1 Department of Obstetrics and Gynecology, 2 Department of Clinical Chemistry and 3 Department of Haematology and Oncology, Großhadern Hospital, Ludwig-Maximilians-University, 81377 Munich, Germany
4 To whom correspondence should be addressed. e-mail: cthaler{at}helios.med.uni-muenchen.de
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: angiotensin I-converting enzyme/fibrinolysis/plasminogen activator inhibitor-1/pregnancy loss/thrombophilic gene polymorphism
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pregnancies appear to require an even balance of coagulation and fibrinolysis in order to avoid excess fibrin accumulation in placental vessels and intervillous spaces as well as to secure fibrin polymerization and stabilization of the placental basal plate (Preston et al., 1996; Buchholz and Thaler, 2003
). A successful implantation, on the other hand, needs endometrial vascular remodelling due to angiogenesis and vascular breakdown (Hickey and Fraser, 2000
). Adequate fine tuning of fibrinolysis is mandatory in order to prevent haemorrhage. For most tissues involved, fibrinolytic activity has been demonstrated (Lockwood, 2001
; Smith et al., 2001
; Chakraborty et al., 2002
). A variety of factors influencing fibrinolysis has been studied. For instance, the fibrin-stabilizing factor (FXIII) has been shown to play an important role in placentation during the first trimester of pregnancy (Kappelmayer et al., 1994
). Deficiency of FXIII with the resulting lack of fibrin stability and tendency towards hemorrhage as well as thrombosis is regarded as a risk factor for miscarriages (Anwar et al., 1999
). Deficiency of FXII, in contrast, impairs fibrinolysis by decreasing plasminogen activation, and this has repeatedly been associated with RM (Braulke et al., 1993
; Ogasawara et al., 2001
). Minimal alterations in the fibrinolysis cascade leading to either hypo- or hyperfibrinolysis are therefore suspected to interfere with placentation and early pregnancy. Several factors influencing fibrinolysis, particularly the plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2), have been shown to be up-regulated during implantation. They modify migration and invasive behaviour of extra villous trophoblast cells, and they also prevent additional haemorrhage during placentation (Feng et al., 2000
; Lockwood, 2001
).
PAI-1 is a key regulating element in the fibrinolysis cascade. Recently, associations between polymorphisms in the PAI-1 and ACE genes and PAI-1 plasma levels have been established. Endothelial PAI-1 expression is modulated by a 4G/5G polymorphism in the PAI-1 promotor, 675 bp upstream from the start site of transcription. PAI-1 expression is also influenced by angiotensin II plasma levels. Angiotensin II, a very potent vasoconstrictor, is generated by the angiotensin I-converting enzyme (ACE), which is well known for its role in blood pressure regulation. ACE expression is associated with a deletion (D)/insertion (I) polymorphism in intron 16 of the ACE gene. Comparable to the PAI-1 4G allele, the ACE D allele leads to an increased PAI-1 expression, resulting in reduced fibrinolysis (Kim et al., 1997).
These two genetic variations have been related to various vascular diseases such as myocardial infarction and deep vein thrombosis (Sartori et al., 1998, 2000; Iacoviello et al., 1998
; Seino et al., 1998
; Gardemann et al., 1999
; Lane and Grant, 2000
) as well as to pregnancy-related disorders such as severe pre-eclampsia, pregnancy-induced hypertension, or serious pregnancy complications such as growth retardation and stillbirth (Zhu et al., 1998
; Fatini et al., 2000
; Glueck et al., 2000
; Yamada et al., 2000
).
In order to evaluate whether ACE and PAI-1 expression levels affect the course of early pregnancies, we determined the prevalence of the ACE D/I and PAI-1 4G/5G polymorphisms in patients with RM.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To analyse the D/I polymorphism in intron 16 of the ACE gene and the 675 4G/5G polymorphism in the promotor region of the PAI-1 gene, genomic DNA was extracted from leukocytes of patients and controls and amplified by PCR using gene-specific primers. Each 50 µl reaction contained 10 mmol/l TrisHCl, pH 8.3, 50 mmol/l KCl, 1.5 mmol/l MgCl2, 0.01% gelatin, 200 µmol/l of each dNTP, 20 µmol/l of each forward and reverse primer, 200 ng of high molecular weight DNA, and 1.25 IU Taq DNA Polymerase (SigmaAldrich). After denaturation at 95°C for 3 min, DNA fragments were amplified for 35 cycles at 95°C for 30 s and at 72°C for 1 min (PAI-1), or for 40 cycles at 95°C for 20 s, at 56°C for 30 s, and at 72°C for 30 s (ACE). The PAI-1 4G/5G promoter genotype was determined according to Margaglione et al. (1994
) by using a mutated oligonucleotide which contains a partial restriction site for the Bsl I enzyme (New England BioLabs), thus making it possible to identify the extra G base by restriction fragment length polymorphism analysis in 2% low melting point agarose gels (Gibco BRL Life Technologies). The ACE D/I genotype was characterized by the length of the PCR product, 190 bp in the case of the deletion and 490 bp in the presence of the insertion (Tiret et al., 1992
). Analyses of the thrombophilic FV Arg506
Gln (factor V Leiden; FVL) and prothrombin G20210A mutations as well as of the MTHFR C677T substitution were performed as described elsewhere (Pihusch et al., 2001
).
