| Abstract|| |
Objectives: The spectrum of thrombophilia in women with recurrent pregnancy loss (RPL) is different in Indian ethnicity as reported by few studies. We aimed to study the prevalence of thrombophilia in RPL patients referred to hematology department of a tertiary centre. Material and Methods: This is an observational study of 112 RPL patients with no apparent cause after extensive workup for non-hematological causes. The investigations performed were routine coagulogram, APLA workup, plasma homocysteine, MTHFRC677T polymorphisms, Protein C, free Protein S, Anti-thrombin III levels, test for Activated Protein C resistance (APC-R) ,Factor V Leiden and Prothrombin gene G20210A mutation. Results: Of 112 patients, at least one thrombophilia was identified in 70.5% and combined thrombophilia in 12.5% patients. Hyperhomocysteinemia (30.4%) and APLA (25.9%) were the commonest thrombophilia whereas anticoagulant defects were seen in 12.5% of the population. Protein C deficiency (5.35%) was the commonest anticoagulant defect followed by APCR (3.6%). Mutational analysis revealed MTHFRC677T polymorphism in 20.5% whereas Factor V Leiden heterozygous in 1.8% patients. None of the patients had homozygous Factor V Leiden or Prothrombin gene G20210A mutation. Hyperhomocysteinemia, MTHFRC677T and Protein C deficiency were more associated with early pregnancy losses whereas Protein S deficiency, Factor V Leiden and APLA caused both early and late losses. Patients with greater number of losses were positive for homozygous MTHFRC677T, factor V Leiden and APLA. Conclusion: The approach to investigating Indian women with RPL should be based on the prevalence of thrombophilia which is unique to Indian ethnicity.
Keywords: Acquired, inherited, recurrent pregnancy loss, thrombophilia
|How to cite this article:|
Mishra P, Singh K, Tyagi S, Juneja R, Mahapatra M. Inherited and acquired thrombophilia in women of Indian ethnicity with recurrent pregnancy loss: An observational study from North India. Indian J Pathol Microbiol 2021;64:741-5
|How to cite this URL:|
Mishra P, Singh K, Tyagi S, Juneja R, Mahapatra M. Inherited and acquired thrombophilia in women of Indian ethnicity with recurrent pregnancy loss: An observational study from North India. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Nov 27];64:741-5. Available from: https://www.ijpmonline.org/text.asp?2021/64/4/741/328520
| Introduction|| |
Recurrent pregnancy loss is defined as two or more losses during pregnancy and has an incidence of 2–3% among women in the reproductive age group. In as much as 50–70% of the cases, the cause remains idiopathic despite an extensive workup to exclude chromosomal abnormalities, endocrine and surgical abnormalities, and preexisting underlying medical comorbidities. Thrombophilia as a cause of recurrent pregnancy loss is controversial. Though it has been reported by a few Indian studies, larger studies are required to investigate the most prevalent hematological abnormalities associated with RPL.,, Our study aims at investigating the prevalence of hereditary and acquired thrombophilias associated with RPL in the Indian ethnic group.
