Aust NZ J Obstet Gynaecol 1992; 3 2 4: 328
Antiphospholipid Antibodies in Pregnancy Mary Birdsall’, Neil Pattison’ and Lawrence Chamley3 Department of Obstetrics and Gynaecologx National Women’s Hospital, Auckland, New Zealand
Summary: We assessed the relationship between antiphospholipid antibodies and recurrent miscarriage, fetal deaths, and the pregnancy complications - placental abruption, fetal growth retardation and preeclampsia. The subjects were 81 women with a history of 3 or more miscarriages, 62 with a history of fetal death in the index pregnancy, 105 with a poor obstetric history or pregnancy complications and 13 with systemic lupus erythematosus. Antiphospholipid antibodies were found in 41% of women with a history of recurrent miscarriages, 29% with a history of recent intermediate fetal death or stillbirth, 19% with a poor obstetric history and 69% with systemic lupus erythematosus. There is a high incidence of antiphospholipid antibodies in complicated pregnancies. Patients presenting with the above pregnancy disorders should be tested for antiphospholipid antibodies because of the risk conferred on a fetus by their presence and to expand the treatment options. Antiphospholipid antibodies are associated with fetal loss, thrombosis and thrombocytopenia (1) but may also be found in otherwise healthy individuals. Although these antibodies are frequently described in patients with systemic lupus erythematosus (SLE) the association between these antibodies and fetal loss without evidence of autoimmune disease is more common - ‘the antiphospholipid syndrome’ (2). The 2 antiphospholipid antibodies, anticardiolipin and lupus anticoagulant, are linked with miscarriage and stillbirth and also intrauterine growth retardation, antepartum haemorrhage and preeclampsia. Their mechanism of action remains unclear but thrombosis of uteroplacental vessels is a reasonable hypothesis. Some recent workers however have challenged the significance of antiphospholipid antibodies (3). The purpose of this study was to assess the relationship between adverse pregnancy outcome and the presence of antiphospholipid antibodies.
METHOD Ethical approval was obtained. Two hundred and sixty-one women who were tested for lupus anticoagulant and anticardiolipin antibodies during 1988 and 1989 at National Women’s Hospital were included in this study. The women were analyzed in 4 groups on the basis of their clinical presentation: 1. Eighty-one women with a history of 3 or more miscarriages. Fifty women conceived in the study period and the outcome of their pregnancies was analyzed. 1 . Registrar, Obstetrics and Gynaecology. 2. Senior Lecturer, Obstetrics and Gynaecology. 3. Research Officer. Address for correspondence: Dr Neil Pattison, National Women’s Hospital, Claude Road, Auckland 3, New Zealand.
Fifteen of those with antiphospholipid antibodies were treated with low dose aspirin (75 mg per day) 2. Sixty-two women following a fetal death. 3. One hundred and five pregnant women with a poor obstetric history (previous fetal loss or severely growth retarded fetus) or with a significant current pregnancy complication (antepartum haemorrhage, early proteinuric preeclampsia or fetal growth retardation). 4. Thirteen pregnant women with SLE. The notes of the 261 patients were reviewed, and complications and outcome in their current pregnancy were analyzed.
