| Abstract|| |
Background: Thrombophilias, both acquired and inherited, have been investigated in the etiopathogenesis of unexplained recurrent pregnancy loss. Aim: To study coagulation inhibitors and activated protein C resistance (APCR) in recurrent pregnancy losses (RPL) occurring in second and third trimesters. Materials and Methods: A total of 30 pregnant women (group A) with two or more recurrent unexplained fetal loses were evaluated for APCR, protein C deficiency, protein S deficiency, antithrombin deficiency, and antiphospholipid antibodies (APLA). Thirty age-matched controls were taken (group B) comprising of pregnant women with at least one live issue. Statistical Analysis: Comparisons between two group frequencies and group means were made using Chi square test and Student's t test, respectively. Results: Protein C and protein S levels were reduced in group A compared with group B and the difference was statistically significant (P=0.005 and P=0.032, respectively). The mean value of antithrombin was slightly reduced in group A compared with group B. APCR was observed in 16.6% cases and 3.3% controls. However, the difference was not statistically significant. APLA was observed in 20% cases and none of the controls. Of these, lupus anticoagulant was positive in 16.6% cases and anticardiolipin antibodies in 10% cases. Combined defects were seen in seven patients. Conclusion: There is a significant risk of RPL in pregnant women with thrombophilias. Therefore, screening for thrombophilias may be justified in pregnant women with unexplained recurrent fetal wastage, especially in second and third trimester.
Keywords: Antiphospholipid antibodies, activated protein C resistance, recurrent pregnancy loss, recurrent abortions, thrombophilias
|How to cite this article:|
Jyotsna P L, Sharma S, Trivedi SS. Coagulation inhibitors and activated protein C resistance in recurrent pregnancy losses in Indian women. Indian J Pathol Microbiol 2011;54:752-5
|How to cite this URL:|
Jyotsna P L, Sharma S, Trivedi SS. Coagulation inhibitors and activated protein C resistance in recurrent pregnancy losses in Indian women. Indian J Pathol Microbiol [serial online] 2011 [cited 2020 Jul 5];54:752-5. Available from: http://www.ijpmonline.org/text.asp?2011/54/4/752/91507
| Introduction|| |
Recurrent pregnancy loss is a heterogeneous condition with several etiological factors. Nearly 70% of spontaneous abortions in the first 12 weeks are due to chromosomal anomalies. However, losses due to cervical incompetence and hematologic disorders are common after twelve weeks. 
Pregnancy is a hypercoagulable state and women with thrombophilias are at increased risk for thrombosis during pregnancy and adverse maternal and fetal sequelae. The hemostatic system plays an important role in the success of pregnancy and the process of implantation and placentation. Implantation of the fertilized egg into the uterine decidua establishes a contact between the fetus, the placenta, and the maternal circulation. This contact is crucial for the success of pregnancy. Prothrombotic changes and thrombosis may interfere with these processes leading to miscarriage.
Severe pregnancy complications such as severe preeclampsia, intrauterine growth retardation (IUGR), abruptio placentae, and still-birth have been shown to be associated with thrombophilias, both acquired and inherited. ,,,,,,,,, The present study was conducted to study natural and acquired coagulation inhibitors and activated protein C resistance (APCR) in recurrent pregnancy losses (RPL).
| Materials and Methods|| |
The study group comprised of thirty pregnant women, with a history of two/more unexplained fetal losses (spontaneous abortions and/or fetal deaths) occurring after the first trimester. The control group comprised of 30 age-matched pregnant women with two or more live issues without any prior history of abortions and/or fetal deaths. A detailed clinical history was taken and physical examination conducted. Patients with any apparent cause of fetal wastage, like genital tract/fetal malformations, infectious causes, ABO and Rh incompatibility, and diabetes mellitus were ruled out using relevant tests like ultrasonography, Venereal Disease Research Laboratory (VDRL) test, TORCH test (Toxoplasma, other infections, Rubella, Cytomegalovirus, Herpes simplex), blood group, and blood glucose tests.
Complete blood counts, screening coagulation tests like Prothrombin Time(PT) and Activated Partial Thromboplastin Time (APTT), tests for natural and acquired coagulation inhibitors, i.e., protein C, protein S, anti-thrombin, antiphospholipid antibodies (APLA), and APCR were performed. Platelet-poor plasma was prepared by centrifuging the blood samples at 2 500 g for 15 to 20 minutes. Estimation of protein C, protein S, APCR, and lupus anticoagulant (LA) was performed using clot-based assays, while the semiquantitative determination of APLA classes (IgG/IgM) was done using ELISA kit and antithrombin was measured using chromogenic assay (DIAGNOSTICA STAGO, France). In all cases showing LA positivity and/or high APLA values, the test was repeated after an interval of 12 weeks.
Statistical analysis was performed using SPSS software. Comparisons between two group frequencies were made using Chi square test. For the comparison of group means, Student's t test was applied.
| Results|| |
The mean age of group A was 24.9 years and that of group B was observed to be 25 years. Most of the subjects in group A had at least two recurrent second trimester abortions and/or stillbirths. The subjects in group B had no pregnancy loss. There was no significant difference between the 2 groups with respect to the routine hematological parameters.
PT and APTT values did not show significant difference between the two groups.
