7. Antiphospholipid syndrome – pathophysiology

Gilad Twig, Yaniv Sherer, Miri Blank, and Yehuda Shoenfeld

INTRODUCTION

ETIOLOGY OF APS

Antiphospholipid syndrome (APS) was first defined as a syndrome in 1983,1 consisting of a triad of manifestations involving arterial and/or venous thrombosis, recurrent fetal loss, accompanied by mild to moderate thrombocytopenia and elevated titers of antiphospholipid (aPL) antibodies: lupus anticoagulant (LA) and/or anticardiolipin antibodies (aCL). Today, this syndrome is known to be systemic and may affect almost every organ and tissue in the body. The cause of APS is still considered a mystery – yet, as in many other autoimmune diseases, a combination of environmental and genetic factors has been proposed. Recent data indicate that infectious agents may play a major role in the etiology of APS. The pathophysiology of APS includes all arms of the coagulation system, as well as other mechanisms not related to hypercoagulability. In fact, aPL have been shown to be directly toxic to the developing fetus, as these antibodies can be passively transferred from humans to naive mice and will induce pregnancy loss in those mice2 (Figure 7.1). Active immunization with human pathogenic monoclonal anticardiolipin antibody induces clinical manifestations of APS in BALB/c mice.3 Additionally, the serum from women with APS is highly teratogenic to rat embryos in culture and also affects embryonic growth.4 Moreover, purification of the immunoglobulin G (IgG) fraction of the sera of women with APS directly affects the embryo and yolk sac, reducing their growth.5 This chapter discusses the etiology and pathophysiology of APS, with special emphasis on the reproductive system.

aPL have long been known to require a cofactor in order to have their effects. Today this co-factor (apolipoprotein H or β2-glycoprotein I (β2GPI) is thought to be the antigen to which aPL bind. Binding of aPL to β2GPI forms divalent IgG–β2GPI complexes that have increased affinity for membrane phospholipids.6 The physiological function of β2GPI is unknown. β2GPI deficiency is not associated with disease; homozygous β2GPI-null mice also appear to suffer no pathological effects.7 The binding of aPL–β2GPI to cell membranes, including trophoblasts, results in injury and/or activation. Like many other autoimmune diseases, APS may have a multifactorial etiology in which genetic susceptibility is made apparent by environmental factors.

One environmental factor that has been intensively investigated in recent years is infection. In a series of 100 APS patients,8 various infections were shown to precede the development of APS, including skin infections (18%), human immunodeficiency virus (HIV) (17%), pneumonia (14%), hepatitis C virus (13%), and urinary tract infection (10%). Helicobacter pylori, a common bacterial pathogen that colonizes the gastric mucosa and induces chronic gastric inflammation, has been associated with APS. In pregnant women, H. pylori infection can cause intrauterine fetal growth retardation,9 and increases the risk of reproductive disorders.10 Molecular mimicry between (β2GPI) and bacterial and viral epitopes is the principal mechanism that links infections to APS. In the case of H. pylori, 34% of patients were positive for anti- β2GPI antibodies that showed high homology to the

107

RECURRENT PREGNANCY LOSS: CAUSES, CONTROVERSIES AND TREATMENT

Figure 7.1 Experimental antiphospholipid syndrome (APS). (a) APS uterine horn showing resorbed pregnancies and live fetuses at day 14 of pregnancy. (b) Control uterine horn showing developing pregnancies with no resorptions at day 14.

target epitopes of H. pylori structures.11,12 Indeed, β2GPI has been found to be immunogenic in vivo. Immunization of BALB/c or PL/J mice or New Zealand white rabbits with β2GPI resulted in generation of anti-β2GPI antibodies.13 The high titers of mouse anti-β2GPI antibodies has been associated with an increased proportion of fetal resorptions (the equivalent of fetal loss in humans), thrombocytopenia, and prolonged activated partial thromboplastin time (aPTT), indicating the

presence of LA, a presentation characteristic of experimental APS.13

Direct experimental evidence for molecular mimicry of bacterial pathogens has emerged from the effect of immunization with certain microbial pathogens that share epitope homology with the β2GPI molecule. Pathogenic anti-β2GPI autoantibodies directed against the TLRVYK epitope were formed in mice immunized with Haemophilus influenzae or Neisseria gonorrhoeae that exhibit

