Portal de Periódicos da CAPES

Lupus anticoagulant and antibodies to β2-glycoprotein I, annexin V, and cardiolipin as a cause of recurrent spontaneous abortion

2007; Elsevier BV; Volume: 88; Issue: 5 Linguagem: Inglês

10.1016/j.fertnstert.2007.01.026

1556-5653

Nabil Mtiraoui, Walid Zammiti, M. Fékih, Samantha Hider, Wassim Y. Almawi, Touhami Mahjoub,

Reproductive System and Pregnancy

The prevalence of lupus anticoagulant (LAC), anticardiolipin (ACA), anti-β2 glycoprotein I (β2GPI), and antiannexin V antibodies were determined in 200 recurrent spontaneous abortion (RSA) patients and 200 age-matched control women. ACA IgG was associated with early, while antiannexin V IgG and LAC were associated with late, and ACA IgG, antiannexin V IgG, and LAC were associated with combined early + late RSA, thereby recommending inclusion of their screening in RSA workout. The prevalence of lupus anticoagulant (LAC), anticardiolipin (ACA), anti-β2 glycoprotein I (β2GPI), and antiannexin V antibodies were determined in 200 recurrent spontaneous abortion (RSA) patients and 200 age-matched control women. ACA IgG was associated with early, while antiannexin V IgG and LAC were associated with late, and ACA IgG, antiannexin V IgG, and LAC were associated with combined early + late RSA, thereby recommending inclusion of their screening in RSA workout. Recurrent spontaneous abortion (RSA), defined as three or more consecutive pregnancy losses before the 20th week of gestation, is a multifactorial disorder with a poor and often undefined etiology. Dysregulated immunity was proposed as a mechanism underlying RSA in the face of unknown causes (1Lee J. Choi B.C. Cho C. Hill J.A. Baek K.H. Kim J.W. Trophoblast apoptosis is increased in women with evidence of Th1 immunity.Fertil Steril. 2005; 83: 1047-1049Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). The common antiphospholipid antibodies (APAs) lupus anticoagulant (LAC), anticardiolipin (ACA), anti-β2 glycoprotein I (β2GPI), and antiannexin V antibodies reportedly precipitated RSA through direct and indirect mechanisms (2Ulcova-Gallova Z. Krauz V. Novakova P. Milichovska L. Micanova Z. Bibkova K. et al.Anti-phospholipid antibodies against phosphatidylinositol, and phosphatidylserine are more significant in reproductive failure than antibodies against cardiolipin only.Am J Reprod Immunol. 2005; 54: 112-117Crossref PubMed Scopus (37) Google Scholar, 3Levy R.A. Avvad E. Oliveira J. Porto L.C. Placental pathology in antiphospholipid syndrome.Lupus. 1998; 7: S81-S85Crossref PubMed Scopus (47) Google Scholar), including antibody-directed trophoblast damage (3Levy R.A. Avvad E. Oliveira J. Porto L.C. Placental pathology in antiphospholipid syndrome.Lupus. 1998; 7: S81-S85Crossref PubMed Scopus (47) Google Scholar) and precipitation of thrombotic events (4Meroni P.L. di Simone N. Testoni C. D'Asta M. Acaia B. Caruso A. Antiphospholipid antibodies as cause of pregnancy loss.Lupus. 2004; 13: 649-652Crossref PubMed Scopus (53) Google Scholar). A link between poor obstetric outcome and high APA levels was proposed, evidenced by their capacity to induce fetal resorption (5Gris J.C. Perneger T.V. Quere I. Mercier E. Fabbro-Peray P. Lavigne-Lissalde G. et al.Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case–control study.Blood. 2003; 102: 3504-3513Crossref PubMed Scopus (86) Google Scholar). Detection of APAs in early pregnancy prevented fetal wastage, and positive β2GPI-dependent ACA reactivity was associated with miscarriage, pre-eclampsia, and fetal growth restriction (6Heilmann L. von Tempelhoff G.F. Kuse S. The influence of antiphospholipid antibodies on the pregnancy outcome of patients with recurrent spontaneous abortion.Clin Appl Thromb Hemost. 2001; 7: 281-285Crossref PubMed Scopus (13) Google Scholar). Although current recommendations call for LAC and ACA testing in RSA, APAs not included in the standard workout were proposed to be included, including those targeting membrane phospholipids (2Ulcova-Gallova Z. Krauz V. Novakova P. Milichovska L. Micanova Z. Bibkova K. et al.Anti-phospholipid antibodies against phosphatidylinositol, and phosphatidylserine are more significant in reproductive failure than antibodies against cardiolipin only.Am J Reprod Immunol. 