Cardiolipin antibody ELISA This ELISA is a modification of our previously published method (4). Briefly, cardiolipin (2.5 ug/well) coated ELISA plates were blocked for 2 hours with 10% newborn calf serum (NBCS) then washed 3 times with PBS. Patient serum diluted in 10% NBCS was added to duplicate wells and incubated for 60 minutes at room temperature. The plates were again washed with PBS and HRP conjugated antihuman IgG or IgM antibodies were added and incubated for 60 minutes. HRP colourdeveloping substrate (0-phenylene diamine 1 mg/ml) was added after further washing with PBS and incubated for 30 minutes in the dark. The reaction was stopped with 3.6% HCI. The optical density at 490 nm was determined. The assay was calibrated against KAPS (Kingston Antiphospholipid Antibody Workshop) standards (the gift of Dr EN Harris, Louisville, Kentucky) which use the units GPL and MPL for IgG and IgM anticardiolipin antibodies respectively. All values of > 5 G or MPL units were considered positive. Lupus anticoagulant Lupus anticoagulant was identified in platelet poor plasma by the KCT% test (6). Briefly, 40 ul of test
329
MARYBIRDSALL ET AL
sample was mixed with 160 ul of pooled normal plateletpoor plasma and incubated at 37 "C for 20s; 100 ul of 2% kaolin, buffered in 0.1M Tris-HCI (pH 7.2) was added and incubated for a further 3 min; 200 ul of prewarmed 0.025/M calcium chloride was added and incubated while shaking at 37 "C until a fibrous 'clot' formed. The KCT% value was calculated by dividing the time taken for a clot to form in the test mixture by the time taken for a clot to form in the control (200 ul of pooled normal platelet-poor plasma): sample clot time x 100 KCT% = control clot time In our population results from screening 1,000 consecutively booking antenatal patients determined that 1.2% of the population had cardiolipin antibodies and 1.0% had lupus anticoagulant. For cardiolipin antibodies, therefore, samples of more than 5 GPL or 5 MPL were considered positive and a KCT% of greater than 118 was considered to be abnormal (6).
RESULTS Antiphospholipid antibodies were found in 31% (80 of 261) of women tested (table 1). Table 1. Incidence of Antiphospholipid Antibodies (Anticardiolipin and Lupus Anticoagulant) in 261 Women with a History of Abnormal Pregnancy Group Indication for testing 1
2 3 4
Recurrent miscarriage Following a fetal death Poor obstetric history or current pregnancy complication Systemic lupus ervthematosus Total
Number Antiphospholipid Tested antibody present Number
Yo
81 62
33 18
41% 29%
105
20
19%
13
9
69%
26 1
80
31%
Forty-one percent (33 of 81) of women with a history of 3 or more recurrent miscarriages were found to have antiphospholipid antibodies (IgG mean 14.9, SD 7.6 GPL units; IgM mean 16.9, SD 12.7 MPL units). All had anticardiolipin antibodies and 2 also had lupus anticoagulant. In the 2-year study period 50 of these 81 women conceived. Forty-two per cent (21 of 50) had antiphospholipid antibodies. Fifteen of these 21 women were treated with low dose aspirin, with 13 successful pregnancies. One patient had a neonatal death after delivery of a normally grown fetus at 27 weeks' gestation and one had a miscarriage at 16 weeks. All 6 women with antiphospholipid antibodies who were not treated with aspirin had successful pregnancies (table 2). Twenty-nine per cent (18 of 62) of women who delivered an intermediate or a late fetal death were found to have antiphospholipid antibodies. Seventeen had anticardiolipin antibodies (IgG mean 20.0, SD 28.8
Table 2. Preliminary Results of Treatment with Low Dose Aspirin for Women with Antiphospholipid Antibodies _____
~
Treated*
Non-treated
15
6
1
0 6 (100%) 0 1 1 0 9 5 8 11+7
~
Total Miscarriage Livebirth Antepartum haemorrhage Gestational proteinuric hypertension Low birth-weight Neonatal death Anticardiolipin level - IgM - IgG
* Not
13 (87%) 3 1 9 1 17 3 13+7
*
randomized or controlled