| Material and Methods|| |
This study is an observational study carried out in the Hematology department of a tertiary care center on 112 pregnant females for 22 months from January 2016 to October 2017. These patients referred to our department were negative after an exhaustive workup to exclude congenital, chromosomal, uterine structural, and endocrine abnormalities, infections, and underlying preexisting comorbidities. The investigations included karyotyping for chromosomal abnormalities such as aneuploidies, 2-D ultrasonography with or without sonohysterography, hysteroscopy, and magnetic resonance imaging (MRI) for congenital structural abnormalities such as septate, unicornuate, bicornuate uterus, or acquired structural abnormalities in the form of fibroid uterus, adhesions, etc., Further investigations included thyroid profile, plasma glucose profile, workup for polycystic ovarian syndrome, endometrial biopsy for chronic endometritis, luteal phase defects, and workup for pelvic infections. Approval from ethical committee was obtained on 20 Oct2020
Routine complete blood counts, coagulation profile in the form of prothrombin time, activated partial thromboplastin time, thrombin time and fibrinogen levels, enzyme-linked immunosorbent assay for protein C, free protein S, anti-thrombin III, plasma homocysteine, workup for lupus anticoagulant which included Activated partial thromboplastin time (APTT)-based test for lupus anticoagulants (PTT-LA), dilute Russell viper venom test—screen and confirm, Enzyme linked immunosorbent assay (ELISA) for IgG anti-β2 glycoprotein I antibodies and automated clot-based assay for activated protein C resistance (APC-R) were carried out as a part of thrombophilia workup 4 months after the last pregnancy loss. Patients with elevated homocysteine levels had previously received vitamin B12, folate, and pyridoxine supplementation before investigating them for thrombophilias. These patients were included in the study after correction of vitamin B12, folate, and pyridoxine levels. The patients were diagnosed positive for LA based on prolonged PTT-LA ≥45 s and normalized Dilute Russell's viper venom time (DRVVT) screen. DRVVT confirms ≥1.2 on two occasions 12 weeks apart. The patients with high IgG anti-β2 glycoprotein I antibodies (≥10 U/mL) on two occasions 12 weeks apart were considered positive for anti-β2 glycoprotein I antibodies. The patients were diagnosed Antiphospholipid antibody (APLA) on the basis of Sydney consensus statement 2006 for the adoption of International Society on Thrombosis and Haemostasis (ISTH) criteria for APLA.
The patients found to have protein C, protein S, and anti-thrombin III deficiency were investigated for acquired causes of these defects such as liver diseases, drugs like warfarin, oral contraceptives in the past, antibiotics, l-asparaginase, vitamin K deficiency, bowel resection, nephrotic syndrome, malignancies, infections, autoimmune diseases, myeloproliferative disorders, microangiopathic hemolytic anemias, and acute thrombotic events in the past 4 months.
DNA extraction of the patients was carried out from anticoagulated whole-blood sample by using a commercially available DNA extraction kit and a quality check of DNA was carried out by Nanodrop 1000 spectrophotometer. Molecular investigations were performed in the form of restriction fragment length polymorphism (RFLP) for Factor V Leiden mutation in patients detected to have APC-R and MTHFRC677T mutations in hyperhomocysteinemia subgroup. Amplification refractory mutation system–polymerase chain reaction (ARMS-PCR) was done to look for prothrombin gene G20210A mutation.
| Results|| |
Of the 112 patients included in our study, the median age was 28 years and ranged from 18 to 40 years. The patient characteristics are shown in [Table 1]. There was at least one underlying hematological abnormality in 70.5% (79/112) of the patients and 12.5% (14/112) had combined thrombophilias. The commonest disorder was hyperhomocysteinemia in 30.4% (34/112) of the patients followed by antiphospholipid antibody syndrome (APLA) in 25.8% (29/112) of the study population. Of the 29 patients with APLA, 13 were positive for lupus anticoagulant, 9 for anti-β2 IgG antibodies by ELISA, and 7 were positive for both. The clinical and laboratory profiles of patients with APLA are shown in [Table 2]. Of the 14 patients with combined thrombophilia, the commonest was combined hyperhomocysteinemia and APLA in 12 patients.
|Table 1: Characteristics of 112 patients with recurrent pregnancy losses|
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|Table 2: Clinical and laboratory characteristics of 29 patients with APLA|
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The patients with hyperhomocysteinemia were subjected to RFLP–PCR for MTHFRC677T mutation analysis. Of the 34 such patients, 6 were found to be homozygous for MTHFRC677T and 2 patients were heterozygous for MTHFRC677T mutation.