Protein C, Protein S, and Antithrombin
The mean value of protein C in group A (69.95 ± 18.33%) when compared with group B (81.33 ± 9.90%) was found to be significantly low (P=0.005). Protein C levels were lowered in 33.3% of group A subjects as compared with 3.3% in group B subjects. The mean value of protein S in group A (74.43 ± 19.8%) was significantly lower than that of group B (83.05 ± 7.44%) (P=0.032). The protein S levels were lowered in 23.3% of group A subjects, while the corresponding values in group B fell within reference range. The mean value of antithrombin in group A (90.4 ± 15.39%) was slightly lower than that of group B (96.9 ± 8.79%). However, the difference was statistically significant. This could be because the single subject of group A showed antithrombin deficiency had a very low level (20% of PNP) [Table 1].
Activated Protein C Resistance
APCR was observed in 5 of 30 (16.6%) subjects in group A, while 1 of 30 (3.3%) subjects in group B showed APCR. Although APCR was present more in patients than in controls, the difference in the group frequencies compared using the Chi-square test was not found to be statistically significant (P=0.195).
APLA were positive in 6/30 cases in group A (20%) and none in controls. The difference was statistically significant ( P=0.024). Of these, LA was found in 5/30 cases (16.6%), while high titers of anticardiolipin antibodies (aCL) were observed in 2/30 subjects (10%) in group A. One of these patients concomitantly had both LA and high titers of aCL. All patients with APLA were tested again after an interval of 12 weeks and showed persistent positivity.
Combined defects were seen in seven patients. Four patients showed combined deficiencies of protein C and protein S. One patient each showed protein C deficiency with APCR, APCR with APLA, and protein C deficiency with APLA, respectively.
| Discussion|| |
Maternal thrombophilia has recently been identified as a major cause of adverse pregnancy outcome, including recurrent pregnancy loss, still-birth, severe pre-eclampsia, placental abruption, and intrauterine growth restriction. ,,,,,,,,,
The most common inherited disorders are deficiencies of antithrombin, protein C, protein S, APCR due to factor V Leiden(FVL) mutation, a function-enhancing mutation of the prothrombin gene (G20210A), and thermolabile mutation for methylene-tetrahydrofolate reductase C677T. The antiphospholipid antibody syndrome (APS) is the most common cause of acquired thrombophilias.  It is associated with complications spanning all trimesters of pregnancy. ,,
The results obtained in this study for protein C and S were comparable with those by de Vries et al. who demonstrated deficiencies of protein C and protein S in patients with complicated pregnancies (intrauterine fetal death, small-for-gestational age, and/or placental abruption). Gris et al. also found an increased prevalence of protein C and protein S deficiency in patients with at least one late still-birth. However, both the above series were unable to identify antithrombin deficiency.
Preston et al. found a significant increase in miscarriages and still-births in patients with protein C, protein S, and antithrombin deficiency. Sanson et al. also found a greater risk for fetal loss after 8 weeks in patients with these deficiencies. However, other studies on women with RPL could not find an increased incidence of protein C/protein S deficiency. 
There are conflicting reports on the association of APCR with RPL. APCR can be inherited or acquired. Inherited APCR is most commonly due to factor V Leiden mutation or other factor V polymorphisms. It is said to be the most common inherited form of thrombophilia. First described in 1993, Ridker et al. did a survey on the ethnic distribution of factor V Leiden mutation in 4 047 American men and women. Their data indicated a higher prevalence of factor V Leiden among Caucasians (5.27%) than minority Americans (0.45% in Asian Americans and 1.23% in African Americans).
Preston et al. found a modest increase of still-births, but not of miscarriage in women with factor V Leiden mutation. Rai et al.  found a significantly higher prevalence of APCR in women with recurrent second trimester miscarriage, thereby suggesting APCR as a mechanism of fetal loss in the second trimester. Dizon-Townson et al. found no association between factor V Leiden and RPL in their studies.
Rai et al., on the other hand, demonstrated acquired APCR, but not factor V Leiden was more common in early/late RPL, thereby concluding that acquired but not congenital APCR is associated with RPL. In the present study, APCR was found in 5/30 (16.6%) cases as compared with 1/30 (3.3%) controls and the difference was not statistically significant ( P = 0.195). APCR (due to factor V Leiden) may not be an important cause of RPL in Asian/Indian population as demonstrated by Kazumasa  and Biswas et al. The APCR positivity obtained in the present study may be due to acquired APCR. Vora et al. found that the risk of fetal loss with protein S deficiency was the highest risk observed for any heritable thrombophilia, followed by protein C, factor V Leiden, endothelial protein C receptor, antithrombin III deficiency, and beta448 fibrinogen polymorphism, in a study on Indian patients. Ayadurai et al. found that thrombophilia was identified in more than one-fourth of the RPL subjects. APCR not caused by factor V Leiden mutation was the most common thrombophilia marker in Malaysians, whereas in Caucasians, it was APCR due to FVL mutation. Larger studies are required to determine the role of APCR in Indian patients with RPL.
Recurrent pregnancy loss is one of the defining diagnostic criteria of APS. APS is the most common cause of acquired thrombophilias. It is associated with complications that span all trimesters of pregnancy, including RPL, severe pre-eclampsia, IUGR, and placental abruption. Various studies have shown LA positivity in RPL ranging from 9 to 17%, while the frequency of aCL in RPL ranged from 11 to 42%. ,,, The results obtained in the present study were similar to those by Narayan et al.
In conclusion, this study suggests that a significant number of Indian patients with RPL show thrombophilic defects. Hence, all patients with unexplained RPL should be investigated for thrombophilic defects. More prospective studies are required to establish the causal link between thrombophilia and adverse pregnancy outcome and assess the effectiveness of thromboprophylaxis in pregnant women with RPL.
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Department of Pathology, D-10, Sector 27, Noida, Uttar Pradesh
Source of Support: None, Conflict of Interest: None