GPL units; IgM mean 14.0, SD 1.3 MPL units). Two of these 17 also had lupus anticoagulant, and 1 had lupus anticoagulant alone. The 7 women whose fetuses were found subsequently to have a congenital abnormality did not have antiphospholipid antibodies. Thirty-three per cent (18 of 55) of women who suffered a fetal loss due to hypertension, antepartum haemorrhage, intrapartum asphyxia, growth retardation, preterm delivery or unexplained fetal loss had antiphospholipid antibodies (table 3). Table 3. Pregnancy Complications in 62 Women with a Fetal Death and Who Were Tested for Antiphospholipid Antibodies Number Antiphospholipid Tested antibodies present
Complication Growth retardation Proteinuric hypertension Antepartum haemorrhage Fetal abnormality
43 14 21 7
Number
70
17 2 7 0
40% 14%
33% 0%
Nineteen per cent (20 of 105) of women with a high risk pregnancy were found to have antiphospholipid antibodies. Sixty-nine percent (9 of 13) of pregnant women with SLE had antiphospholipid antibodies. There were 230 women from the above 4 groups whose pregnancy outcome was able to be analyzed (31 women with a history of recurrent miscarriage did not conceive in the study period). The incidence of antiphospholipid antibodies associated with pregnancy complications is listed in table 4. Table 4. Incidence of Antiphospholipid Antibodies in 230 Complicated Preenancies
_____
~~
Complication Stillbirth or intermediate fetal death Antepartum haemorrhage Preterm delivery (before 34 completed weeks) Low birth-weight (< 10th percentile) Gestational proteinuric hypertension One or more previous: - growth retarded infants(s) - perinatal death(s)
Number Antiphospholipid Tested antibodies present Number
%
62 28
18 6
29% 21%
64 62 22
22 13 6
25% 21% 27%
36 25
6 7
16% 28%
330
AUST.AND N.Z. JOURNAL OF OBSTETRICS AND GYNAECOLOGY
DISCUSSION A population study in 1987 of 1,000 consecutively booked antenatal patients at National Women’s Hospital revealed a prevalence of either anticardiolipin antibodies or lupus anticoagulant at 2% (6). Anticardiolipin antibodies were deemed to be positive if levels were greater than 5 G or MPL units. In the selected high risk population reported here the prevalence was 31%, confirming the strong relationship between adverse pregnancy outcome and the presence of antiphospholipid antibodies. Forty-one per cent (33 of 81) of women being investigated for recurrent unexplained first trimester loss had antiphospholipid antibodies which is similar to that reported by other authors (range 14% [7] to 42% [S]). Anticardiolipin antibodies in low titre (5-20 GPL units) was the usual finding. There was a high incidence of antiphospholipid antibodies found in patients having a fetal loss. Twentynine per cent of women with a fetal loss (between 20 weeks and term) had antiphospholipid antibodies. This is important as many of these patients had previously been told their loss was unexplained. The strong association between the presence of antibody and fetal loss, growth retardation, antepartum haemorrhage and preeclampsia adds support to the theory that the mechanism of action of these antibodies is an effect on placental vasculature. The mechanism of action of antiphospholipid antibodies remains unclear although a decidual vasculopathy has been demonstrated with intravascular deposition of IgG and fibrin (9). This may indicate that the antibody binds directly to the vascular endothelium which then undergoes immune mediated damage; however this remains to be proven. It is also unclear whether platelets are directly bound by antibodies or whether they are secondarily involved subsequent to the proposed endothelial damage. The injection of mice with IgG purified from patients with antiphospholipid antibodies led to abortion (9) which adds support to the suggestion that these antibodies are pathogenic and not merely an association. These mechanisms could explain the adverse pregnancy outcomes described in this series - from an increased incidence of preeclampsia to a high likelihood of intrauterine growth retardation subsequent to the decidual vasculopathy. The 25 To incidence of preterm delivery which includes both elective and spontaneous births may be secondary to this decidual pathology. With such a strong association between adverse pregnancy outcome and antiphospholipid antibodies, any woman presenting with recurrent miscarriage, late
