Venous thromboembolism (VTE), manifested as deep vein thrombosis (DVT) and/or pulmonary embolism (PE), is one of the major causes of pregnancy-related mortality and morbidity [1]. The annual incidence of VTE ranges from one to five per 1000 individuals; however, its diagnosis remains challenging and difficult [2].
Venous thromboembolism may occur due to inherited disorders or be associated with acquired clinical condition(s). The prominent etiologies of VTE in pregnancy are inherited thrombophilias, which include deficiencies of protein C (PC), protein S (PS) and antithrombin (AT). Factor V Leiden (FVL G1691A) polymorphism, prothrombin G20210A, and methylenetetrahydrofolate reductase gene mutations also cause VTE in pregnancy [3,4]. Acquired clinical conditions that may result in VTE in pregnancy include autoimmune and inflammatory disorders, increased body mass index (BMI), obesity, prolonged immobilization, miscarriages, surgical intervention, contraceptive use and multiparity [4,5,6].
During pregnancy, which is a physiological hypercoagulable state, there is also increased activity of coagulation factors I, II, VII, VIII, IX and X and tissue factor [7]. Plasma level of PS, activated PC levels, and plasminogen activator inhibitor levels decrease in pregnancy [7]. Thrombophilia further aggravates the preexisting hypercoagulable state in pregnancy [3]. In pregnant women, the risk of VTE has been reported to be 5- to 6-fold compared with that in nonpregnant age-matched controls [1,8]. This risk further increases from 3.7- to 8.5-fold in women with a family history of VTE and up to 34-fold in women with hereditary thrombophilia [1]. The clinical manifestations associated with the FVL G1691A polymorphism in pregnant women include severe placental abruption, fetal growth disturbances, placental infarction, stillbirth, increased risk for gestational hypertension, hemolysis, elevated liver enzymes, and low platelet syndrome [9]. Considering the clinical manifestations of thrombophilia, this study aimed to determine the frequencies of FVL G1691A polymorphism and prothrombin G20210A mutations and measure the plasma levels of PC, PS and AT in pregnant women with VTE and healthy pregnant women.
This prospective case-control study was conducted between January 2018 and August 2019 at the Khartoum Teaching Hospital in Khartoum State, Sudan, recruiting 396 pregnant women. Ethics approval was obtained from the University of Science and Technology, Sudan, and the study was conducted according to the Declaration of Helsinki. Informed consent was obtained from all women before the collection of blood samples. Blood samples were collected before the initiation of the anticoagulant therapy. The 396 participants were equally divided into pregnant women diagnosed with VTE (
Data were collected using a predesigned and pre-tested questionnaire. Participants were asked about demographic data and VTE risk factors, such as familial history of thrombosis, fractures, recent surgery, kidney disease, malignant tumors, immobilization, history of hypertension, diabetes mellitus, smoking habits, and use of oral contraception or hormone replacement therapy. The BMI was calculated to evaluate obesity [body weight (kg) divided by the square of height (m)].
Blood samples were collected in EDTA-containing vacutainers for DNA analysis and in sodium citrate-anticoagulated tubes for PC, PS and AT measurements. Citrated plasma was obtained by centrifugation of blood samples at 3000 rpm for 30 min. Plasma was extracted and stored at −80 °C until analysis. Protein C and PS levels were measured by a clot-based assay using the reagents of TECHNOCLOT® (Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, Vienna, Austria). These levels were determined from a calibration curve in which prolongation of clotting time is proportional to the protein concentration. The AT level was determined using an automated coagulation analyzer (Sysmex CA-1500) using the TECHNOCHROM® AT III Kit (Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH). Reference values of PC, PS and AT in the range of 55.0–125.0, 50.0–130.0 and 62.0–120.0 ng/mL, respectively, were considered as normal based upon the preestablished normal levels. A decreased level of any of these natural anticoagulants from the reference values were considered to be deficient.
DNA was extracted from the blood samples collected in the EDTA vacutainers and purified using the QIAamp DNA Blood Mini Kit according the manufacturer's standard protocol (Qiagen Sciences Inc., Germantown, MD, USA). The FVL G1691A polymorphism and prothrombin G20210A mutation were detected by allele-specific polymerase chain reaction (ASPCR) using the Bio-Rad DNA Engine Dyad Peltier Thermal Cycler (Bio-Rad Laboratories, Foster City, CA, USA) as described previously [10].
