1 Institut Clínic of Gynecology, Obstetrics and Neonatology, 2 Hemotherapy and Hemostasis Unit and 3 Unit of Systemic Autoimmune Diseases, Faculty of Medicine, University of Barcelona, Barcelona, Spain
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Abstract |
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Key words: antiphospholipid antibodies/ß2 glycoprotein-I/implantation failure/IVF/recurrent abortion
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Introduction |
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The APS is characterized by the occurrence of aPL in association with clinical features such as thrombo-embolic phenomena, thrombocytopenia and pregnancy wastage. This condition was first recorded in patients with systemic lupus erythematosus and later in otherwise healthy subjects (secondary and primary APS, respectively) (Hughes, 1993, 1998
). Recently (Cabral et al., 1996
) a peculiar syndrome was described for the first time in six patients with recurrent venous and/or arterial thromboses without aPL, as detected by standard antiphospholipid assays, nor clinical or serological evidence of other autoimmune disease, but in the presence of antibodies to ß2-glycoprotein-I (aß2GP-I). These authors considered this condition as a variant in the so-called `primary antiphospholipid/cofactor syndromes' (Alarcón-Segovia and Cabral, 1996
; Cabral et al., 1996
).
ß2GP-I, also known as apolipoprotein H, is a protein involved in the binding of aPL to phospholipid surfaces. It is an absolute requirement (cofactor) for the binding of autoimmune aCL to cardiolipin. In addition, LA has been found to be directed towards either ß2GP-I or prothrombin bound to anionic phospholipids (Arnout and Vermylen, 1998). Interestingly, some authors suggest that aPL and aß2GP-I are two different antibody populations and that a new pathological entity associated with the presence of only aß2GP-I may exist (Alarcón-Segovia and Cabral, 1996
; Cabral et al., 1996
). Thus, patients with clinical features suggestive of APS but LA and aCL negative should be tested for aß2GP-I (Harris et al., 1998
). On the contrary, others stress that there is little proof to date that measuring antibodies to ß2GP-I is superior to conventional LA and aCL assays in making the diagnosis of APS in patients with appropriate clinical features (Branch and Hatasaka, 1998
).
It has previously been reported that the presence of circulating aPL is associated with recurrent abortion and may play a role in failure of implantation after in-vitro fertilization (IVF) and embryo transfer (Balasch et al., 1996). The present study, which is a retrospective analysis of prospectively collected material, shows that these conditions are not associated with the occurrence of aß2GP-I in patients lacking aPL in standard antiphospholipid assays.
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Materials and methods |
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Group one included patients (mean 31.2 years, range 2240) with two or more (range 212, mean 3.1) consecutive spontaneous abortions. Of the 100 patients in this group, 14 (14%) had been delivered of an infant once prior to their series of abortions. All patients in group one were routinely screened and found to be normal for systemic diseases, diabetes mellitus, thyroid dysfunction, chromosome assessment of the woman and her partner, uterine abnormalities, endometrial and hormonal luteal phase defects, and endometrial infection.
Group two comprised 75 patients experiencing repeated unexplained failure of IVF and embryo transfer including at least three transfers replacing two to four fresh or frozenthawed embryos each. The mean age of these patients was 33.1 years (range 2839) and the indications for IVF were tubal factor in 33 patients (44%), endometriosis in 16 (21%), male in 15 (20%), and unexplained in 11 (14%).
Controls included three groups of women. Group three comprised 100 normal healthy women (mean age 33.2 years, range 2239) having at least one child (range 15, mean 2.1) but no previous abortion and who were donors from the blood bank of our hospital. Group four included 60 infertile patients achieving a live birth with their first IVF/embryo transfer attempt carried out during the same time period as patients in group two. Indications for IVF in patients in group four were tubal factor (29 women, 48%), endometriosis (15 women, 25%), male (10 women, 16%), and unexplained (six women, 10%). Finally, group five or positive controls comprised 50 patients (mean 32.2 years, range 2539) with recurrent abortion (mean 3.2 miscarriages, range 27) who tested positive for aPL (LA and/or aCL) on at least two occasions, separated by a minimum interval of 8 weeks.