pregnancy loss, intrauterine growth retardation, early onset proteinuric hypertension or antepartum haemorrhage should be tested for these antibodies. The presence of these antibodies suggests an aetiology for the problem and leads to consideration of treatment options. The appropriate therapy however remains unclear. There have been successful pregnancy outcomes reported with the use of steroids, heparin, aspirin, plasmaphoresis, high dose immunoglobulin, no treatment or any combination of the above. Our current treatment regimens are low dose aspirin (75 mg/day) for those patients with anticardiolipin antibodies alone or low levels of lupus anticoagulant (KCT 100%
Antiphospholipid Antibodies in Pregnancy Mary Birdsall’, Neil Pattison’ and Lawrence Chamley3 Department of Obstetrics and Gynaecologx National Women’s Hospital, Auckland, New Zealand
Summary: We assessed the relationship between antiphospholipid antibodies and recurrent miscarriage, fetal deaths, and the pregnancy complications - placental abruption, fetal growth retardation and preeclampsia. The subjects were 81 women with a history of 3 or more miscarriages, 62 with a history of fetal death in the index pregnancy, 105 with a poor obstetric history or pregnancy complications and 13 with systemic lupus erythematosus. Antiphospholipid antibodies were found in 41% of women with a history of recurrent miscarriages, 29% with a history of recent intermediate fetal death or stillbirth, 19% with a poor obstetric history and 69% with systemic lupus erythematosus. There is a high incidence of antiphospholipid antibodies in complicated pregnancies. Patients presenting with the above pregnancy disorders should be tested for antiphospholipid antibodies because of the risk conferred on a fetus by their presence and to expand the treatment options. Antiphospholipid antibodies are associated with fetal loss, thrombosis and thrombocytopenia (1) but may also be found in otherwise healthy individuals. Although these antibodies are frequently described in patients with systemic lupus erythematosus (SLE) the association between these antibodies and fetal loss without evidence of autoimmune disease is more common - ‘the antiphospholipid syndrome’ (2). The 2 antiphospholipid antibodies, anticardiolipin and lupus anticoagulant, are linked with miscarriage and stillbirth and also intrauterine growth retardation, antepartum haemorrhage and preeclampsia. Their mechanism of action remains unclear but thrombosis of uteroplacental vessels is a reasonable hypothesis. Some recent workers however have challenged the significance of antiphospholipid antibodies (3). The purpose of this study was to assess the relationship between adverse pregnancy outcome and the presence of antiphospholipid antibodies.
METHOD Ethical approval was obtained. Two hundred and sixty-one women who were tested for lupus anticoagulant and anticardiolipin antibodies during 1988 and 1989 at National Women’s Hospital were included in this study. The women were analyzed in 4 groups on the basis of their clinical presentation: 1. Eighty-one women with a history of 3 or more miscarriages. Fifty women conceived in the study period and the outcome of their pregnancies was analyzed. 1 . Registrar, Obstetrics and Gynaecology. 2. Senior Lecturer, Obstetrics and Gynaecology. 3. Research Officer. Address for correspondence: Dr Neil Pattison, National Women’s Hospital, Claude Road, Auckland 3, New Zealand.
Fifteen of those with antiphospholipid antibodies were treated with low dose aspirin (75 mg per day) 2. Sixty-two women following a fetal death. 3. One hundred and five pregnant women with a poor obstetric history (previous fetal loss or severely growth retarded fetus) or with a significant current pregnancy complication (antepartum haemorrhage, early proteinuric preeclampsia or fetal growth retardation). 4. Thirteen pregnant women with SLE. The notes of the 261 patients were reviewed, and complications and outcome in their current pregnancy were analyzed.