2005; 54: 112-117Crossref PubMed Scopus (37) Google Scholar), or phospholipid-binding proteins—β2GPI (7Ulcova-Gallova Z. Bouse V. Krizanovska K. Balvin M. Rokyta Z. Netrvalova L. Beta 2-glycoprotein I is a good indicator of certain adverse pregnancy conditions.Int J Fertil Womens Med. 2001; 46: 304-308PubMed Google Scholar) and annexin V (8Matsubayashi H. Arai T. Izumi S. Sugi T. McIntyre J.A. Makino T. Anti-annexin V antibodies in patients with early pregnancy loss or implantation failures.Fertil Steril. 2001; 76: 694-699Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The present study investigated the contribution of the traditional (LAC and ACA) and nontraditional (annexin V and β2GPI) APAs in women with idiopathic RSA. This was a matched case–control study performed in 2002 to 2004 at the Maternity Center of Hôpital Farhat Hached of Sousse, Tunisia. Cases comprised 200 women with three or more unexplained consecutive pregnancy losses, 5–20 weeks of gestation (mean age 28.6 ± 5.6 years); pregnancy losses were classified as early (5–10 weeks) and late (11–20 weeks). Patients underwent a complete RSA assessment, which included physical examination, hysteroscopy, LAC, APAs, and cervical cultures. Exclusion criteria included induced abortions, infections, systemic disease, use of combined oral contraceptive pills, uterine structural abnormalities, and/or personal or family history of thrombosis. As control, 200 women with uncomplicated pregnancies were recruited from the same maternity center, and were matched according to age (28.2 ± 5.5 years), and total number of pregnancies. Participants were asked to sign an informed consent form agreeing to participate in the study, which was conducted after all institutional ethics requirements were met and institutional review board approval was obtained. Blood samples were collected in trisodium citrate tubes 8–12 weeks after the last pregnancy, and plasma was stored frozen (−80°C). Anti--β2 glycoprotein I, antiannexin V, and ACA IgG and IgM levels were performed on two separate occasions by ELISA, using the Zymutest kit (Hyphen Biomed, France). The cutoff value of each APA [in phospholipid arbitrary units (AU)], was defined as the mean optical density (OD) ± 3 SD of controls. Sample were scored positive for anti-β2GPI IgG (10.8 AU) or IgM (7.3 AU), antiannexin V IgG (13.7 AU) and IgM (12.8 AU), and ACA IgG (21.5 AU) and IgM (22.8 AU) when their OD values for IgG and IgM measurement were higher than the calculated cutoff points. Lupus anticoagulant activity was detected by dilute Russell viper venom time using a Behring LA kit (Dade Boehring, Germany), kaolin clotting time, and a tissue thromboplastin inhibition test. Initially positive tests were retested after 6–8 weeks, with repeatedly positive specimens retested again after 3 months. For samples with a prolonged clotting time, a confirmation assay (dilute Russell viper venom confirm [ratio]) was performed with added phospholipids. Quantitative data are described by their mean ± SD. Statistical analysis was performed on SPSS v. 13.0 statistics software. Pearson's chi-square and Student's t test were employed for testing qualitative and continuous variables, respectively. Logistic regression analysis was performed in univariant then multivariant way, with control parous women being the reference group, was performed to determine odds ratios (OR) and 95% confidence intervals (95% CI) associated with RSA risk. Statistical significance was set at P<.05. Patients were comparable to controls with regard to age, body mass index (BMI), number of smokers, alcohol consumers, and oral contraceptive users. Although the pregnancy rate was comparable between patients and controls, the mean number of live births per women was higher among controls, with patients reporting 726 first-or second-trimester pregnancy losses. Positive LAC was seen in 82 (41.0%) cases and 14 (7.0%) controls (P<.001), and positive ACA IgG was seen in 41 (20.5%) patients and 4 (2.0%) controls (P<.001). Positive antiannexin V IgG was also seen in 9 (4.5%) patients, but in none of the controls (P=.004) (Table 1). Significant but weak correlations were