Anticoagulant defects were present in 14.3% (16/112) of the study population and of the six patients with APC-R, 3.6% (4/112) patients had acquired APC-R and 1.8% (2/112) were heterozygous for Factor V Leiden mutation. None of the patients were found to be homozygous for Factor V Leiden mutation. [Table 3] and [Table 4] summarize the thrombophilias detected in our study population.
|Table 3: Prevalence of thrombophilia in 112 patients with recurrent pregnancy losses|
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The prevalence of inherited thrombophilias in our study was 17.9% (20/112) and that of acquired thrombophilias was 52.7% (59/112) approximately. The inherited thrombophilias included 8 cases of MTHFRC677T mutation, 2 cases of heterozygous Factor V Leiden mutation, and 10 cases of inherited anticoagulant defect as shown in [Table 3]. These cases of anticoagulant deficiency were found to be negative for all acquired cases associated with these defects and hence classified as 'inherited' by exclusion. The acquired thrombophilias included 26 cases of hyperhomocysteinemia negative for MTHFRC677T mutation and 29 cases of APLA and 4 cases of acquired protein C deficiency.
| Discussion|| |
Thrombophilias cause thrombosis of decidual and placental microvasculature and defective trophoblastic proliferation and invasiveness causing increased apoptosis of trophoblasts. There are various European studies and limited Indian studies on the prevalence of inherited and acquired thrombophilias in patients with recurrent pregnancy losses.,,,, Our study was designed to study the prevalence of various thrombophilias in Indian women experiencing recurrent pregnancy losses.
The prevalence of thrombophilias in various studies is different depending on the ethnicity and inclusion criteria for RPL., The prevalence of thrombophilia in our study population was 70.5% which is slightly higher than 64.15% reported by an Indian study on 53 patients. Another study on 587 Indian women with RPL reported a 65% prevalence of thrombophilia, however, they did not investigate their patients for hyperhomocysteinemia.
Hyperhomocysteinemia in 30.4% of our study population was the commonest hematological abnormality followed by antiphospholipid antibody syndrome. A similar prevalence of 32% was reported by an Indian study on 100 patients experiencing frequent pregnancy losses. Another Indian study on 100 women experiencing RPL showed a statistically significant correlation between homocysteine levels and early pregnancy losses. Of the 112 patients, 7.1% (8/112) had MTHFR mutation of which 6 were homozygous. This prevalence is much lower than 21.4% reported by Sah et al. and 12% by van der Molen et al.
All the six patients with homozygous MTHFRC677T polymorphism had early pregnancy losses and belonged to the group with a greater number of pregnancy losses (≥3). An Indian study by Nair et al. in 106 pregnant women demonstrated a 6.3009-fold higher risk of RPL in homozygosity for MTHFR C677T polymorphism and 1.9591-fold increased risk of recurrent miscarriages in women heterozygous for MTHFR C677T polymorphism. However, a large number of previous studies do not report any statistically significant difference in the prevalence of MTHFRC677T polymorphisms in patients with RPL and control group., Data from meta-analysis from various Asian studies showed a strong correlation between MTHFRC677T polymorphism and RPL indicating ethnic differences in effect of MTHFRC677T polymorphism.
The second most common thrombophilia in our study group was antiphospholipid antibody syndrome seen in 25.9% (29/112) of the population. The prevalence of anti-β2 glycoprotein I antibody (17.6%) and LA (14.2%) in our study is more than that reported in an Indian study on 587 women with recurrent pregnancy losses (11 and 8%, respectively). Patients with dual positivity for lupus anticoagulant and anti-β2 glycoprotein I antibodies constituted 6.3% of the study group and had a higher number of RPLs pregnancy losses compared to those positive for anti-β2 glycoprotein I antibodies and lupus anticoagulant alone. This is in concordance with the previous studies., The highest association of RPL has been found with anticardiolipin antibodies according to various studies, however, the data regarding anticardiolipin antibodies was not available to us which is a major limitation of our study.,
Factor V Leiden mutation and prothrombin G20210A mutation have been implicated as an important cause of recurrent pregnancy loss in various western studies., However, the frequency of heterozygous Factor V Leiden mutation was only 1.8% in our study and none of our cases were homozygous for Factor V Leiden mutation. Another Indian study on 78 pregnant women demonstrated a 2% prevalence of heterozygous Factor V Leiden mutation and none of their patients with RPL was homozygous for Factor V Leiden mutation. An Indian study by Patil et al. reported a 3.5% prevalence of Factor V Leiden mutation in 587 patients with RPL and none of the patients were homozygous for Factor V Leiden mutation.