Cardiolipin antibody ELISA This ELISA is a modification of our previously published method (4). Briefly, cardiolipin (2.5 ug/well) coated ELISA plates were blocked for 2 hours with 10% newborn calf serum (NBCS) then washed 3 times with PBS. Patient serum diluted in 10% NBCS was added to duplicate wells and incubated for 60 minutes at room temperature. The plates were again washed with PBS and HRP conjugated antihuman IgG or IgM antibodies were added and incubated for 60 minutes. HRP colourdeveloping substrate (0-phenylene diamine 1 mg/ml) was added after further washing with PBS and incubated for 30 minutes in the dark. The reaction was stopped with 3.6% HCI. The optical density at 490 nm was determined. The assay was calibrated against KAPS (Kingston Antiphospholipid Antibody Workshop) standards (the gift of Dr EN Harris, Louisville, Kentucky) which use the units GPL and MPL for IgG and IgM anticardiolipin antibodies respectively. All values of > 5 G or MPL units were considered positive. Lupus anticoagulant Lupus anticoagulant was identified in platelet poor plasma by the KCT% test (6). Briefly, 40 ul of test
329
MARYBIRDSALL ET AL
sample was mixed with 160 ul of pooled normal plateletpoor plasma and incubated at 37 "C for 20s; 100 ul of 2% kaolin, buffered in 0.1M Tris-HCI (pH 7.2) was added and incubated for a further 3 min; 200 ul of prewarmed 0.025/M calcium chloride was added and incubated while shaking at 37 "C until a fibrous 'clot' formed. The KCT% value was calculated by dividing the time taken for a clot to form in the test mixture by the time taken for a clot to form in the control (200 ul of pooled normal platelet-poor plasma): sample clot time x 100 KCT% = control clot time In our population results from screening 1,000 consecutively booking antenatal patients determined that 1.2% of the population had cardiolipin antibodies and 1.0% had lupus anticoagulant. For cardiolipin antibodies, therefore, samples of more than 5 GPL or 5 MPL were considered positive and a KCT% of greater than 118 was considered to be abnormal (6).
RESULTS Antiphospholipid antibodies were found in 31% (80 of 261) of women tested (table 1). Table 1. Incidence of Antiphospholipid Antibodies (Anticardiolipin and Lupus Anticoagulant) in 261 Women with a History of Abnormal Pregnancy Group Indication for testing 1
2 3 4
Recurrent miscarriage Following a fetal death Poor obstetric history or current pregnancy complication Systemic lupus ervthematosus Total
Number Antiphospholipid Tested antibody present Number
Yo
81 62
33 18
41% 29%
105
20
19%
13
9
69%
26 1
80
31%
Forty-one percent (33 of 81) of women with a history of 3 or more recurrent miscarriages were found to have antiphospholipid antibodies (IgG mean 14.9, SD 7.6 GPL units; IgM mean 16.9, SD 12.7 MPL units). All had anticardiolipin antibodies and 2 also had lupus anticoagulant. In the 2-year study period 50 of these 81 women conceived. Forty-two per cent (21 of 50) had antiphospholipid antibodies. Fifteen of these 21 women were treated with low dose aspirin, with 13 successful pregnancies. One patient had a neonatal death after delivery of a normally grown fetus at 27 weeks' gestation and one had a miscarriage at 16 weeks. All 6 women with antiphospholipid antibodies who were not treated with aspirin had successful pregnancies (table 2). Twenty-nine per cent (18 of 62) of women who delivered an intermediate or a late fetal death were found to have antiphospholipid antibodies. Seventeen had anticardiolipin antibodies (IgG mean 20.0, SD 28.8
Table 2. Preliminary Results of Treatment with Low Dose Aspirin for Women with Antiphospholipid Antibodies _____
~
Treated*
Non-treated
15
6
1
0 6 (100%) 0 1 1 0 9 5 8 11+7
~
Total Miscarriage Livebirth Antepartum haemorrhage Gestational proteinuric hypertension Low birth-weight Neonatal death Anticardiolipin level - IgM - IgG
* Not
13 (87%) 3 1 9 1 17 3 13+7
*
randomized or controlled
GPL units; IgM mean 14.0, SD 1.3 MPL units). Two of these 17 also had lupus anticoagulant, and 1 had lupus anticoagulant alone. The 7 women whose fetuses were found subsequently to have a congenital abnormality did not have antiphospholipid antibodies. Thirty-three per cent (18 of 55) of women who suffered a fetal loss due to hypertension, antepartum haemorrhage, intrapartum asphyxia, growth retardation, preterm delivery or unexplained fetal loss had antiphospholipid antibodies (table 3). Table 3. Pregnancy Complications in 62 Women with a Fetal Death and Who Were Tested for Antiphospholipid Antibodies Number Antiphospholipid Tested antibodies present