Qualitative data, which are expressed as proportions, were analyzed using the χ2 test. Quantitative data are expressed as mean ± SD. The z-test was used for testing the significance of quantitative data. The association between two quantitative parameters was evaluated by correlation analysis, and adjusted odds ratios (ODs) were calculated for determining the strength of association between qualitative parameters. Statistical significance was set at
Among the 396 study participants, 198 were pregnant women with VTE (VTE group) and the remaining 198 were healthy pregnant women (control group), with the mean (±SD) age being 28.21 ± 7.69 and 28.42 ± 5.93 years (
Clinical features of pregnant women with venous thromboembolism (
Deep vein thrombosis | 156 | 78.8 |
Pulmonary thromoboembolism | 20 | 10.1 |
Upper limb venous thrombosis | 10 | 5.1 |
Portal vein thrombosis | 8 | 4.0 |
Cerebral vein thrombosis | 4 | 2.0 |
A total of 161 patients (81.3%) showed an association with various known risk factors of VTE, whereas 37 patients (18.6%) had no known risk factor of VTE (Table 2). Patients with a history of contraceptive use were more susceptible to developing VTE (33.8%). Moreover, high BMI (12.6%), termination (11.6%) and family history (10.1%), were the most common risk factors of VTE in the study population. The frequencies of other risk factors are shown in Table 2.
Risk factors of venous thromboembolism in pregnant women (
Contraceptive use | 67 | 33.80 |
High body mass index | 25 | 12.60 |
Termination | 23 | 11.60 |
Family history | 20 | 10.10 |
Combined risk factors | 14 | 7.07 |
Hypertension | 6 | 3.03 |
Surgery | 3 | 1.52 |
Renal disease | 2 | 1.01 |
Immobility | 1 | 0.51 |
Smoking | 0 | 0.00 |
Trauma | 0 | 0.00 |
No known risk factors | 37 | 18.69 |
Thrombophilia profiles revealed highly significant differences in the VTE group compared with those in the control group (
Levels and frequencies of protein C, protein S and antithrombin in the study participants (
Protein C | 74.93±40.8 | 97.83±23.75 | 0.0001 | deficient |
13 (6.7) |
2 (1.01) |
6.89 (153–30.95) | 0.012 |
Protein S | 87.47±25.24 | 102.46±22.73 | 0.0001 | deficient |
20 (10.1) |
3 (1.50) |
7.30 (2.134–24.996) | 0.002 |
Antithrombin | 80.19±30.33 | 85.46±18.23 | 0.037 | deficient |
23 (11.6) |
10 (5.10) |
2.47 (1.144–5.338) | 0.021 |
VTE: venous thromboembolism; OR (95% CI): odds ratio (95% confidence interval).
Regarding FVL G1691A, the frequency of the homozygous normal genotype (G/G) was lower in the VTE group (96.0%) than in the control group (100.0%). Heterozygous (G/A) and homozygous (A/A) mutations were detected in the VTE group, with frequencies of 3.5% (
Frequencies of Factor V Leiden G1691A polymorphism and prothrombin G2021A mutation in all participants (
FVL G1691A | Homozygous (G/G) | 190 (96.0) | 198 (100.0) | 17.89 (1.026–312.150) | 0.048 |
Heterozygous (G/A) | 7 (3.5) | 0 (0.0) | 15.70 (0.890–276.880) | 0.060 | |
Homozygous (A/A) | 1 (0.5) | 0 (0.0) | 3.05 (0.123–75.220) | 0.496 | |
Prothrombin G20210A | Homozygous (G/G) | 197 (99.5) | 198 (100.0) | 0.332 (0.013–08.191) | 0.499 |
Heterozygous (G/A) | 1 (0.5) | 0 (0.0) | 3.05 (0.123–75.220) | 0.496 | |
Homozygous (A/A) | 0 (0.0) | 0 (0.0) | 0.00 (0.000–0.000) | 0.000 |
FVL G1691A: Factor V Leiden; Homozygous (G/G): homozygous normal genotype; VTE: venous thromboembolism; OR (95% CI): odds ratio (95% confidence interval); Heterozygous (G/A): heterozygous mutant form; Homozygous (A/A): homozygous mutant form.
The FVL G1691A mutation was detected in 4.49% of patients (
Frequencies of factor V Leiden G1691A polymorphism and protein C, protein S and antithrombin deficiencies in various observed clinical features of pregnant women with venous thromboembolism (
Deep vein thrombosis | 7/156 (4.49) | 11/156 (7.05) | 16/156 (10.26) | 18/156 (11.54) |
Pulmonary thromboembolism | 1/20 (5.00) | 1/20 (10.00) | 1/20 (0.50) | 2 (10.00) |
Upper limb venous thrombosis | 0/10 (0.00) | 0/10 (0.00) | 2/10 (20.00) | 1/10 (10.00) |
Cerebral vein thrombosis | 0/4 (0.00) | 0/4 (0.00) | 1/4 (25.00) | 1/4 (25.00) |
Portal vein thrombosis | 0/8 (0.00) | 1/8 (12.50) | 0/8 (0.00) | 1/8 (12.50) |
FVL G1691A: Factor V Leiden; PC: protein C, PS: protein S; AT: antithrombin
Table 6 depicts the correlation of DVT, FVL G1691A polymorphism, and PC and PS deficiencies with risk factors, including age, family history, high BMI, contraceptive use and termination. In the study population, DVT was found to be correlated negatively with age (
Correlation of deep vein thrombosis, factor V Leiden G1691A polymorphism, and protein C, protein S and antithrombin deficiencies with risk factors among pregnant women (
Deep vein thrombosis | −0.122 | 0.015 | 0.054 | 0.286 | 0.189 | 0.000 | 0.042 | 0.047 | 0.274 | 0.000 |
Factor V Leiden G1691A | −0.033 | 0.517 | 0.024 | 0.635 | 0.111 | 0.027 | −0.047 | 0.352 | −0.047 | 0.325 |
Protein C deficiency | 0.036 | 0.471 | −0.053 | 0.292 | 0.020 | 0.693 | 0.227 | 0.000 | 0.036 | 0.467 |
Protein S deficiency | 0.105 | 0.035 | 0.038 | 0.456 | 0.059 | 0.241 | 0.065 | 0.197 | 0.148 | 0.003 |
Antithrombin deficiency | −0.110 | 0.005 | 0.012 | 0.751 | 0.199 | 0.001 | 0.126 | 0.253 | 0.274 | 0.001 |
BMI: body mass index.