shown between antiannexin V IgG and ACA IgG (Spearman's correlation coefficient r2 = .122, P<.05) and LAC and ACA IgG (r2 = .294, P<.001). The prevalence of other antibodies and/or isotypes was comparable between cases and controls.Table 1Antibody distribution among cases and controls.aStudy subjects comprised 200 recurrent spontaneous abortion cases and 200 age-matched controls.AntibodyIsotypePercentileCasesbNumber of subjects (percent of total within group).ControlsbNumber of subjects (percent of total within group).PcPearson chi-square test.OR (95%CI)ACAIgMAll7 (3.5)1 (0.5).070.88–59IgGAll41 (20.5)4 (2.0)<.0014.4–36 P9520 (10.0)0 (0.0)<.001N/AdN/A = not applicable.Anti-β2GPIIgGAll9 (4.5)5 (2.5).420.6–5.6IgMAll9 (4.5)6 (3.0).600.5–4.4Antiannexin VIgMAll5 (2.5)0 (0.0).06N/AIgGAll9 (4.5)0 (0.0).004N/A P9514 (7.0)2 (1.0).0057.50 (1.60–2.40)Positive LAC82 (41.0)14 (7.0)<.0015.00–17.00Note: OR = odds ratios; CI = confidence interval; LAC = lupus anticoagulant; ACA = anticardiolipin; anti-β2GPI = anti-β2 glycoprotein I.Mtiraoui. Autoantibodies in recurrent pregnancy loss. Fertil Steril 2007.a Study subjects comprised 200 recurrent spontaneous abortion cases and 200 age-matched controls.b Number of subjects (percent of total within group).c Pearson chi-square test.d N/A = not applicable. Open table in a new tab Note: OR = odds ratios; CI = confidence interval; LAC = lupus anticoagulant; ACA = anticardiolipin; anti-β2GPI = anti-β2 glycoprotein I. Mtiraoui. Autoantibodies in recurrent pregnancy loss. Fertil Steril 2007. Increasing OR values were seen with increasing cutoff values for both antibodies, with a mean fivefold increased risk (OR: 4.8, 95% CI: 1.5–12.7) at antibody titers exceeding the 90th percentile for ACA IgG, and a sevenfold increased risk (OR 7.5, 95% CI: 1.6–24) at titers exceeding the 95th percentile for antiannexin V IgG (Table 1). Elevated ACA IgG was seen in 29 (35.4%) and in 2 (14.3%) LAC-positive cases and controls, respectively, and no increased frequency of antiannexin V IgG were associated with a positive LAC among cases (6.1%) and controls (0.0%). Interestingly, positive ACA IgG/negative LAC resulted in a 10-fold increased risk (OR = 10.4, 95% CI: 2.3–47). Positive LAC was seen in 7 of the 33 exclusively early (OR = 3.6, 95% CI: 1.3–9.7), 25 of the 43 exclusively late (OR = 18.5, 95% CI: 8.2–41.6), and in 50 of the 124 combined early + late (OR = 9.0, 95% CI: 4.7–17.2) RSA cases. Similarly, positive ACA IgG was seen in 5 (15.2%) exclusively early (OR = 8.8, 95% CI: 2.2–34.5), in 12 (27.9%) exclusively late (OR = 19.0, 95% CI: 5.8–62.6), and in 24 (19.4%) (OR = 11.8, 95% CI: 4.0–35.0) combined early + late RSA cases. Regression analysis demonstrated that BMI >25 kg/m2 (OR = 0.88, 95% CI = 0.79–0.99), and positive ACA IgG (OR = 11.1, 95% CI = 2.2–55) were associated with exclusively early, antiannexin V IgG (OR = 10.9, 95% CI = 1.2–98) and LAC (OR = 12, 95% CI = 4.6–31) with exclusively late RSA, and BMI >25 kg/m2 (OR = 0.93, 95% CI = 0.87–0.99), positive ACA IgG (OR = 7, 95% CI = 2.1–22.7), antiannexin V IgG (OR = 10.3, 95% CI = 1.2–87) and LAC (OR = 7.5, 95% CI = 3.7–15.1) were associated with combined (early + late) RSA. Insofar as autoimmune factors were implicated in RSA pathogenesis, an association between antiphospholipid (cardiolipin, phosphatidylserine) or antiphospholipid-binding proteins (β2GPI and annexin V) and RSA was demonstrated for both animal models and in prospective clinical studies (5Gris J.C. Perneger T.V. Quere I. Mercier E. Fabbro-Peray P. Lavigne-Lissalde G. et al.Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case–control study.Blood. 