Complication Growth retardation Proteinuric hypertension Antepartum haemorrhage Fetal abnormality
43 14 21 7
Number
70
17 2 7 0
40% 14%
33% 0%
Nineteen per cent (20 of 105) of women with a high risk pregnancy were found to have antiphospholipid antibodies. Sixty-nine percent (9 of 13) of pregnant women with SLE had antiphospholipid antibodies. There were 230 women from the above 4 groups whose pregnancy outcome was able to be analyzed (31 women with a history of recurrent miscarriage did not conceive in the study period). The incidence of antiphospholipid antibodies associated with pregnancy complications is listed in table 4. Table 4. Incidence of Antiphospholipid Antibodies in 230 Complicated Preenancies
_____
~~
Complication Stillbirth or intermediate fetal death Antepartum haemorrhage Preterm delivery (before 34 completed weeks) Low birth-weight (< 10th percentile) Gestational proteinuric hypertension One or more previous: - growth retarded infants(s) - perinatal death(s)
Number Antiphospholipid Tested antibodies present Number
%
62 28
18 6
29% 21%
64 62 22
22 13 6
25% 21% 27%
36 25
6 7
16% 28%
330
AUST.AND N.Z. JOURNAL OF OBSTETRICS AND GYNAECOLOGY
DISCUSSION A population study in 1987 of 1,000 consecutively booked antenatal patients at National Women’s Hospital revealed a prevalence of either anticardiolipin antibodies or lupus anticoagulant at 2% (6). Anticardiolipin antibodies were deemed to be positive if levels were greater than 5 G or MPL units. In the selected high risk population reported here the prevalence was 31%, confirming the strong relationship between adverse pregnancy outcome and the presence of antiphospholipid antibodies. Forty-one per cent (33 of 81) of women being investigated for recurrent unexplained first trimester loss had antiphospholipid antibodies which is similar to that reported by other authors (range 14% [7] to 42% [S]). Anticardiolipin antibodies in low titre (5-20 GPL units) was the usual finding. There was a high incidence of antiphospholipid antibodies found in patients having a fetal loss. Twentynine per cent of women with a fetal loss (between 20 weeks and term) had antiphospholipid antibodies. This is important as many of these patients had previously been told their loss was unexplained. The strong association between the presence of antibody and fetal loss, growth retardation, antepartum haemorrhage and preeclampsia adds support to the theory that the mechanism of action of these antibodies is an effect on placental vasculature. The mechanism of action of antiphospholipid antibodies remains unclear although a decidual vasculopathy has been demonstrated with intravascular deposition of IgG and fibrin (9). This may indicate that the antibody binds directly to the vascular endothelium which then undergoes immune mediated damage; however this remains to be proven. It is also unclear whether platelets are directly bound by antibodies or whether they are secondarily involved subsequent to the proposed endothelial damage. The injection of mice with IgG purified from patients with antiphospholipid antibodies led to abortion (9) which adds support to the suggestion that these antibodies are pathogenic and not merely an association. These mechanisms could explain the adverse pregnancy outcomes described in this series - from an increased incidence of preeclampsia to a high likelihood of intrauterine growth retardation subsequent to the decidual vasculopathy. The 25 To incidence of preterm delivery which includes both elective and spontaneous births may be secondary to this decidual pathology. With such a strong association between adverse pregnancy outcome and antiphospholipid antibodies, any woman presenting with recurrent miscarriage, late
pregnancy loss, intrauterine growth retardation, early onset proteinuric hypertension or antepartum haemorrhage should be tested for these antibodies. The presence of these antibodies suggests an aetiology for the problem and leads to consideration of treatment options. The appropriate therapy however remains unclear. There have been successful pregnancy outcomes reported with the use of steroids, heparin, aspirin, plasmaphoresis, high dose immunoglobulin, no treatment or any combination of the above. Our current treatment regimens are low dose aspirin (75 mg/day) for those patients with anticardiolipin antibodies alone or low levels of lupus anticoagulant (KCT 100%
