Abstract
To compare the levels of IgG and IgM anti beta-2 glycoprotein I antibodies and IgG and IgM anti prothrombin antibodies among women with unexplained recurrent pregnancy losses and women with at least 2 live issues. To compare the prevalence of newer anti beta-2 glycoprotein I & anti prothrombin antibodies with conventional Lupus anticoagulant & anticardiolipin antibodies. 50 women with recurrent pregnancy losses & 50 matched controls were evaluated for the presence of: Lupus anticoagulant—screened by LA sensitive aPTT& DRVV and confirmatory Staclot Assay. ELISA kits were used for detecting IgG & IgM anticardiolipin, anti beta-2 glycoprotein I & anti prothrombin antibodies. 11/50 (22 %) women in study group and none in control group had circulating antiphospholipid antibodies. 2 cases (4 %) had lupus anticoagulant. 1 case (2 %) had anticardiolipin antibody & 6 cases (12 %) were positive for anti beta-2 Glycoprotein I antibody (p value = 0.027). 3 cases (6 %) had anti prothrombin antibody. All were mutually exclusive except for one. Women with recurrent pregnancy losses should be tested for anti beta-2 Glycoprotein I antibodies & anti prothrombin antibodies in addition to conventional lupus anticoagulant and anticardiolipin antibodies. This approach can decrease the incidence of SNAP (seronegative antiphospholipid syndrome) cases while establishing the true prevalence of antiphospholipid syndrome.
Keywords: Anticardiolipin, Antiphospholipid syndrome, Lupus coagulation inhibitor, Pregnancy, Prothrombin, Beta 2-glycoprotein I
Introduction
Recurrent pregnancy losses (RPL) are defined as three or more consecutive pregnancy losses with not more than one pregnancy successfully reaching into the third trimester. 1–2 % of women in reproductive age group experience 3 or more pregnancy losses [1, 2]. Pregnancy losses can be attributed to a wide variety of causes but after evaluating patients for the etiology of pregnancy loss and performing all routine investigations, about 50 % of the cases still remain undiagnosed [3]. Immunologic causes are a diagnosis of exclusion. Foremost among them is antiphospholipid syndrome which comprises about 5–20 % cases of recurrent pregnancy loss [4].
Antiphospholipid syndrome is characterised by thromboembolic events, pregnancy losses along with presence of circulating antiphospholipid antibodies. The antiphospholipid antibodies are a heterogeneous group of autoantibodies that bind to different protein epitopes (beta-2 glycoprotein I, prothrombin, annexin V etc.) complexed to phospholipids. The two best known antiphospholipid antibodies are lupus anticoagulant and anticardiolipin antibodies. It is diagnosed according to modified Sapporo criteria [5]. It requires presence of any one of three antibodies viz. lupus anticoagulant (LA), anti cardiolipin (aCL) and anti beta-2 glycoprotein I (anti beta-2 GP I) antibody on 2 occasions 12 weeks apart. At present anti prothrombin antibody is not a part of the modified Sapporo criteria. This study aims to compare the presence of anti beta-2 GP I, anti prothrombin antibodies with classical antiphospholipid antibodies (LA, aCL).
Materials and Methods
Place of Study
The study was conducted in the Department of Pathology and Department of Obstetrics and Gynaecology, Lady Hardinge Medical College and associated Shrimati Sucheta Kriplani Hospital, New Delhi-110001.
Study Subjects
The case group was formed by 50 women with recurrent pregnancy losses after excluding known patients with Rh/ABO incompatibility, blighted ovum, diabetes mellitus, thyroid disease, positive TORCH/Hepatitis/HIV serology, anatomical defects of uterus on USG, chromosomal anomaly in the participant, spouse or previous abortus/fetus. The control group comprised 50 women with 2 live births and no history of pregnancy loss.
Laboratory Tests
All cases were tested for (a) Routine tests: Complete blood count with peripheral blood smear. (b) Screening tests for coagulation: PT (Prothrombin time) & APTT (Activated partial thromboplastin time) (c) Tests for antiphospholipid antibodies: Lupus anticoagulant (PTT-LA- STACLOT assay & Dilute Russell Viper Venom time screen + confirm STACLOT assay), Anticardiolipin antibody IgG/IgM (ELISA, Asserachrom Diagnostics), Anti Beta-2 Glycoprotein I antibody IgG/IgM (ELISA, Asserachrom Diagnostics), Anti-prothrombin antibody IgG/IgM (ELISA, DRG Diagnostics). A positive test was confirmed by repeat testing after a period of 12 weeks and was considered positive only when both samples were positive.
Statistical Analysis
The data was expressed as mean ± 1 standard deviation. Comparisons between two group frequencies were made using Chi Square Test. For the comparison of two group means, Student t test was applied. When comparing means of more than two parameters, ANOVA (A one-way analysis of variance) test was applied. The statistical software used was SPSS & SS.
Observations and Results
All cases and controls were age matched. The age group ranged from 19 to 30 years with a mean of 24.84 ± 3.026 years for cases and 25.28 ± 2.214 years for controls.
A total of 177 pregnancy losses occurred in 50 Cases of the study group. There was no pregnancy loss among the control group. In the study group all 50/50 (100 %) cases experienced first trimester pregnancy losses while 14/50 (28 %) cases also experienced second trimester pregnancy losses. Only 3/50 (6 %) cases experienced third trimester pregnancy losses while 1 patient had pregnancy losses in all trimesters.
There was no statistically significant difference in the CBC parameters, PT & APTT between study and control groups.