In our study, among the anticoagulant defects, protein C deficiency was the commonest followed by activated protein C resistance and anti-thrombin-III deficiency. A large Indian study on 578 women with RPL reported a higher prevalence of protein S deficiency (16%) than protein C deficiency (6%). The prevalence of protein C deficiency in our patients was similar to their study, however, only 1.7% of our patients had protein S deficiency. The prevalence of Antithrombin III (AT-III) deficiency in our study is 3.5% which is similar to a prevalence of 2.6% in their study. Another Indian study reported a statistically significant correlation between RPL, protein C, and protein S deficiency, however, there was no significant correlation between AT-III deficiency and APC-R. A study by Rai et al. found that acquired APC-R was more common in RPL than Factor V Leiden mutation in Caucasian females. In our study, 3.5% of the patients had APC-R of which only 1.8% had Factor V Leiden mutation. This is much lower than a prevalence of 16.6% of APC-R in a study by Jyotsna et al. The availability of data with regard to the prevalence of acquired APC-R in India is limited.
The prevalence of combined thrombophilias in our study is 12.5% (14/112) which is lower than 28.3% by another Indian study. The commonest combined thrombophilia was hyperhomocysteinemia with APLA. Previous studies have reported hyperhomocysteinemia in around a quarter of patients with primary APLA syndrome. Previous studies have reported an association of elevated homocysteine levels and detection of LA., Homocysteine interacts with LA to promote thrombotic effects. Of the 14 patients with coexisting hyperhomocysteinemia and APLA, 12 were positive for LA and 2 dual positive for LA and IgG anti-β2 glycoprotein I antibodies. All 14 patients with combined thrombophilias had a greater number of pregnancy losses and all were first trimester losses.
In our study, hyperhomocysteinemia, MTHFRC677T polymorphisms, and protein C deficiency were more associated with early pregnancy losses whereas protein S deficiency, Factor V Leiden mutation, and APLA caused both early and late pregnancy losses. Patients with a greater number of pregnancy losses were positive for MTHFRC677T mutation, Factor V Leiden mutation, and antiphospholipid antibodies. Our study is concordant with a previous study by Patil et al. except for the lack of statistically significant correlation between MTHFRC677T polymorphism, heterozygous Factor V Leiden mutation, and RPL.
We realize that our study has many limitations. First, it is an observational study of patients referred to the Hematology department. Hence, the control population was not a part of our study, so the odds ratio and the relative risk of these thrombophilias for RPL could not be studied. Second, we do not have the data on anticardiolipin antibodies for our patients carried out in a separate department.
| Conclusion|| |
Thrombophilias are common in women with RPL, however, whether a workup for thrombophilias in such patients is required is controversial. Acquired causes of thrombophilia are more common in women with recurrent miscarriages and inherited thrombophilias like Factor V Leiden and prothrombin G20210A mutation are uncommon in Indian ethnicity. MTHFRC677T polymorphisms are common in patients with hyperhomocysteinemia and recurrent miscarriages. Prospective studies with a large sample size are required in the Indian ethnic group to decide whether thrombophilia workup should be a part of the antenatal workup of patients with recurrent pregnancy losses.
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Conflicts of interest
There are no conflicts of interest.
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Department of Laboratory Sciences and Molecular Medicine, Army Hospital (R and R), Delhi Cantt, New Delhi - 110 010
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4]