108

the TLRVYK sequence, or with tetanus toxoid that does not present linearly the sequence TLRVYK but can still serve as a mimotope. The formed anti- β2GPI autoantibodies have been shown to be pathogenic and capable of inducing the clinical picture of experimental APS, manifested by a high percentage of fetal loss, thrombocytopenia, and a prolonged aPTT.14 Moreover, the pathogenic effect of monoclonal antibodies to β2GPI is inhibited by the addition of synthetic peptides including the TLRVYK sequence. The latter prevented the development of APS in mice injected with monoclonal antibodies to β2GPI, or decreased the degree of endothelial cell activation, monocyte adhesion, and expression of adhesion molecules in vitro.15 Infection may be one of the mechanisms giving rise to APS. In humans, infection with varicella zoster virus (VZV) has been associated with APS.16 Although HIV, hepatitis A, hepatitis B, and hepatitis C are also associated with an increased prevalence of aCL antibodies, most of these are not β2GPIdependent.17 The difference between APS and the mere presence of aPL may be due to diseases such as syphilis and Lyme disease raising antibodies that recognize phospholipids directly, whereas in APS the infections raise antibodies that recognize epitopes on phospholipid-binding proteins such as β2GPI.

MECHANISMS OF REPRODUCTIVE

FAILURE IN APS

THROMBOSIS

As mentioned above, systemic thromboembolism is the principal manifestation of APS. Evidence for thrombi in the placental circulation and the beneficial effect of antithrombotic therapy in APS patients suffering from recurrent pregnancy loss (RPL) suggest a central role for this mechanism in reproductive failure. The underlying basis for the hypercoagulable state in APS is complex, and involves altered activity of all three major components that govern hemostasis: platelets, fibrinolysis, and the coagulation cascade. The coagulation system in APS

ANTIPHOSPHOLIPID SYNDROME – PATHOPHYSIOLOGY

was shown to be altered at different levels. aPL inhibit both protein C activation and the function of activated protein C (APC), thereby preventing the inactivation of activated factors V and VIII.18 This inhibition is conditional upon the presence of β2GPI which is a pre-requisite for the binding of aPL to protein C. In addition, autoantibodies directed against protein C, protein S, and thrombomodulin have been detected in some APS patients.19

Tissue factor (TF), an initiator of the extrinsic coagulation cascade, which is not normally expressed by intravascular cells, has been shown to be altered in APS patients. It has been shown that TF-related procoagulant activity and TF mRNA levels in monocytes are increased in primary APS patients with thrombosis when compared with those without thrombosis.20 Injection of purified IgG aCL from APS patients with previous thrombotic episodes induced a significant increase in both monocyte procoagulant activity and TF expression, as compared with purified IgG or IgM aCL from two systemic lupus erythematosus (SLE) patients without thrombosis.21 In addition, functional anti-TF pathway inhibitor activity has been detected in the sera of a subset of APS patients, showing a correlation between the degree of inhibition and associated occurrence of arterial thrombosis and stroke.22

Endothelial cells are affected by aPL autoantibodies. Potentiation of human umbilical vein endothelial cells (HUVEC) procoagulant activity by aPL contained in sera from SLE patients is strongly decreased after depleting IgG from the sera.23 Human anti-β2GPI IgM monoclonal antibodies and polyclonal anti-β2GPI antibodies have been shown to induce tissue factor at both protein and mRNA level in HUVEC monolayers in vitro.24 aPL can further up-regulate adhesion molecules (E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1)) expression and secretion of the proinflammatory cytokines interleukin-1b (IL-1b) and IL-6.25 Increased plasma levels of soluble VCAM-1 have been found in primary APS patients with recurrent thrombotic events, and elevated levels of tissue plasminogen activator and von Willebrand

109

RECURRENT PREGNANCY LOSS: CAUSES, CONTROVERSIES AND TREATMENT

factor (vWF) (as endothelial perturbation markers) have been associated with aPL in SLE.

Decreased endothelial cell prostacyclin (PGI2), the principal inhibitor of platelet aggregation, and increased thromboxane A2 (TXA2) production by platelets have both been implicated as mechanisms predisposing to thrombosis in patients with APS. aPL enhance platelet TXA2 production, and allow platelet activation to occur without a compensatory increment in the vascular biosynthesis of PGI2.26

Hypofibrinolysis can further aggravate the prothrombotic state in APS. Endothelial cell dysfunction can increase plasma levels of plasminogen activator inhibitor type-1 (PAI-1) and tissue-type plasminogen activator (tPA) antigens.27 In addition the hypofibrinolytic state can be further aggravated by the presence of autoantibodies against components of the fibrinolytic system such as as antiplasmin/plasminogen28 and anti-tPA.29

