment included anovulation,
unexplained infertility and male and tubal factor.
All patients in the IVF programme received a standardized protocol
of controlled ovarian stimulation using a combined regimen of GnRH
agonist (triptorelin, Ipsen Pharma Biotech, Signes, France) and human
menopausal gonadotrophin (Menogon, Ferring GmBH, Wittland II,
Germany), as described elsewhere (Qublan et al., 2005).
Serum samples were obtained 5–7 days before the commencement
of the ovarian stimulation from women in groups A and B and at least
2 months after delivery or abortion in group C. Peripheral blood was
collected in Na2 EDTA vacuum tubes (Becton Dickinson L, Rutherford,
NJ, USA). The genotype of the three mutations—FVL mutation
G1691A, prothrombin mutation (PTH) G20210A and MTHFR—was
detected by genomic DNA isolation from 300 ml of buffy coat using
the Wizard Genomic DNA Purification kit (Promega, Madison, WI,
USA). Biotinylated primers were used for PCR amplification. This
was followed by hybridization of amplification products to a test strip
containing allele-specific oligonucleotide probes that were immobilized as an array of parallel lines. Bound biotinylated sequences were
detected using streptavidin–alkaline phosphatase and colour substrate.
All genotypes of the three mutations were detected on the same strip
according to a predetermined staining pattern.
Functional protein C levels were measured using a chromogenic
assay (Diagnostica Stago, Paris, France). Protein C deficiency was
diagnosed when levels were <60% (normal range, 60–140%). Functional protein S levels were measured by using a clotting assay, and
free antigenic protein S was detected using latex immunoassay (turbidimetry) method (Diagnostica Stago). Levels of <50% (normal range,
50–160%) were considered as being deficient. Antithrombin levels
were measured by using a chromogenic assay (Diagnostica Stago),
and deficiency was diagnosed when functional AT levels were <80%
(normal range, 80–120%). Immunoglobulin M (IgM) and IgG ACL
were assayed using validated enzyme-linked immunosorbent assay
(ELISA) calibrated against international standards, as described elsewhere (Coulam et al., 1997). ACL antibodies were reported in international units (positive when >10 IU/ml). Screening for LA was
performed by the kaolin cephalin clotting time utilizing sensitive reagents and by the dilute Russell’s viper venom time with a neutralization procedure using frozen–thawed platelets. Results for LA were
expressed as positive or negative. Serum levels of homocystine were
determined using a fluorescence polarization immunoassay (Abbott
Laboratory, Abbott Park, IL, USA). Normal values for homocystine
ranged between 4 and 11 μmol/l.
With the level of significance of 0.05 for testing one-sided hypothesis of the difference between proportions reported by Grandone et al.
(2001), our sample size yielded a power of 90.7%.
The results of statistical analysis are presented as means ± SD and
percentages. Chi-square test, Fisher’s exact test and unpaired Student’s t-test were used as appropriate. Bonferroni’s correction was
used to adjust the P-values by multiplying the observed P-values from
the significance tests by the number of tests, K; any K times P that
exceeded 1 was ignored. Then if any KP was <0.05, the two groups
were considered significantly different at the 0.05 level. The Statistics
Package for Social Sciences software (version 11.5, SPSS, Chicago,
IL, USA) and Microsoft Office Excel 2003 were used for data processing and data analysis.