Detection of aß2GP-I by enzyme-linked immunosorbent assay (ELISA)
aß2GP-I were determined as previously described (Navarro et al., 1996). Briefly, microtitre plates (Maxisorp®; Nunc, Roskilde, Denmark) were coated (16 h, 4°C) with 25 µg/ml purified human ß-2 glycoprotein I (Stago, Asnières, France) in carbonate buffer (pH = 9.6). After washing, coated plates were blocked (1 h, 22°C) with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS)-Tween, and then incubated (1 h, 22°C) with samples diluted in PBS-Tween plus 1% BSA. After a new washing, plates were incubated (1 h, 22°C) with horseradish peroxidase-conjugated anti-human IgG and anti-human IgM (Dako, Glostrup, Denmark) diluted in PBS-Tween-BSA. For colour developing, ortho-phenylenediamine dihydrochloride in phosphate citrate buffer (pH 5.0) plus H2O2 was added, incubated (10 min, 22°C) and optical density read at 492 nm (OD492). In each assay, 10 negative sera were used as controls and OD492 higher than 3 SD above the mean value were considered positive.
Detection of aCL by ELISA
aCL were measured using an ELISA as previously described (Balasch et al., 1996). A reference serum sample was used to standardize the assay (Harris et al., 1987
).
Detection of LA
The following tests were carried out in all patients to detect LA: prothrombin time, activated partial thromboplastin time, kaolin clotting time, diluted Russell's viper venom time and tissue thromboplastin inhibition test according to methods previously reported (Balasch et al., 1996). In order to rule out a deficit in a coagulation factor, each assay was also performed in mixtures with control plasmas or phospholipids as recommended (Brandt et al., 1995
). Patients were considered to have LA activity when at least two of these assays were shown to be positive.
Statistical analysis
For statistical analysis the 2 test with the Yates correction was used.
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Results |
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Discussion |
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Recent evidence indicates that aPL do not bind directly to phospholipid but rather to a phospholipid-binding protein or to a combination of phospholipid and phospholipid-binding protein. One of these proteins, the plasma ß2GP-I, has been extensively studied as the `cofactor' for antibody-mediated pathology. It binds to anionic phospholipids and acts as a natural inhibitor of blood coagulation (Meroni et al., 1998). This opens the possibility that aPL may be pathogenic by disrupting the function of ß2GP-I rather than membrane phospholipid (Chamley, 1997
). Although there is general agreement that LA and aCL are associated with a clinical syndrome (the APS), it is now believed by most authorities in the field that the relevant antigen involves ß2GP-I as either the primary antigen or a cofactor for expression of the antigen. The impact of this on the diagnosis of the APS remains uncertain, however (Branch and Hatasaka, 1998
).
The LA and aCL tests are generally accepted confirmatory tests for APS. The aCL test is more sensitive and is positive in more than 8090% of patients with the disorder. The problem has been that this test is not specific and may be positive in a number of disorders other than APS (Harris et al., 1998). The LA is less frequently positive in APS and is regarded as the more specific test. This specificity derives from the fact that the LA reaction is found much less frequently in non-APS disorders. However, this test is not specific to APS and, in haematology laboratories where LA testing is performed, APS often represents only a minority of patients with positive tests. Since about 1020% of patients with APS have positive LA reactions alone and are aCL negative, performance of both aCL and LA tests is required (Harris et al., 1998
). Therefore, for more confident diagnosis of APS more specific tests are required that are also sensitive. The most extensively studied antigen in this regard is ß2GP-I, and directly measuring antibodies to ß2GP-I to detect clinically relevant aPL in patients with clinical features suggestive of APS but who are aCL and LA negative (Roubey, 1994
; Harris et al., 1998
) has been suggested. However, data in the literature with respect to the potential role of aß2GP-I on reproductive failure are controversial.