Platelets play a central role in primary hemostasis and are involved in the prothrombotic state of APS patients. Monoclonal aCL obtained from patients with APS increased platelet interaction with the subendothelium.21 It has been proposed that a minor degree of platelet activation can lead to exposure of phospholipids, which can potentially be amplified to a much larger degree in the serum of APS patient than in controls.21 β2GPI initially binds to these phospholipids, then binds aPL to form β2GPI–phospholipid complexes. The latter can further activate platelet aggregation by allowing the interaction between the Fc portion and the platelet surface FcγRII receptors (the only FcγR molecules present on platelets).21,30 In addition to activation of the FcγRII receptors, the β2GPI–phospholipid complexes can also exert their action through complement activation, as complement generated in the presence of aPL binds to negatively charged phospholipids and activates platelets.31

In addition to the systemic prothrombotic effects, APS autoantibodies may alter the placental circulation by attacking certain placental epitopes. Annexin A5, a potent anticoagulant protein that has a thrombomodulatory role in the placental circulation, is such a target.32 Annexin V is found on the apical surface of placental syncytiotrophoblasts, and forms

clusters on exposed phospholipids, thereby forming a protective shield on the phospholipid surface. Annexin V blocks phospholipids from becoming available for coagulation reactions. The annexin V protective shield could be damaged by either binding to anti-annexin V or preventing its binding to the phospholipid membrane, or by blocking autoantibodies against annexin V/phospholipids.33 Antiannexin V autoantibodies have been detected in patients with SLE and APS associated with pregnancy loss, while reduced levels of annexin V have been observed on the placental villi of women having aPL and RPL and a thrombogenic background.34

Recently, complement activation was reported as essential and causative of APL-induced fetal injury. Treatment with heparin prevented this activation both in-vivo and in-vitro.35

ARACHIDONIC ACID AND PROSTACYCLIN

aPL inhibit arachidonic acid release.36 Arachidonic acid is an essential prerequisite for PGI2 production (PGI2 is a physiological inhibitor of thrombocyte aggregation, and a potent vasodilatator). aPL have been shown to increase the concentration of TXA2, thus altering the PGI2/ TXA2 balance.37 The alteration in the PGI2/TXA2 balance has two effects: vasoconstriction, which impedes the blood supply to the fetus, and platelet activation, with the procoagulant effects described above.

In a mouse model of experimental APS, Shoenfeld and Blank38 infused aCL into pregnant mice in order to induce APS. Mice that were cotreated with a thromboxane receptor antagonist had a significant reduction in the fetal resorption rate from 45% to 19.8% and an increase in mean placental and embryo weights. There was also an increased platelet count (from 597 100 to 1 075 000 platelets/mm3) in treated mice, indicating the effect of thrombocyte aggegation in APS.

ANTICYTOKINE EFFECT

The anti-inflammatory cytokine IL-3 is important for the maintenance of normal pregnancy. IL-3 enhances placental and fetal development while increasing the

110

number of megakaryoctes. The serum level of IL-3 in pregnant patients with primary APS or APS secondary to SLE was found to be lower than in controls.39 In vitro studies have revealed that low-dose aspirin (10 mg/μl) stimulates IL-3 production through its ability to raise leukotriene production, while higher doses of aspirin failed to induce IL-3 generation.39,40 Furthermore, ciprofloxacin treatment significantly decreased the rate of pregnancy loss in BALB/c mice with experimental APS. This effect correlated with an increases in the serum IL-3 level and in bone marrow megakaryocytes.41 Other cytokines may also be involved. The level of the proinflammatory and prothrombotic cytokine tumor necrosis factor α (TNF-α), has been shown to be significantly higher in patients with APS than in healthy controls.42

INDUCTION OF PLACENTAL CELL APOPTOSIS

Most the literature describes placental pathology in terms of aPL being directed against negatively charged phospholipids, leading to placental infarction and eventually pregnancy loss. aPL may affect the adhesion molecules between the elements of the syncytiotrophoblast. Cellular activation increases the expression of cell adhesion molecules,43,44 which may promote leukocyte adhesion to the endothelial surface. Although the thrombogenic effects of aPL are mediated by ICAM-1, VCAM-1, and P-selectin,45 aPL may damage the trophoblast in a manner unrelated to thrombosis, as the cytotrophoblast cells express phospholipid on their surface. This concept is supported by histological evidence from patients with aPL and fetal death. Women with aPL have decreased vasculosyncitial membranes, increased syncytial knots, and substantially more fibrosis, hypovascular villi, and infarcts than women without APS.46 The changes in syncytial membranes may be secondary to thrombosis, but thrombosis could also be secondary to placental damage that allows free transplacental passage of maternal aPL. Addition of IgG purified from women with SLE/APS, positive for aCL/anti-DNA antibodies, reduced yolk sac and embryonic growth more than sera negative for aPL but positive for anti-phosphatidylserine and anti-laminin.47 The sera of SLE/APS patients has