2003; 102: 3504-3513Crossref PubMed Scopus (86) Google Scholar, 9Rand J. Eerden P.V. Wu X.X. Chazotte C. Defective annexin A5 crystallization: a mechanism for pregnancy losses in the antiphospholipid syndrome.Thromb Res. 2005; 115: 77-81PubMed Google Scholar). Unlike ACA and LAC, the role of antiannexin V and anti-β2GPI antibodies in RSA remains to be established, and it is likely to involve precipitation of a prothrombotic state resulting in defective implantation and placentation (9Rand J. Eerden P.V. Wu X.X. Chazotte C. Defective annexin A5 crystallization: a mechanism for pregnancy losses in the antiphospholipid syndrome.Thromb Res. 2005; 115: 77-81PubMed Google Scholar). Unlike LAC, ACA IgG, and antiannexin V IgG, anti-β2GPI antibodies did not contribute to the RSA risk, in agreement with previous studies that also found no association of anti-β2GPI antibodies in RSA women, and in APA-positive women with equal to or greater than two fetal losses (10Rai R.S. Regan L. Clifford K. Pickering W. Dave M. Mackie I. et al.Antiphospholipid antibodies and beta 2-glycoprotein-I in 500 women with recurrent miscarriage: results of a comprehensive screening approach.Hum Reprod. 1995; 10: 2001-2005PubMed Google Scholar). Patients were age matched to controls so as to circumvent potential influence of age on RSA. The discrimination between positive and negative antibody profile was based on a threshold defined by the mean antibody values ±3 SD of healthy controls, adopted so as to circumvent the confusion inherent in the use of commercial kits among different populations (11Branch D.W. Silver R.M. Pierangeli S.S. van Leeuwen I. Harris E.N. Antiphospholipid antibodies in women with recurrent pregnancy loss, fertile controls, and antiphospholipid syndrome.Obstet Gynecol. 1997; 89: 549-555Crossref PubMed Scopus (119) Google Scholar), thereby recommending controlling variability between commercially available serologic tests (5Gris J.C. Perneger T.V. Quere I. Mercier E. Fabbro-Peray P. Lavigne-Lissalde G. et al.Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case–control study.Blood. 2003; 102: 3504-3513Crossref PubMed Scopus (86) Google Scholar, 11Branch D.W. Silver R.M. Pierangeli S.S. van Leeuwen I. Harris E.N. Antiphospholipid antibodies in women with recurrent pregnancy loss, fertile controls, and antiphospholipid syndrome.Obstet Gynecol. 1997; 89: 549-555Crossref PubMed Scopus (119) Google Scholar). By using >95th percentile, heightened prevalence of ACA IgG and antiannexin V IgG was seen in cases, which translated into higher RSA risk. Positive ACA IgG were found in 2% of control subjects, which was comparable to rates reported elsewhere (5Gris J.C. Perneger T.V. Quere I. Mercier E. Fabbro-Peray P. Lavigne-Lissalde G. et al.Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case–control study.Blood. 2003; 102: 3504-3513Crossref PubMed Scopus (86) Google Scholar, 6Heilmann L. von Tempelhoff G.F. Kuse S. The influence of antiphospholipid antibodies on the pregnancy outcome of patients with recurrent spontaneous abortion.Clin Appl Thromb Hemost. 2001; 7: 281-285Crossref PubMed Scopus (13) Google Scholar), but was significantly higher among RSA cases (11Branch D.W. Silver R.M. Pierangeli S.S. van Leeuwen I. Harris E.N. Antiphospholipid antibodies in women with recurrent pregnancy loss, fertile controls, and antiphospholipid syndrome.Obstet Gynecol. 1997; 89: 549-555Crossref PubMed Scopus (119) Google Scholar). Whereas LAC levels change during pregnancy, persistently elevated levels were strongly associated with nonearly fetal loss, as shown also elsewhere (4Meroni P.L. di Simone N. Testoni C. D'Asta M. Acaia B. Caruso A. Antiphospholipid antibodies as cause of pregnancy loss.Lupus. 2004; 13: 649-652Crossref PubMed Scopus (53) Google Scholar), likely through precipitation of a prothrombotic state, as suggested (4Meroni P.L. di Simone N. Testoni C. D'Asta M. Acaia B. Caruso A. Antiphospholipid antibodies as cause of pregnancy loss.Lupus. 2004; 13: 649-652Crossref PubMed Scopus (53) Google Scholar, 12Sugi T. Matsubayashi H. Inomo A. Dan L. Makino T. Antiphosphatidylethanolamine antibodies in recurrent early pregnancy loss and mid-to-late pregnancy loss.J Obstet Gynaecol Res. 2004; 30: 326-332Crossref PubMed Scopus (70) Google Scholar). The simultaneous occurrence of LAC with antiannexin V IgG, but not with ACA IgG, led to a further increase in RSA risk, similar to what was found for venous thrombosis (13Pengo V. Biasiolo A. Brocco T. Tonetto S. Ruffatti A. Autoantibodies to phospholipid-binding plasma proteins in patients with thrombosis and phospholipids reactive antibodies.Thromb Haemost. 