Results
The clinical data of the study and control groups are summarized in Table I. Age was similar in the three groups. There
were no significant differences between groups A and B in the
duration and aetiology of infertility. Table II shows the frequency of the thrombophilic factors in the three groups, including the mutations in FVL, C677T MTHFR, FII G20210A,
proteins S and C, ATIII and antiphospholipid antibodies
(APLA) (LA and ACL antibodies). At least one inherited or
acquired thrombophilic factor was detected in 68.9% (62/90)
of women with repeated IVF failure compared with 25.6% (23/
90) and 25% (25/100) in the successful IVF treatment and
healthy controls, respectively. This difference was statistically
significant (P < 0.01). The incidence of FVL mutation was
higher in the study group compared with the two control
groups. In women with repeated IVF failure, homozygosity for
FVL mutation was found in 4.4% (4/90) of patients compared
with none in the other two control groups. Similarly, 10%
(9/90) of women in the study group were heterozygous for FVL
mutation versus 1.1 and 2% in control groups B (P = 0.108)
Table I. Clinical data of the study and control groups
Age (year)
Duration of infertility (year)
Aetiology of infertility
Anovulation
Unexplained infertility
Tubal factor
Male factor
Study group
Control group
P-Value
Group A (n = 90)
Group B (n = 90)
Group C (n = 100)
31 ± 4.2
6 ± 2.1
30 ± 3.1
5.7 ± 2.4
30 ± 2.8
16 (17.8)
28 (31.1)
12 (13.3)
34 (37.8)
18 (20)
26 (28.9)
15 (16.7)
31 (34.4)
0.899
0.336
0.664
Values are expressed as mean ± SD and percentages.
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H.S.Qublan et al.
Table II. Frequency of thrombophilic factors in the study groups
Thrombophilic factors
Factor V Leiden
Heterozygous
Homozygous
Methylenetetrahydrofolate reductase (C677T) mutation
Heterozygous
Homozygous
Prothrombin G20210A gene
Heterozygous
Homozygous
Protein C deficiency
Protein S deficiency
Antithrombin III deficiency
Lupus anticoagulant
Anticardiolipin
Combined thrombophilia
Study group
Control group
P-Value
Group A (n = 90)
Group B (n = 90)
Group C (n = 100)
9 (10)
4 (4.4)
1 (1.1)
0
2 (2)
0
0.108
0.049
0.162
0.294
7 (7.8)
13 (14.4)
8 (8.9)
3 (3.3)
9 (9)
2 (2)
1.00
0.046a
1.00
0.012a
5 (5.6)
1 (1.1)
2 (2.2)
3 (3.3)
1 (1.1)
8 (8.9)
9 (10)
32 (35.6)
3 (3.3)
1 (1.1)
1 (1.1)
2 (2.2)
0
2 (2.2)
2 (2.2)
4 (4.4)
3 (3)
0
0
3 (3)
1 (1)
2 (2)
3 (3)
3 (3)
0.720
1.00
1.00
1.00
1.00
0.294
0.222
<0.0001a
0.460
0.474
0.223
1.00
1.00
0.204
0.294
<0.0001a
A versus B
A versus C
P-Values are adjusted using Bonferroni’s correction. Values within parentheses are percentage.
a
Significant at a = 0.05.
and C (P = 0.162), respectively. MTHFR C677T mutation was
found in 22.2% (20/90) of women in the study group compared
with 12.2 and 11% in control groups B and C, respectively.
Furthermore, 65% (13/20) of women in the study group were
homozygous for C677T MTHFR mutation compared with 27.3
and 18.2% in either of the control groups. In contrast, heterozygous C677T MTHFR was similar in the three groups. In
women with C677T MTHFR mutation, hyperhomocystinaemia
was found to be significantly higher in the study group [60%
(12/20) of women] compared with each control group [9.1%
(1/11) each] (P < 0.0001). Homozygous C677T MTHFR mutation with hyperhomocystinaemia was found in 55% (11/20) of
patients in the study group compared with none in both control
groups (P < 0.042). The incidence of heterozygous C677T
MTHFR mutation with hyperhomocystinaemia was similar in
the three groups.
Regarding the other thrombophilic factors, ACL antibodies
and LA antibodies were more common in women with
repeated IVF failures (18.9%) compared with 4.4 and 5% in
either of the control groups. However, these differences were
not statistically significant. Furthermore, there were no statistically significant differences between group A and group B and
between group A and group C in the prevalence of G20210A
mutation and deficiencies of ATIII and proteins C and S.
Finally, combined thrombophilia (two or more thrombophilic
factors) was significantly higher in women who have had
repeated IVF failure as compared with the control groups (35.6
versus 4.4 and 3%) (P < 0.0001).