ANTIPHOSPHOLIPID SYNDROME – PATHOPHYSIOLOGY

also been shown to inhibit the rate of trophoblastic cell growth and to accelerate the rate of apoptosis of cultured human placental cells.47,48

aPL can alter the secretion of human chorionic gonadotropin (hCG). Our team has shown that the addition of human polyclonal purified aCL, which were shown to induce experimental APS, suppressed the pulsatile secretion of hCG.49 In vitro, monoclonal aPL halved trophoblastic hCG and human phospholipid production.50 These results may be secondary to the effect of aPL on trophoblast differentiation and invasion.

aPL do not seem to be teratogenic to the embryo, however. The offspring of SLE/APS patients do not have a higher rate of anomalies, although the pregnancies are often complicated by intrauterine growth retardation.51

CONCLUSIONS

APS is a systemic syndrome whose etiology involves both environmental and genetic factors. Infections may be highly important in the etiology of this syndrome, and molecular mimicry is probably the mechanism by which infectious agents induce aPL. aPL exert their pathogenic effects via various mechanisms, including the induction of a hypercoagulable state. Indeed, therapy of APS is usually directed towards eliminating the enhanced thrombosis. In RPL, the combination of low-molecular-weight heparin and low-dose aspirin is considered to be the treatment of choice. However, when therapy fails, other interventions aimed at controlling the levels of autoantibodies rather than their effects should be considered, as other mechanisms involving autoantibodies are also important in APS. Hence, immunomodulation might theoretically block some of the more detrimental effects of aPL rather than anticoagulant therapy alone.

ACKNOWLEDGMENTS

This study was supported in part by the Federico Foundation Ernesto Hecht Research Grant (to Y Sherer).

111

RECURRENT PREGNANCY LOSS: CAUSES, CONTROVERSIES AND TREATMENT

REFERENCES

1.Hughes GR. Thrombosis, abortion, cerebral disease, and the lupus anticoagulant. BMJ 1983; 287:1088–9.

2.Blank M, Cohen J, Toder V, et al, Induction of anti-phospholipid syndrome in naive mice with mouse lupus monoclonal and human polyclonal anti-cardiolipin antibodies. Proc Natl Acad Sci USA. 1991; 88:3069–73.

3.Bakimer R, Fishman P, Blank M, et al. Induction of primary antiphospholipid syndrome in mice by immunization with a human monoclonal anticardiolipin antibody (H-3). J Clin Invest 1992; 89:1558–63.

4.Ornoy A, Yacobi S, Avraham S, et al. The effect of sera from women with SLE and/or antiphospholipid syndrome on rat embryos in culture. Reprod Toxicol 1998; 12:185–91.

5.Ornoy A, Yacobi S, Tartakover Matalon S, et al. The effects of antiphospholipid antibodies obtained from women with SLE APS and associated pregnancy loss on rat embryos and placental explants in culture. Lupus 2003; 12:573–8.

6.Rand JH. The antiphospholipid syndrome. Annu Rev Med 2003; 54:409–24.

7.Sheng Y, Reddel SW, Herzog H, et al. Impaired thrombin generation in β2-glycoprotein I null mice. J Biol Chem 2001; 276:13817–21.

8.Cervera R, Asherson RA, Acevedo ML, et al. Antiphospholipid syndrome associated with infections: clinical and microbiological characteristics of 100 patients. Ann Rheum Dis 2004: 63:1312–17.

9.Eslick GD, Yan P, Xia HH, et al. Foetal intrauterine growth restrictions with Helicobacter pylori infection. Aliment Pharmacol Ther 2002: 16:1677–82.

10.Figura N, Piomboni P, Ponzetto A, et al. Helicobacter pylori infection and infertility. Eur J Gastroenterol Hepatol 2002: 14:663–9.