In the study group, overall 11/50 cases were positive for circulating antiphospholipid antibodies (Fig. 1). Of these 2 cases (4 %) were positive for LA & 1 case (2 %) was positive for anticardiolipin antibody. There was no statistically significant difference between study and control groups with respect to LA and anticardiolipin antibodies (Table 1).
Fig. 1.
Antiphospholipid antibody positive cases (all figures in percentage)
Table 1.
Cases testing positive for Lupus anticoagulant & anticardiolipin antibody
| Cases (n = 50) (Mean ± SD) | Controls (n = 50) (Mean ± SD) | p value | |
|---|---|---|---|
| LA positive | 2/50 (4 %) | 0/50 | 0.495 |
| aCL positive | 1/50 (2 %) | 0/50 | 1.000 |
In the study group 6 cases (12 %) tested positive for anti beta-2 GP I antibody, all controls were negative and there was a statistically significant difference (p value < 0.05) between study and control groups (Table 2). A total of 3 cases (6 %) were positive for anti prothrombin antibody while all controls were negative, however p value was not significant (p value > 0.05) (Table 2). A single case showed positivity for both anti beta-2 GP I and anti prothrombin antibody. In the control group all the cases tested negative for lupus anticoagulant, anticardiolipin, anti beta-2 GP I & antiprothrombin antibodies (Table 1, 2).
Table 2.
Cases testing positive for anti Beta-2 GP I & antiprothrombin antibodies
| Cases (n = 50) (Mean ± SD) | Controls (n = 50) (Mean ± SD) | p value | |
|---|---|---|---|
| Anti Beta-2 GP I positive | 6/50 (12 %) | 0/50 | 0.027 |
| Anti prothrombin positive | 3/50 (6 %) | 0/50 | 0.242 |
Discussion
Antibodies against beta 2 GP I were identified in 1990 by 3 different groups when they found that among the patients of antiphospholipid syndrome, anticardioipin antibodies are directed against a plasma protein cofactor beta 2 glycoprotein I [6–8]. It has been found that anticardiolipin antibodies which correlate with thrombotic complications and pregnancy losses are actually antibodies which recognise beta 2 GP I and have an affinity for cardiolipin (anticardiolipin dependent beta 2 GP I antibodies) [9]. Antigen/antibody complexes of prothrombin and beta-2 glycoprotein I are largely responsible for the lupus anticoagulant activity detected in in vitro tests for coagulation. These antibodies are responsible for the pathogenic manifestations of antiphospholipid syndrome, namely thrombotic events and pregnancy loss [10–13].
In our study among 11 antiphospholipid antibody positive women, 9 were negative for commonly used screening tests for the presence of antiphospholipid antibodies i.e. LA (using LA sensitive APTT & Dilute Russell viper venom time) and aCL. Among these 9 cases, 6/9 showed the presence of anti beta-2 GP I antibodies & 3/9 had circulating anti prothrombin antibodies with 1 case showing presence of both anti beta-2 GP I and anti prothrombin antibodies (Fig. 1). Hence 9 LA & aCL negative cases showed the presence of newer antiphospholipid antibodies. In contrast to other studies, in our study all control subjects tested negative for antiphospholipid antibodies. A review of literature shows a widely varying prevalence of antiphospholipid antibodies in patients with recurrent pregnancy loss. There is paucity of literature from the Indian subcontinent regarding anti beta-2 GP I & antiprothrombin antibodies. Vora et al. [14] studied 198 women with RPL & found LA positivity in 10.11 % patients, aCL IgM in 19.6 % patients, aCL IgG in 22.2 % cases. Anti Beta-2 GP I IgM was positive in 6.3 % cases, anti beta-2 GP I IgG was positive in 9.7 % cases. Bizzaro et al. [15] studied 77 women with RPL of whom 6 % cases & 4 % controls were positive for anti beta-2 GP I & aCL IgG/IgM antibodies. 16 % cases & 8 % controls were positive for anti prothrombin IgG/IgM antibodies. Zammiti et al. [16] studied 200 women with RPL and found anti beta-2 GP I IgG to be positive in 4.5 % cases, anti beta-2 GP I IgM to be elevated in 1.5 % cases. To the best of our knowledge antiprothrombin antibodies have previously not been studied in Indian females with recurrent pregnancy losses. Sater et al. [17] studied 277 recurrent pregnancy loss cases and compared them with 288 controls. They found 12 % positivity for anti-prothrombin antibodies among cases and also 6.1 % in controls. Marozio et al. [18] studied 56 women with late fetal death (>26 weeks) who were negative for lupus anticoagulants and anticardiolipin. 28.6 % patients tested positive for antiprothrombin antibodies as compared to 3.6 % controls. In patients with seronegative antiphospholipid syndrome with pregnancy loss, Conti et al. [19] found antiprothrombin antibody to be positive in 2 of 8 patients. Recent studies also indicate that Domain I of anti beta-2 glycoprotein I antibodies to be highly associated with antiphospholipid syndrome [20].
Therefore women with recurrent pregnancy losses should be routinely tested for all antiphospholipid antibodies viz. anti beta-2 Glycoprotein I antibodies, anti prothrombin antibodies, lupus anticoagulant and anticardiolipin antibodies. By doing so, we can reduce the diagnosis of seronegative antiphospholipid syndrome. A larger cohort study should be done to find out the true prevalence of anti beta-2 glycoprotein I & antiprothrombin antibodies in patients with recurrent pregnancy losses.
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