Hemostasis is a delicate balance between procoagulant and anticoagulant factors that maintains blood flow within the blood vessels. Abnormal bleeding tendency or thrombosis occurs when the level of any of the pro coagulant factors and/or anticoagulants is in excess, deficient, or functionally abnormal. Protein C, PS, and AT are the most important natural anticoagulants. Factor V and prothrombin (factor II) are pivotal in the coagulation cascade. Thrombin also plays a crucial role in the conversion of fibrinogen into fibrin and factor XIII into XIIIa to form a stable clot. Simultaneously, thrombin forms a complex with thrombomodulin that activates PC in the presence of PS. The activated PC inhibits the activated coagulation factors V and VIII, thereby limiting the clot formation [11]. Deficiency of PC or PS results in increased susceptibility to thrombosis. Antithrombin inactivates the activated coagulation factors XI, IX, X and II. Single-nucleotide polymorphism of the
In this study, the frequency of the FVL G1691A heterozygous mutation (G/A) was 3.5%, whereas that of the homozygous mutation (A/A) was 0.5% [OR = 17.71, 95% confidence interval (9%% CI): = 1.015–309.05], which is consistent with the results of previous studies [17,18]. The rate of the FVL G1691A polymorphism is almost similar in various pregnancy-associated clinical manifestations [19]. In a group of Sudanese women with preeclampsia, the frequency of such polymorphism was found to be 9.6% [20]. Moreover, only one (0.5%, OR = 0.332, 95% CI: = 0.013–8.191) case was heterozygous (G/A) for prothrombin G20210A polymorphism. These findings are consistent with some studies but in contrast with others. However, a recent study did not detect this mutation in a selected group of pregnant women [20]. In another study conducted in Sudan, the prevalence of prothrombin G20210A mutation was reported as 3.0%, which is higher than that observed in this study [17]. Other researchers have also reported a high prevalence of prothrombin G20210A heterozygous mutations in pregnant women with DVT or PE [19]. By reviewing the ODs, it is evident that although statistically insignificant, patients with VTE were more susceptible to carry the FVL G1691A polymorphism than controls. According to the clinical presentation, of 156 patients with DVT, seven (4.49%) had the heterozygous FVL G1691A (G/A) polymorphism and one of 20 patients with pulmonary thromboembolism had a homozygous (A/A) polymorphism. These data suggest that among the VTE cases, FVL G1691A polymorphism was the most common finding in patients with DVT, which was consistent with other studies that reported a high prevalence of this polymorphism in patients with DVT [16,19,21,22,23,24]. Only one case had a heterozygous prothrombin G202210A mutation, representing 0.5% of the study population, in contrast with other studies that reported a slightly high prevalence [19,25]. The results of this study showed that AT deficiency was the most common finding in patients with DVT. The rates of PC and PS deficiencies were slightly lower than the rate of AT deficiency. These findings suggest that AT deficiency is more commonly associated with DVT than FVL G1691A polymorphism and PC and PS deficiencies among patients with VTE. Similarly, AT deficiency was the most common finding in patients with PE and venous thrombosis of the upper limb. The correlation analysis disclosed a statistically significant (
In conclusion, among the VTE cases, FVL G1691A polymorphism and PC, PS, and AT deficiencies were the most common findings in patients presenting with DVT. The AT deficiency was more common in the study population than PC and PS deficiencies. Contraceptive use, high BMI, and termination correlated strongly with FVL G1691A polymorphism and PC and PS deficiencies in patients with VTE. Major limitation of the study was no follow-up of the pregnant patients with VTE to observe the effect of FVL G1691A polymorphism and low levels of PC, PS and AT on the outcome of pregnancy. We intend to conduct further studies to explore the role of these factors in the pregnancy outcome.