11.Sorice M, Pittoni V, Griggi T, et al. Specificity of anti-phospholipid antibodies in infectious mononucleosis: a role for anti-cofactor protein antibodies. Clin Exp Immunol 2000; 120:301–6.

12.Blank M, Shoenfeld Y. β2-Glycoprotein-I, infections, antiphospholipid syndrome and therapeutic considerations. Clin Immunol 2004; 112:190–9.

13.Blank M, Faden D, Tincani A, et al. Immunization with anticardiolipin cofactor (β2-glycoprotein I) induces experimental antiphospholipid syndrome in naive mice. J Autoimmun 1994; 7:441–55.

14.Blank M, Krause I, Fridkin M, et al. Bacterial induction of autoantibodies to β2-glycoprotein-I accounts for the infectious etiology of antiphospholipid syndrome. J Clin Invest 2002; 109:797–804.

15.Blank M, Shoenfeld Y, Cabilly S, et al. Prevention of experimental antiphospholipid syndrome and endothelial cell activation by synthetic peptides. Proc Natl Acad Sci USA 1999; 96:5164–8.

16.Manco Johnson MJ, Nuss R, Key N, et al. Lupus anticoagulant and protein S deficiency in children with postvaricella purpura fulminans or thrombosis. J Pediatr 1996; 128:319–23.

17.Guglielmone H, Vitozzi S, Elbarcha O, et al. Cofactor dependence and isotype distribution of anticardiolipin antibodies in viral infections. Ann Rheum Dis 2001; 60:500–4.

18.de Groot PG, Horbach DA, Derksen RH. Protein C and other cofactors involved in the binding of antiphospholipid antibodies: relation to the pathogenesis of thrombosis. Lupus 1996; 5:488–93.

19.Pengo V, Biasiolo A, Brocco T, et al. Autoantibodies to phospholipidbinding plasma proteins in patients with thrombosis and phospho- lipid-reactive antibodies. Thromb Haemost 1996; 75:721–4.

20.Cuadrado MJ, Lopez-Pedrera C, Khamashta MA, et al. Thrombosis in primary antiphospholipid syndrome: a pivotal role for monocyte tissue factor expression. Arthritis Rheum 1997; 40:834–41.

21.Reverter JC, Tassies D, Font J, et al. Effects of human monoclonal anticardiolipin antibodies on platelet function and on tissue factor expression on monocytes. Arthritis Rheum 1998; 41:1420–7.

22.Forastiero RR, Martinuzzo ME, Broze GJ. High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome. J Thromb Haemost 2003: 1:718–24.

23.Oosting JD, Derksen RH, Blokzijl L, et al. Antiphospholipid antibody positive sera enhance endothelial cell procoagulant activity – studies in a thrombosis model. Thromb Haemost 1992; 68:278–84.

24.Kornberg A, Renaudineau Y, Blank M, et al. Anti-β2-glycoprotein I antibodies and anti-endothelial cell antibodies induce tissue factor in endothelial cells. Isr Med Assoc J 2000; 2(Suppl):27–31.

25.Meroni PL, Raschi E, Camera M, et al. Endothelial activation by aPL: a potential pathogenetic mechanism for the clinical manifestations of the syndrome. J Autoimmun 2000: 15:237–40.

26.Lellouche F, Martinuzzo M, Said P, et al. Imbalance of thromboxane/ prostacyclin biosynthesis in patients with lupus anticoagulant. Blood 1991; 78:2894–9.

27.Jurado M, Paramo JA, Gutierrez-Pimentel M, et al. Fibrinolytic potential and antiphospholipid antibodies in systemic lupus erythematosus and other connective tissue disorders. Thromb Haemost 1992; 68:516–20.

28.Yang CD, Hwang KK, Yan W, et al. Identification of anti-plasmin antibodies in the antiphospholipid syndrome that inhibit degradation of fibrin. J Immunol 2004; 172:5765–73.

29.Cugno M, Cabibbe M, Galli M, et al. Antibodies to tissue-type plasminogen activator (tPA) in patients with antiphospholipid syndrome: evidence of interaction between the antibodies and the catalytic domain of tPA in 2 patients. Blood 2004; 103:2121–6.

30.Font J, Espinosa G, Tassies D, et al. Effects of β2-glycoprotein I and monoclonal anticardiolipin antibodies in platelet interaction with subendothelium under flow conditions. Arthritis Rheum 2002; 46:3283–9.

31.Shibata S, Sasaki T, Hirabayashi Y, et al. Risk factors in the pregnancy of patients with systemic lupus erythematosus: association of hypocomplementaemia with poor prognosis. Ann Rheum Dis 1992; 51:619–23.