For statistical analysis, results of the two groups were compared with the t-test or with the Pearsons 2-test for categorical variables. We performed a KolmogorovSmirnov test and the hypothesis that the data are normally distributed was not rejected. Association of RM with the ACE D/I and PAI-I 4G/5G polymorphisms and the MTHFR mutation was also tested with the CochranArmitage trend test. Stepwise logistic regression analysis was performed with the patients and the controls as the dependent variable and the mutations/polymorphisms in the factor V, prothrombin, MTHFR, PAI-1 and ACE genes as the explanatory variable. Statistical analyses were performed with the Statistical Package for Social Sciences (SPSS for Windows 9.0, SPSS Inc., USA) and the SAS statistical software (V8.1, SAS Institute Inc., USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
None of these studies, however, investigated the cumulative risk associated with a combination of the two polymorphisms which are both known to influence PAI-1 levels (Figure 1). Both proteins affect the same downstream mechanism and combinatorial effects have been proposed to increase the incidence of diabetic macroangiopathy (Kimura et al., 1998). Our study demonstrated that the combination of homozygosity for the PAI-1 4G and ACE D alleles was significantly more prevalent among RM patients. Both the 4G allele and the D allele lead to an increased PAI-1 expression (Figure 1), which may result in hypofibrinolysis. Gris et al. (1997
) postulated in a study of 500 patients that high PAI-1 levels may be a risk factor for RM. Excess fibrin accumulating in spiral arteries and within the intervillous space may well impede perfusion and prevent normal development of the pregnancy.
FVL and the MTHFR C677T mutation, in contrast, do not appear to significantly predispose to first or second trimester RM in our group of patients (Pihusch et al., 2001). Using logistic regression analysis, we were also unable to find an association between heterozygosity for FVL and homozygosity for the PAI-1 4G allele (P = 0.25). This is in conflict with data reported by Glueck et al. (2001
), showing a higher incidence of this combination in patients with severe pregnancy complications.
In conclusion, our results demonstrate that homozygosity for the ACE D allele is a risk factor for RM. Homozygosity for the ACE D and PAI-1 4G alleles additionally amplifies the RM risk and this may very well be exerted by their common effect to increase PAI-1 expression. Analysis of these two polymorphisms should therefore be included in the routine work-up of patients with RM. The clinical implications of these data need to be addressed in a prospective study to answer the question whether or not double homozygous individuals should be treated with low molecular weight heparin.
![]() |
Acknowledgements |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blumenfeld, Z. and Brenner, B. (1999) Thrombophilia-associated pregnancy wastage. Fertil. Steril., 72, 765774.[CrossRef][ISI][Medline]
Braulke, I., Pruggmayer, M., Melloh, P. et al. (1993) Factor XII (Hageman) deficiency in women with habitual abortion: new subpopulation of recurrent aborters? Fertil. Steril., 59, 98101.[ISI][Medline]
Buchholz, T. and Thaler, C.J. (2003) Inherited thrombophilia: impact on human reproduction. Am. J. Reprod. Immunol., 49, 113.[CrossRef][ISI][Medline]
Chakraborty, C., Gleeson, L.M., McKinnon, T. et al. (2002) Regulation of human trophoblast migration and invasiveness. Can. J. Physiol. Pharmacol., 80, 116124.[CrossRef][ISI][Medline]
Coulam, C.B., Clark, D.A., Beer, A.E. et al. (1997) Current clinical options for diagnosis and treatment of recurrent spontaneous abortion. Clinical Guidelines Recommendation Committee for Diagnosis and Treatment of Recurrent Spontaneous Abortion. Am. J. Reprod. Immunol., 38, 5774.[ISI][Medline]
Coumans, A.B., Huijgens, P.C., Jakobs, C. et al. (1999) Haemostatic and metabolic abnormalities in women with unexplained recurrent abortion. Hum. Reprod., 14, 211214.