Discussion
This study showed that at least one thrombophilic factor was
detected in 68.9% of women who have had previously three or
more IVF–embryo transfer failures. This incidence is higher
than that reported by others (Geva et al., 1995; Grandone et al.,
2001; Martinelli et al., 2003; Azem et al., 2004). There are
many possible explanations for this discrepancy. First, all
patients in our study were of the same ethnic origin. Differences
2696
in the prevalence of thrombotic mutations according to ethnicity are likely to result in various strengths of associations in
different populations. Second, strict criteria for selecting
patients in our study were applied. All women were apparently
healthy with no previous history of thyroid disease, diabetes
mellitus or thromboembolic disease. Furthermore, women with
endometriosis, hydrosalpinx and abnormal uterine cavity on
the hysterosalpingogram and those who were receiving hormonal treatment were not included in the study group. In the IVF–
embryo transfer cycles, only cycles in which grade 1 and 2
embryos were included. Such a selection may be responsible
for the increased prevalence of thrombophilia in our study. The
polymorphism of MTHFR C677T represented the most common inherited thrombophilic factor in our study (22.2%). This
finding is consistent with previous reports. Martinelli et al.
(2003) evaluating 162 women with failed IVF/ICSI treatment
reported an incidence of 19%. Moreover, Azem et al. (2004)
who investigated 45 women with a history of four or more
failed IVF cycles reported an incidence of 17.8%. Homozygosity for the thermolabile variant of MTHFR predisposes to the
development of hyperhomocystinaemia (Engbersen et al.,
1995). It has been reported that homozygosity for a common
C677T mutation in the 5,10-MTHFR gene that was associated
with hyperhomocystinaemia leads to a 3-fold increase in risk
for early miscarriages (Unfried et al., 2002). Heby (1995)
reported that elevated maternal plasma homocystine levels
were associated with impaired DNA methylation and gene
expression that lead to defective chorionic villous vascularization with subsequent early embryonic death. Moreover, investigating the chorionic villous vascularization by both
histopathology and an image analysis system in spontaneous
miscarriage tissues from 19 women with recurrent early pregnancy losses, Nelen et al. (2000) observed significantly smaller
median area, perimeters and diameter per chorionic vascular
element in women with elevated total homocystine levels.
FVL is a genetic disorder that is characterized by an
impaired anticoagulant response to activated protein C. The
distribution of FVL varies greatly in ethnic groups (De Stefano
Thrombophilia and IVF failure
et al., 1998). Mutation in FVL gene increases thrombin generation, with a 4- to 8-fold increased risk of thrombosis in the heterozygous mutation and 80-fold increased risk in the homozygous
mutation (Kujovich, 2004). In a recent meta-analysis, 3- to 4-fold
increased risk of recurrent early pregnancy loss was found in
women with FVL mutation (Rey et al., 2003). Vascular placental insufficiency has been suggested as a potential cause of
these early pregnancy losses. Results of our study showed that
the prevalence of FVL mutation was higher in women with
repeated IVF failure (14.4%) than was found in women with
successful IVF–embryo transfer (1%) and the healthy controls
(2%). However, these differences did not reach statistical significance. This finding is consistent with that reported by
Grandone et al. (2001), who found an incidence of 11.1% in
women with greater than or equal to three IVF failures compared to none in the control groups. Others have failed to find
such association (Martinelli et al., 2003; Azem et al., 2004).
Moreover, Gopel et al. (2001), investigating 102 mother–child
pairs who had had successful IVF, reported improved implantation rate as an advantage of having FVL mutation. Further
research in this area is required to reconcile these different
findings.
