1 Department of Obstetrics and Gynaecology and the Research Unit of Autoimmune Diseases and 2 Department of Medicine B Sheba Medical Centre, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Hashomer, Israel 52621
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Abstract |
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Key words: amniotic fluid/anticardiolipin antibodies/antiphospholipid syndrome/fetal cord blood
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Introduction |
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Materials and methods |
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Samples of maternal blood were collected from all patients in the study and control groups during labour. Fetal cord blood was collected immediately after the cord was clamped during labour in both groups. Among five patients in each group, amniotic fluid was collected during labour, by performing amniotomy through an amnioscope and after disposal of the first 1020 ml of forewater. The other six amniotic fluid samples in each group were collected during Caesarean section, by aspiration from the amniotic sac before it had been ruptured. All patients signed an informed consent form before joining the study.
Maternal and fetal sera, and amniotic fluid, were treated and measured for the level of aCL IgG using enzyme-linked immunoabsorbent assay (ELISA) in the presence of ß2GPI (ß2-glycoprotein I/apolipoprotein H) as follows: 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with cardiolipin (50 µg/ml in ethanol). The plates were dried overnight at 40°C and then blocked for 2 h with 5% (vol/vol) bovine serum. The sera and amniotic fluid were centrifuged at 60 g for 10 min and diluted 1:2001:3600 in 2% bovine serum/Tris-buffered saline (TBS). After overnight incubation at 40°C, a 1:1000 dilution of alkaline phosphatase-conjugated anti-human IgG in 2% bovine serum was added for 4 h at room temperature with shaking. The reaction was developed with p-nitrophenyl phosphate as substrate. Colour was read in a Titertek (S.L.T. Laboratory Instruments, Vienna, Austria) ELISA reader at 405 nm. Inhibition of anticardiolipin binding in amniotic fluid was carried out as follows: amniotic fluid at dilution giving 50% of maximal binding to the cardiolipin antigen was preincubated with the antigen, in different concentrations (100 µg/ml). The preincubation was carried out with cardiolipin and an irrelevant protein (BSA), in order to confirm the specific binding, or to show cross-reactivity of antibodies. After incubation of the amniotic fluid with the different inhibitors (cardiolipin, BSA) overnight at 4°C, the remaining activity was tested by ELISA as detailed above. As control, we used the amniotic fluid incubated without the antigen, but with appropriate volume of PBS instead.
The percentage of inhibition was calculated as follows:
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where OD = optical density. Samples demonstrating aCL presence in amniotic fluid were tested for percent inhibition (Blank et al., 1991).
Statistical analysis was done using Student's t-test, and Fisher's exact test for comparison between groups.
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Results |
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Discussion |
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Several other authors (Briley et al., 1989; Brey et al., 1990
; Levine et al., 1990
), described that aCL had emerged as a result of unexplained stroke in young people.
In the present study, we firstly describe a group of 11 APS pregnant patients demonstrating ~27.3% aCL presence in the cord blood of their offspring, and 54.5% aCL presence in their amniotic fluid. No evidence of such transference was encountered in the control group.
A possible mechanism for the transference of aCL could be speculated in the following manner: aCL IgG could cross the placenta, enter the fetal circulation, and produce its immediate harbinger effect at some stage of embryonal, fetal, or neonatal life. The presence of aCL in the infant might be due to silent presence in the reticuloendothelial system, and possibly could merge into the circulation at later stages of life, but this theory is not yet proven in the literature. These autoantibodies might migrate to the amniotic fluid either through the fetal circulation via the kidney and to the fetal urine and amniotic fluid. This silent stay within the reticuloendothelial system might explain why only half of the neonates demonstrating aCL in amniotic fluid had aCL in their cord blood. Another possibility is that the fetus is capable of self-production of these autoantibodies (El-Roeiy et al., 1987). A possible explanation for the aggravation of this autoimmune phenomenon might be due to the stimulating effect of oestrogens (Piura and Shoenfeld, 1986
). In the control group in our study, no increment in the levels of aCL was encountered as expected (Hess and Baum, 1971
; Hinkle et al., 1979
; Reyes-Lopez et al., 1980
; Patton et al., 1987
). The exact mechanism by which aCL causes thrombotic lesions is not clear. The epitope for aCL anticoagulant activity is probably the ß2-glycoprotein-1 complex with anionic phospholipid in the membranes, inhibiting the intrinsic clotting cascade (Henry et al., 1988
), scavenging procoagulant anionic macromolecules released into the circulation by tissue damage (Schousboe, 1985
); inhibiting prothrombinase activity (Nimpf et al., 1986
), and/or inhibiting platelet aggregation (Nimpf et al., 1987
). Recently, immunostaining for annexin V (human annexin V, also termed placental anticoagulant protein I and endinexin II) which like ß2GPI binds to exteriorized anionic phospholipids, was found to be diminished on the placental villous syncytiotrophoblast of patients with high concentration of aCL and a history of recurrent stillbirths or late pregnancy losses (Rand et al., 1993
). In a recently published review (Caruso et al., 1999
), several other mechanisms for the actions of aCL have been put forward. Among these were aberrations in the generation of eicosanoids such as thromboxane and prostacycylin, decreased production of IL-3 in maternal plasma, and finally, possible interference of aPL with signal transduction in trophoblast cells and prevention of hormone production by the placenta.
We have shown in this study that aCL found in amniotic fluid of patients with APS are immunologically highly active, since relatively high percentages of the cardiolipin activity were inhibited by the amniotic fluid containing aCL (Figure 1). Interestingly, none of the offspring of the APS patients in our study demonstrated adverse outcome, in spite of the clear transference of aCL to the cord blood and amniotic fluid. This finding could be explained either by the small number of patients in the study, or the treatment the study group had received. In two other series of offspring to mothers with APS, no adverse neonatal outcomes that could be attributed to the presence of aCL were encountered (Blank et al., 1991
; Botet et al., 1997
).
Further studies are required for the long-term follow-up of these offspring, especially regarding neurological and haemostatic sequelae. Larger series of patients with APS are required with regard to the possible role of detection of aCL in amniotic fluid during amniocentesis as a predictor of fetal outcome combined with Doppler surveillance (Caruso et al., 1999), which might change the pregnancy management.
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Notes |
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References |
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Submitted on October 6, 1999; accepted on January 24, 2000.