Thus, one study has shown that mice immunized with human ß2GP-I produce high titre antibodies against this protein and aCL, but do not suffer fetal loss or thrombocytopenia (Silver et al., 1995). On the contrary, other similar studies have shown increased fetal loss in mice immunized with ß2GP-I (Garcia et al., 1997
). In human gestation, the presence of aß2GP-I in sera of patients lacking aCL and LA has been associated with late pregnancy complications such as pre-eclampsia, fetal growth retardation, and gestational diabetes (Ailus et al., 1996
; Faden et al., 1997
) but others disagree (Aoki et al., 1994
; Katano et al., 1996
). Similarly, controversial data exist with respect to recurrent abortion. Thus, it has been reported (Stern et al., 1998
) that aß2GP-I are associated with recurrent miscarriage but the same was true for aCL in their series of patients and no data regarding aß2GP-I in aCL negative women were provided in that study. On the contrary, no relationship between aß2GP-I and fetal losses has been suggested in small series of patients (El-Kadi et al., 1995
; Martinuzzo et al., 1995
). In addition, in a large series (Rai et al., 1995b
) of 500 women with recurrent miscarriage no difference was found in the plasma ß2GP-I concentrations between aPL (LA and/or aCL) positive and aPL negative women with miscarriages and normal women although aß2GP-I were not investigated in that report. The association of ß2GP-I IgM antibody with IVF implantation failure has been previously suggested (Stern et al., 1998
). However, such an association did not exist when aß2GP-I IgG isotype was considered. Again, the confounding effect of aPL antibodies was not taken into account in that study (Stern et al., 1998
). In addition, these authors used patients newly referred for IVF treatment (i.e., who had not yet commenced treatment) as a control group rather than women being successful at IVF as done in the present study.
In fact, neither of the two previous reports (Rai et al., 1995b; Stern et al., 1998
) specifically investigated the incidence of aß2GP-I positive sera in recurrent aborters or IVF implantation failure patients with no detectable antibodies in standard antiphospholipid assays (aCL and/or LA). This was done in the present study where a large series of 100 patients with recurrent abortion and 75 IVF women with repeated embryo transfer together with appropriate controls were included. The results of this study clearly suggest that aß2GP-I screening in first trimester recurrent abortion or in failure of implantation after IVF is not warranted in aCL and LA negative patients. Finally, aß2GP-I were detected in 23 out of 50 (46%) patients with recurrent abortion and testing positive for the LA and/or aCL. The reported incidences of aß2GP-I in APS are highly variable, ranging from 0% (Gharavi et al., 1993
) to 90% (Cabiedes et al., 1995
) which may be explained by differences in patient populations and in laboratory methodology (Tsutsumi et al., 1998
).
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Acknowledgments |
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Notes |
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References |
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Alarcón-Segovia, D. and Cabral, A.R. (1996) The antiphospholipid/cofactor syndromes. J. Rheumatol., 21, 13191322.
Aoki, K., Matsuura, E., Sasa, H. et al. (1994) ß2-Glycoprotein I-dependent and -independent anticardiolipin antibodies in healthy pregnant women. Hum. Reprod., 9, 18491851.[Abstract]
Arnout, J. and Vermylen, J. (1998) Mechanism of action of ß2-glycoprotein I-dependent lupus anticoagulants. Lupus, 7 (suppl. 2), 2328.[ISI][Medline]
Asherson, R.A. (1991) `Primary' anti-phospholipid syndrome. In Harris, E.N., Exner, T., Hughes, G.R.V. and Asherson, R.A. (eds), Phospholipid-binding Antibodies. CRC Press, Boca Raton, pp. 377386.
Balasch, J. and Font, J. (1996) Prophylaxis of fetal losses in the antiphospholipid syndrome. In Asherson, R.E., Cervera, R., Piette, J.C. and Shoenfeld, Y. (eds), The Antiphospholipid Syndrome. CRC Press, Boca Raton, pp. 299307.