Fatini, C., Gensini, F., Battaglini, B. et al. (2000) Angiotensin-converting enzyme DD genotype, angiotensin type 1 receptor CC genotype, and hyperhomocysteinemia increase first-trimester fetal-loss susceptibility. Blood Coagul. Fibrinolysis, 11, 657662.[CrossRef][ISI][Medline]
Feng, Q., Liu, Y., Liu, K. et al. (2000) Expression of urokinase, plasminogen activator inhibitors and urokinase receptor in pregnant rhesus monkey uterus during early placentation. Placenta, 21, 184193.[CrossRef][ISI][Medline]
Gardemann, A., Lohre, J., Katz, N. et al. (1999) The 4G4G genotype of the plasminogen activator inhibitor 4G/5G gene polymorphism is associated with coronary atherosclerosis in patients at high risk for this disease. Thromb. Haemost., 82, 11211126.[ISI][Medline]
Girling, J. and de Swiet, M. (1998) Inherited thrombophilia and pregnancy. Curr. Opin. Obstet. Gynecol., 10, 135144.[CrossRef][ISI][Medline]
Glueck, C.J., Wang, P., Fontaine, R.N. et al. (1999) Plasminogen activator inhibitor activity: an independent risk factor for the high miscarriage rate during pregnancy in women with polycystic ovary syndrome. Metabolism, 48, 15891595.[ISI][Medline]
Glueck, C.J., Phillips, H., Cameron, D. et al. (2000) The 4G/4G polymorphism of the hypofibrinolytic plasminogen activator inhibitor type 1 gene: an independent risk factor for serious pregnancy complications. Metabolism, 49, 845852.[CrossRef][ISI][Medline]
Glueck, C.J., Kupferminc, M.J., Fontaine, R.N. et al. (2001) Genetic hypofibrinolysis in complicated pregnancies. Obstet. Gynecol., 97, 4448.
Gris, J.C., Ripart-Neveu, S., Maugard, C. et al. (1997) Respective evaluation of the prevalence of haemostasis abnormalities in unexplained primary early recurrent miscarriages. The Nimes Obstetricians and Haematologists (NOHA) Study. Thromb. Haemost., 77, 10961103.[ISI][Medline]
Hickey, M. and Fraser, I.S. (2000) Clinical implications of disturbances of uterine vascular morphology and function. Baillières Best. Pract. Res. Clin. Obstet. Gynecol., 14, 937951.[CrossRef][ISI][Medline]
Iacoviello, L., Burzotta, F., Di Castelnuovo, A. et al. (1998) The 4G/5G polymorphism of PAI-1 promoter gene and the risk of myocardial infarction: a meta-analysis. Thromb. Haemost., 80, 10291030.[ISI][Medline]
Kappelmayer, J., Bacsko, G., Kelemen, E. et al. (1994) Onset and distribution of factor XIII-containing cells in the mesenchyme of chorionic villi during early phase of human placentation. Placenta, 15, 613623.[ISI][Medline]
Kim, D.K., Kim, J.W., Kim, S. et al. (1997) Polymorphism of angiotensin converting enzyme gene is associated with circulating levels of plasminogen activator inhibitor-1. Arterioscler. Thromb. Vasc. Biol., 17, 32423247.
Kimura, H., Gejyo, F., Suzuki, Y. et al. (1998) Polymorphisms of angiotensin converting enzyme and plasminogen activator inhibitor-1 genes in diabetes and macroangiopathy 1. Kidney Int., 54, 16591669.[CrossRef][ISI][Medline]
Kupferminc, M.J., Eldor, A., Steinman, N. et al. (1999) Increased frequency of genetic thrombophilia in women with complications of pregnancy. New Engl. J. Med., 340, 913.
Kutteh, W.H., Park, V.M. and Deitcher, S.R. (1999) Hypercoagulable state mutation analysis in white patients with early first-trimester recurrent pregnancy loss. Fertil. Steril., 71, 10481053.[CrossRef][ISI][Medline]
Lane, D.A. and Grant, P.J. (2000) Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease. Blood, 95, 15171532.