Elevated plasma prothrombin levels with 2- to 4-fold
increased risk of venous thrombosis were found in association
with a single-nucleotide substitution (G20210A) in the 3′
untranslated regions of the prothrombin gene (Kujovich,
2004). In the meta-analysis of Rey et al. (2003), mutation in
prothrombin gene was associated with a 2- to 3-fold increased
risk of recurrent early miscarriages. The prevalence of prothrombin gene mutation in our series was similar in the study
and the control groups, a finding that is in contrast to that of
Grandone et al. (2001) and in agreement with that of Azem
et al. (2004) and Martinelli et al. (2003). The disagreement
with Grandone et al. (2001) might be related to the smaller
sample size of their series, where they investigated 18 women
with greater than three IVF failures and 24 with successful
IVF–embryo transfer, comparing them with 216 women who
conceived spontaneously.
In Jordan, the background genotype frequency for FVL is
15%, for FII G20210A 2% and for MTHFR C677T 24% (Eid
and Rihani, 2004). This is not in accordance with what we have
found in the two control groups. The reason for this discrepancy may be that most of the study subjects of the cited paper
were males. These gene mutation frequencies were significantly higher among thrombotic Jordanian patients (25.7% for
FVL, 6% for FII G20210A and 31.7% for MTHFR C677T)
(Eid and Shubeilat, 2005).
Deficiencies in the natural anticoagulant proteins C and S
and ATIII occur much less frequently. Several studies have
evaluated the potential role of these deficiencies in recurrent
early pregnancy losses (Sanson et al., 1996; Raziel et al.,
2001). Conflicting results were reported in this regard. This
might be related to the rarity of these deficiencies and the
smaller-size studies. Only one study has examined the prevalence and the role of these deficiencies in women with repeated
IVF failure (Azem et al., 2004). Azem et al. (2004) found no
significant association between these deficiencies and repeated
IVF failures, which is consistent with the findings of our study.
APLAs are a nonorgan-specific antibodies directed against
the membrane phospholipids or phospholipid-binding plasma
proteins (Galli et al., 1990). The importance of these antibodies as an aetiological factor of implantation failure after IVF–
embryo transfer is now well established (Birkenfeld et al.,
1994; Kaider et al., 1996; Coulam et al., 1997). It has been
suggested that APLAs reduce the levels of Annexin V, which
is a potent anticoagulant, leading to thrombosis and possible
implantation failure or early pregnancy loss (Lockwood and
Rand, 1994; Matsubayashi et al., 2001). In our study, 18.9%
(17/90) of women with repeated IVF failure were positive for
APLAs compared with 4.4 and 5% in either of the control
groups, but this difference was not statistically significant. This
is not in agreement with previous reports (Birkenfeld et al.,
1994; Kaider et al., 1996; Coulam et al., 1997). Birkenfeld et al.
(1994) evaluated 56 women with implantation failure and 14
women who successfully conceived after IVF–embryo transfer.
They found that 32% of women with implantation failure versus
10% in the control group were positive for APLAs (P = 0.02).
Moreover, Kaider et al. (1996), investigating 42 women with
IVF failure and 42 who had conceived successfully after IVF–
embryo transfer, found that 26.2% of women in the study group
compared with 4.8% in the control group were positive for
APLAs (P = 0.01). These authors recommended testing for the
presence of APLAs in all patients undergoing IVF before initiating treatment. More recently, Coulam et al. (1997), in a largersize study, found that 69 women of 312 (22%) with implantation
failure after IVF treatment were positive for APLAs compared
with five women of 100 (5%) fertile control group (P < 0.01).
The results of our study showed that the combination of two
or more thrombophilic factors was significantly higher in
women who had repeated IVF failures compared with the controls. These findings are in agreement with those reported
recently by Coulam et al. (2006), who observed that 74% of
women with recurrent implantation failure after IVF–embryo
transfer had three or more gene mutations compared with 20%
of healthy fertile controls.
In summary, the results of our study indicate that thrombophilia
may have a significant role in IVF–embryo transfer implantation
failure. Women with repeated IVF–embryo transfer failure should
be screened for thrombophilia. Prospective randomized controlled
interventional studies with large numbers are needed to determine
the effect of thromboprophylaxis in such cases.
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Submitted on March 23, 2006; resubmitted on April 20, 2006, May 2, 2006;
accepted on May 10, 2006