Balasch, J., Creus, M., Fábregues, F. et al. (1996) Antiphospholipid antibodies and human reproductive failure. Hum. Reprod., 11, 23102315.[Abstract]
Balasch, J., Creus, M., Fábregues, F. et al. (1998) Antiphospholipid antibodies and the outcome of pregnancy after the first in-vitro fertilization embryo transfer cycle. Hum. Reprod., 13, 11801183.[Abstract]
Berry, C.W., Brambati, B., Eskes, T.K.A.B. et al. (1995) The Euro-team early pregnancy (ETEP) protocol for recurrent miscarriage. Hum. Reprod., 10, 15161520.[Abstract]
Branch, D.W. and Hatasaka, K.K. (1998) Antiphospholipid antibodies and infertility: fact or fallacy. Lupus, 7 (suppl. 2), 9094.
Brandt, J.T., Triplett, D.A., Alving, B. and Scharrer, I. (1995) Criteria for the diagnosis of lupus anticoagulant: an update. Subcommittee on lupus anticoagulant/antiphospholipid antibody of the Scientific and Standardisation Committee of the ISTH. Thromb. Haemost., 74, 11851190.[ISI][Medline]
Bronson, R. (1995) Immunology and reproductive medicine. Hum. Reprod., 10, 755757.[ISI][Medline]
Brown, H.L. (1991) Antiphospholipid antibodies and recurrent pregnancy loss. Clin. Obstet. Gynecol., 34, 1726.[ISI][Medline]
Cabiedes, J., Cabral, A.R. and Alarcón Segovia, D. (1995) Clinical manifestations of antiphospholipid syndromes in patients with systemic lupus erythematosus associate more strongly with anti-ß2-glycoprotein-I than antiphospholipid antibodies. J. Rheumatol., 22, 18991906.[ISI][Medline]
Cabral, A.R., Amigo, M.C., Cabiedes, J. and Alarcón-Segovia, D. (1996) The antiphospholipid/cofactor syndromes: a primary variant with antibodies to ß2-glycoprotein-I but no antibodies detectable in standard antiphospholipid assays. Am. J. Med., 101, 472481.[ISI][Medline]
Chamley, L.W. (1997) Antiphospholipid antibodies or not? The role of ß2 glycoprotein 1 in autoantibody-mediated pregnancy loss. J. Reprod. Immunol., 36, 123142.[ISI][Medline]
Edelman, P., Rouquette, A.M., Verdy, E. et al. (1986) Autoimmunity, fetal losses, lupus anticoagulant: beginning of systemic lupus erythematosus or new autoimmune entity with gynaeco-obstetrical expression? Hum. Reprod., 1, 295297.[Abstract]
El-Kadi, H.S., Keil, L.B. and DeBari, V.A. (1995) Analytical clinical relationships between human IgG autoantibodies to ß2-glycoprotein-I and anticardiolipin antibodies. J. Rheumatol., 22, 22332237.[ISI][Medline]
Faden, D., Tincani, A., Tanzi, P. et al. (1997) Anti-beta 2 glycoprotein I antibodies in a general obstetric population: preliminary results on the prevalence and correlation with pregnancy outcome. Anti-ß2 glycoprotein I antibodies are associated with some obstetrical complications, mainly preeclampsia-eclampsia. Eur. J. Obstet. Gynecol. Reprod. Biol., 73, 3742.[ISI][Medline]
Garcia, C.O., Kanbour-Shakir, A., Tang, H. et al. (1997) Induction of experimental antiphospholipid antibody syndrome in PL/J mice following immunisation with ß2GPI. Am. J. Reprod. Immunol., 37, 118124.[ISI][Medline]
Gharavi, A.E., Harris, E.N., Sammaritano, L.R. et al. (1993) Do patients with antiphospholipid syndrome have autoantibodies to ß2-glycoprotein I? J. Lab. Clin. Med., 122, 426431.[ISI][Medline]
Gleicher, N. (1998) Autoantibodies in infertility: current opinion. Hum. Reprod. Update, 4, 169176.