Lockwood, C.J. (2001) Regulation of plasminogen activator inhibitor 1 expression by interaction of epidermal growth factor with progestin during decidualization of human endometrial stromal cells. Am. J. Obstet. Gynecol., 184, 798804.[CrossRef][ISI][Medline]
Margaglione, M., Di Minno, G., Grandone, E. et al. (1994) Abnormally high circulation levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with a history of ischemic stroke. Arterioscler. Thromb., 14, 17411745.[Abstract]
Morgan, L., Foster, F., Hayman, R. et al. (1999) Angiotensin-converting enzyme insertiondeletion polymorphism in normotensive and pre-eclamptic pregnancies. J. Hypertens., 17, 765768.[CrossRef][ISI][Medline]
Nelen, W.L., Blom, H.J., Steegers, E.A. et al. (2000) Hyperhomocysteinemia and recurrent early pregnancy loss: a meta-analysis. Fertil. Steril., 74, 11961199.[CrossRef][ISI][Medline]
Nelen, W.L. (2001) Hyperhomocysteinaemia and human reproduction. Clin. Chem. Lab. Med., 39, 758763.[ISI][Medline]
Ogasawara, M.S., Aoki, K., Katano, K. et al. (2001) Factor XII but not protein C, protein S, antithrombin III, or factor XIII is a predictor of recurrent miscarriage. Fertil. Steril., 75, 916919.[CrossRef][ISI][Medline]
Pihusch, R., Buchholz, T., Lohse, P. et al. (2001) Thrombophilic gene mutations and recurrent spontaneous abortion: prothrombin mutation increases the risk in the first trimester. Am. J. Reprod. Immunol., 46, 124131.[ISI][Medline]
Preston, F.E., Rosendaal, F.R., Walker, I.D. et al. (1996) Increased fetal loss in women with heritable thrombophilia. Lancet, 348, 913916.[CrossRef][ISI][Medline]
Quenby, S., Vince, G., Farquharson, R. et al. (2002) Recurrent miscarriage: a defect in natures quality control? Hum. Reprod., 17, 19591963.
Rai, R., Backos, M., Elgaddal, S. et al. (2002) Factor V Leiden and recurrent miscarriageprospective outcome of untreated pregnancies. Hum. Reprod., 17, 442445.
Sarig, G., Younis, J.S., Hoffman, R. et al. (2002) Thrombophilia is common in women with idiopathic pregnancy loss and is associated with late pregnancy wastage. Fertil. Steril., 77, 342347.[CrossRef][ISI][Medline]
Sartori, M.T., Wiman, B., Vettore, S. et al. (1998) 4G/5G polymorphism of PAI-1 gene promoter and fibrinolytic capacity in patients with deep vein thrombosis. Thromb. Haemost., 80, 956960.[ISI][Medline]
Sartori, M.T., Patrassi, G.M., Rigotti, P. et al. (2000) Improved fibrinolytic capacity after withdrawal of steroid immunosuppression in renal transplant recipients. Transplantation, 69, 21162121.[ISI][Medline]
Seino, Y., Ikeda, U., Maeda, Y. et al. (1998) Angiotensin-converting enzyme gene polymorphism and plasminogen activator inhibitor 1 levels in subjects with cerebral infarction. J. Thromb. Thrombolysis, 5, 263267.[CrossRef][ISI][Medline]
Smith, A.N., Carter, Q.L., Kniss, D.A. et al. (2001) Characterization of a TGFbeta-responsive human trophoblast-derived cell line. Placenta, 22, 425431.[CrossRef][ISI][Medline]
Tiret, L., Rigat, B., Visvikis, S. et al. (1992) Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels. Am. J. Hum. Genet., 51, 197205.[ISI][Medline]
Yamada, N., Arinami, T., Yamakawa-Kobayashi, K. et al. (2000) The 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene is associated with severe preeclampsia. J. Hum. Genet., 45, 138141.[CrossRef][ISI][Medline]
Younis, J.S., Brenner, B., Ohel, G. et al. (2000) Activated protein C resistance and factor V Leiden mutation can be associated with first- as well as second-trimester recurrent pregnancy loss. Am. J. Reprod. Immunol., 43, 3135.[ISI][Medline]
Zhu, M., Xia, Y. and Cheng, W. (1998) [Study on a deletion polymorphism of the angiotensin converting enzyme gene in pregnancy induced hypertension]. Chung. Hua. Fu. Chan. Ko. Tsa. Chih., 33, 8385.[Medline]
Submitted on July 8, 2002; resubmitted on May 30, 2003; accepted on August 15, 2003.