32.Wang X, Campos B, Kaetzel MA, et al. Annexin V is critical in the maintenance of murine placental integrity. Am J Obstet Gynecol 1999: 180:1008–16.

33.Rand JH, Wu XX, Andree HA, et al. Pregnancy loss in the antiphos- pholipid-antibody syndrome – a possible thrombogenic mechanism. N Engl J Med 1997; 337:154–60.

34.Matsubayashi H, Arai T, Izumi S, et al. Anti-annexin V antibodies in patients with early pregnancy loss or implantation failures. Fertil Steril 2001; 76:694–9.

35.Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid antibody-induced fetal loss by inhibiting complement activation. Nat Med 2004; 10:1222–6.

36.Carreras LO, Vermylen JG. ‘Lupus’ anticoagulant and thrombosis – possible role of inhibition of prostacyclin formation. Thromb Haemost 1982; 48:38–40.

37.Robbins DL, Leung S, Miller-Blair DJ, et al. Effect of anticardiolipin/β2-glycoprotein I complexes on production of thromboxane A2 by platelets from patients with the antiphospholipid syn-

drome. J Rheumatol 1998; 25:51–6.

38Shoenfeld Y, Blank M. Effect of long-acting thromboxane receptor antagonist (BMS 180,291) on experimental antiphospholipid syn-

drome. Lupus 1994; 3:397–400.

39.Fishman P, Falach-Vaknin E, Sredni B, et al. Aspirin–interleukin-3 interrelationships in patients with anti-phospholipid syndrome. Am J Reprod Immunol 1996; 35:80–4.

112

40.Fishman P, Falach-Vaknin E, Sredni B, et al. Aspirin modulates inter- leukin-3 production: additional explanation for the preventive effects of aspirin in antiphospholipid antibody syndrome. J Rheumatol 1995; 22:1086–90.

41.Blank M, George J, Fishman P, et al. Ciprofloxacin immunomodulation of experimental antiphospholipid syndrome associated with elevation of interleukin-3 and granulocyte–macrophage colonystimulating factor expression. Arthritis Rheum 1998; 41:224–32.

42.Bertolaccini ML, Atsumi T, Lanchbury JS, et al. Plasma tumor necrosis factor α levels and the 238A promoter polymorphism in

patients with antiphospholipid syndrome. Thromb Haemost 2001; 85:198–203.

43.Simantov R, LaSala JM, Lo SK, et al. Activation of cultured vascular endothelial cells by antiphospholipid antibodies. J Clin Invest 1995; 96:2211–19.

44.Meroni PL, Raschi E, Camera M, et al. Endothelial activation by aPL: a potential pathogenetic mechanism for the clinical manifestations of the syndrome. J Autoimmun 2000; 15:237–40.

45.Pierangeli SS, Espinola RG, Liu X, et al. Thrombogenic effects of antiphospholipid antibodies are mediated by intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and P-selectin. Circ Res 2001; 88:245–50.

ANTIPHOSPHOLIPID SYNDROME – PATHOPHYSIOLOGY

46.Out HJ, Kooijman CD, Bruinse HW, et al. Histo-pathological findings from patients with intrauterine fetal death and antiphospholipid antibodies. Eur J Obstet Gynecol 1991; 41:179–86.

47.Ornoy A, Yacobi S, Matalon ST, et al. The effects of antiphospholipid antibodies obtained from women with SLE/APS and associated pregnancy loss on rat embryos and placental explants in culture. Lupus 2003; 12:573–8.

48.Matalon ST, Shoenfeld Y, Blank M, et al. Antiphosphatidylserine antibodies affect rat yolk sacs in culture: a mechanism for fetal loss in antiphospholipid syndrome. Am J Reprod Immunol 2004; 51:144–51.

49.Shurtz-Swirski R, Inbar O, Blank M, et al. In vitro effect of anticardiolipin autoantibodies upon total and pulsatile placental hCG secretion during early pregnancy. Am J Reprod Immunol 1993; 29:206–10.

50.Katsuragawa H, Kanzaki H, Inoue T, et al. Monoclonal antibody against phosphatidylserine inhibits in vitro human trophoblastic hormone production and invasion. Biol Reprod 1997; 56:50–8.

51.Bernstein PS, Divon MY. Etiologies of fetal growth restriction. Clin Obstet Gynecol 1997; 40:723–9.

113