1996; 75: 721-724PubMed Google Scholar). Interestingly, a positive LAC without elevated antiannexin V IgG or ACA IgG did not significantly affect RSA risk. Although explanation for this remains speculative, it may reside in the low number of LAC-positive control subjects, which may have masked potential interaction between LAC and other antibodies. A larger sample will be needed to confirm, or alternatively rule out, this assumption. In our hands, the prothrombotic capacity of LAC, but not ACA IgG or antiannexin V IgG (14Tsakiris D.A. Yasikoff M.L. Wolf F. Marbet G.A. Anticardiolipin antibodies do not seem to be associated with APC resistance in vivo or in vitro.Ann Hematol. 1995; 71: 195-198Crossref PubMed Scopus (7) Google Scholar), was linked with activated protein C resistance (APCR), after controlling for inherited causes of APCR (factor V-Leiden). However, it remains to be seen as to whether this factor V-Leiden-independent APCR is true or spurious, being linked to the progression of pregnancy itself, as was suggested (15Bokarewa M.I. Wramsby M. Bremme K. Blomback M. Variability of the response to activated protein C during normal pregnancy.Blood Coagul Fibrinolysis. 1997; 8: 239-244Crossref PubMed Scopus (23) Google Scholar). The presence of APAs was associated RSA, via a mechanism that requires participation of immune and nonimmune factors, which acted in inducing a prothrombotic state favoring fetal loss (16Gleicher N. Some thoughts on the reproductive autoimmune failure syndrome (RAFS) and Th-1 versus Th-2 immune responses.Am J Reprod Immunol. 2002; 48: 252-254Crossref PubMed Scopus (29) Google Scholar). For APAs to be linked to pregnancy loss, they must be present at moderate or high levels (5Gris J.C. Perneger T.V. Quere I. Mercier E. Fabbro-Peray P. Lavigne-Lissalde G. et al.Antiphospholipid/antiprotein antibodies, hemostasis-related autoantibodies, and plasma homocysteine as risk factors for a first early pregnancy loss: a matched case–control study.Blood. 2003; 102: 3504-3513Crossref PubMed Scopus (86) Google Scholar), raising the possibility that they could be the consequence, and not necessarily the cause of RSA. By inducing an alloresponse, sequestration of or crossreactivity with endogenous molecules during an inflammatory response may facilitate the generation of antibodies against self-antigens (16Gleicher N. Some thoughts on the reproductive autoimmune failure syndrome (RAFS) and Th-1 versus Th-2 immune responses.Am J Reprod Immunol. 2002; 48: 252-254Crossref PubMed Scopus (29) Google Scholar), exemplified by the APA crossreactivity with oxidized low-density lipoprotein (17Horkko S. Miller E. Dudl E. Reaven P. Curtiss L.K. Zvaifler N.J. et al.Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids. Recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein.J Clin Invest. 1996; 98: 815-825Crossref PubMed Scopus (323) Google Scholar). By reducing annexin V levels in trophoblasts, and the production of (vasodilating) prostacyclin, APAs reportedly precipitate attenuated fibrinolysis and APCR (18Gharavi A.E. Pierangeli S.S. Levy R.A. Harris E.N. Mechanisms of pregnancy loss in antiphospholipid syndrome.Clin Obstet Gynecol. 2001; 44: 11-19Crossref PubMed Scopus (54) Google Scholar). Insofar as pregnancy loss associated with APS was fetal death because of placental insufficiency, it is likely that APS-associated hypercoagulability directly impacted on the utero–placental circulation, evidenced by the finding of thrombotic, fibrotic, and/or marked infarction in placentas of APS pregnancies. In conclusion, our findings suggest that the assessment of Tunisian women with RSA from the 10th week of gestation on should include, in addition to LAC and ACA, screening for antiannexin V and potentially other antibodies (2Ulcova-Gallova Z. Krauz V. Novakova P. Milichovska L. Micanova Z. Bibkova K. et al.Anti-phospholipid antibodies against phosphatidylinositol, and phosphatidylserine are more significant in reproductive failure than antibodies against cardiolipin only.Am J Reprod Immunol. 2005; 54: 112-117Crossref PubMed Scopus (37) Google Scholar, 12Sugi T. Matsubayashi H. Inomo A. Dan L. Makino T. Antiphosphatidylethanolamine antibodies in recurrent early pregnancy loss and mid-to-late pregnancy loss.J Obstet Gynaecol Res. 2004; 30: 326-332Crossref PubMed Scopus (70) Google Scholar).