Gleicher, N., Pratt, D., Dudkiewicz, A. et al. (1993) What do we really know about autoantibody abnormalities and reproductive failure: a critical review. Autoimmunity, 16, 115140.[ISI][Medline]
Harris, E.N., Gharavi, A.E., Patel, S.P. and Hughes, G.R.V. (1987) Evaluation of the anticardiolipin antibody test: report of a standardization workshop held in April 1986. Clin. Exp. Immunol., 68, 215222.[ISI][Medline]
Harris, E.N., Pierangeli, S.S. and Gharavi, A.E. (1998) Diagnosis of the antiphospholipid syndrome: a proposal for use of laboratory tests. Lupus, 7 (suppl. 2), 144148.
Hatasaka, H.H., Branch, D.W., Kutteh, W.H. and Scott, J.R. (1997) Autoantibody screening for infertility: explaining the unexplained? J. Reprod. Immunol., 34, 137153.[ISI][Medline]
Hughes, G.R.V. (1993) The antiphospholipid syndrome: ten years on. Lancet, 342, 341344.[ISI][Medline]
Hughes, G.R.V. (1998) Hughes' syndrome: the antiphospholipid syndrome. A historical view. Lupus, 7 (suppl. 2), 14.[ISI][Medline]
Katano, K., Aoki, K., Sasa, H. et al. (1996) ß2-Glycoprotein I-dependent anticardiolipin antibodies as a predictor of adverse pregnancy outcomes in healthy pregnant women. Hum. Reprod., 11, 509512.[Abstract]
Lockshin, M.D. (1998) Pregnancy loss and antiphospholipid antibodies. Lupus, 7 (suppl. 2), 8689.[ISI][Medline]
Martinuzzo, M.E., Forastiero, R.R. and Carreras, L.O. (1995) Anti-ß2-glycoprotein I antibodies; detection and association with thrombosis. Br. J. Haematol., 89, 397402.[ISI][Medline]
Meroni, P.L., Del Papa, N., Raschi, E. et al. (1998) ß2-glycoprotein I as a `cofactor' for anti-phospholipid reactivity with endothelial cells. Lupus, 7 (suppl. 2), 4447.
Navarro, M., Cervera, R., Teixidó, M. et al. (1996) Antibodies to endothelial cells and to ß-2-Glycoprotein I in the antiphospholipid syndrome: prevalence and isotype distribution. Br. J. Rheumatol., 35, 523528.[ISI][Medline]
Rai, R.S., Clifford, K., Cohen, H. and Regan, L. (1995a) High prospective fetal loss rate in untreated pregnancies of women with recurrent miscarriage and antiphospholipid antibodies. Hum. Reprod., 10, 33013304.[Abstract]
Rai, R.S., Regan, L., Clifford, K. et al. (1995b) Antiphospholipid antibodies and ß2-glycoprotein-I in 500 women with recurrent miscarriage: results of a comprehensive screening approach. Hum. Reprod., 10, 20012005.[Abstract]
Roubey, R.A.S. (1994) Autoantibodies to phospholipid-binding plasma proteins: a new view of lupus anticoagulants and other `antiphospholipid' antibodies. Blood, 84, 28542867.
Silver, R.M., Pierangeli, S.S., Gharavi, A.E. et al. (1995) Induction of high levels of anticardiolipin antibodies in mice by immunization with ß2-glycoprotein I does not cause fetal death. Am. J. Obstet. Gynecol., 173, 14101415.[ISI][Medline]
Simpson, J.L., Carson, S.A., Chesney, C. et al. (1998) Lack of association between antiphospholipid antibodies and first trimester spontaneous abortion: prospective study of pregnancies detected within 21 days of conception. Fertil. Steril., 69, 814820.[ISI][Medline]
Stern, C., Chamley, L. and Hale, L. (1998) Antibodies to ß2 glycoprotein I are associated with in vitro fertilization implantation failure as well as recurrent miscarriage: results of a prevalence study. Fertil. Steril., 70, 938944.[ISI][Medline]
Tsutsumi, A., Ichikawa, K., Matsuura, E. et al. (1998) Anti-ß2-glycoprotein I antibodies. Lupus, 7 (suppl. 2), 98102.
Submitted on January 22, 1999; accepted on April 22, 1999.