Introduction
Recurrent miscarriage (RM), unlike sporadic pregnancy loss, necessitates additional attention, monitoring, and follow-up in subsequent pregnancies to be successful [
1,
2]. RM is a disappointing experience that negatively impacts couples’ psychological status and worsens their social discomfort [
3]. RM is classified into primary and secondary: primary RM is the repeated miscarriage without viable previous babies, but secondary RM is the repeated pregnancy loss with a live birth at some time [
3]. Secondary RM has a better prognosis than primary RM, in fact, primary RM is a poorly understood condition, which affects 1–3% up to 5% of couples trying to have children worldwide [
3‐
14]. RM is defined by the American Society of Reproductive Medicine (ASRM) and the European Society for Human Reproductive and Embryology (ESHRE) as the loss of two or more consecutive pregnancies from conception to 24 weeks of gestation [
1,
4‐
9,
11,
13‐
17]. While the World Health Organization (WHO) and the Royal College of Obstetricians and Gynecologists (RCOG) define RM as three or more miscarriages verified by ultrasonography or histology up to 24 weeks of gestation [
2,
4,
5,
15,
16,
18‐
20]. As a result, several measurements and recommendations were developed and implemented to reach standardization for appropriate inquiry and treatment approaches [
1,
15].
In normal pregnancy, the hemostatic system is modified to a hypocoagulable condition to preserve oxygen and nutrition transmission via the placenta for fetal survival, then changed to a hypercoagulable state to prevent excessive bleeding following birth [
2,
6,
10,
21‐
23]. Because of the interaction of various risk factors that play a role in RM such as maternal age, lifestyle behaviors (stress, smoking, and excessive alcohol consumption), history of miscarriage, antiphospholipid syndrome, uterine malformation, endometritis, endocrinological, abnormal parental karyotypes, obesity, genetic factors, and thrombophilia with other unknown factors, RM is considered a multifactorial condition [
1‐
5,
7‐
9,
11,
12,
14,
15,
17,
19,
24,
25]. Inherited thrombophilia (IT) is a hypercoagulopathy state that increases the risk of thrombosis. The combination of physiological alterations in hemostatic systems with IT in pregnancy has been reported to increase the risk of pregnancy complications in 40–50% [
6,
24] or even 80% of cases [
12].
RM is a multi-etiological syndrome with more than 50% of cases remaining unexplained, with the underlying processes remaining unknown, limiting the diagnosis and treatment protocols, and creating a challenge to patients and doctors [
1,
3,
4,
6‐
8,
12,
14‐
16,
18,
20,
24,
26]. RM demonstrates significant causative variability in known and unknown etiology groups [
15,
16]. Despite substantial research on the subject, studies are still limited, suffer from population selection bias, use diverse diagnostic criteria, and yield conflicting conclusions [
4].
Inherited or acquired thrombophilia causes irregular blood coagulation and an increased risk of venous thromboembolism (VTE). IT is caused by mutations that affect gene function in the anticoagulant mechanism, causing the hemostatic system to become thrombotic [
11,
21]. Thrombophilia affects about 5% of the general population [
3]. Prothrombin gene (FII) mutation (G20210A) and factor V Leiden (FVL) were detected in around 50–70% of VTE cases diagnosed with inherited thrombophilia [
21]. Pregnancy is associated with around 20% of thrombotic events; additionally, pregnancy raises the incidence of VTE by 5 times [
12,
18,
21]. It is estimated that around 40–50% of VTE caused by IT occurs during pregnancy [
3,
12,
21]. The association between thrombophilia and RM has been investigated by many studies with controversial conclusions [
3,
11,
12]. Therefore, in cases of RM, a panel of laboratory tests belonging to hemostatic, and IT are usually recommended [
3].
Clinically, most carriers of thrombophilia mutations will not show symptoms and will go untreated, but exposure to additional risk factors such as pregnancy may raise the likelihood of life-threatening complications to become clinically clear [
12,
26]. IT panel includes FVL R506Q mutation, FII gene G20210A mutation, Methyltetrahydrofolate Reductase gene mutations (MTHFR C1677T; A1298C), B-fibrinogen gene − 455G > A, Plasminogen activator inhibitor 1 (PAI1) 5G/4G, Factor XIIIA (FXIIIA) V34L, and FV HR2 A4070G. FII G20210A, FVL and MTHFR C677T and A1298C are the most extensively studied thrombophilia mutations in RM, and most studies reported wide ranges of prevalence for each mutation among the respective study population with controversial findings [
6,
10‐
16,
24,
26‐
30].
In the past 13 years, eight studies have been conducted regarding RM among Palestinian women in Palestine (West Bank and Gaza) [
6‐
9,
25,
27,
31‐
33]. These reports examined one [
9,
25,
27], two [
7] three [
6,
8,
33], or seven IT mutations [
32] and reported controversial results.
About 2.4% of married Palestinian women experience primary infertility, and about 6.0% experience secondary infertility [
34]. The incidence of miscarriage in the Palestinian community was estimated at 4–8% that is considered relatively high and necessitates more investigations to minimize miscarriage and increase the opportunity of normal pregnancy and delivery of live baby [
27]. In Palestine, miscarriage cases have been attributed to several factors including thrombophilia (32.5%), followed by IVF pregnancies (8.8%), and twin pregnancies (8.6%) among other causes [
34]. Government high-risk pregnancy clinics received referrals from 17.4% of pregnant Palestinian women in 2018 [
34]. In the investigation of unexplained RM cases, analysis of IT has been a common approach. Therefore, the aim of our study was to investigate the association between the common eight IT mutations and their combinations among Palestinian women with unexplained RM.
Discussion
The impact of lifestyle factors on sporadic miscarriage is well-established, however, their contribution to RM remains unclear. This study aimed to investigate this point by examining a group of patients (aged 20–42 years) and controls (aged 21–45 years) who were of similar ethnic and social backgrounds and residing in different districts of the West Bank. The sample was selected using statistical methods to ensure it was representative of the West Bank population. Anthropometric data and various lifestyle factors were analyzed for both groups, consisting of 100 patients and 100 controls. The study found no significant differences between patients and controls in terms of family history of abortion, consanguineous marriage, diabetes mellitus, hypertension, family history of cardiovascular disease, anticoagulant use, coffee consumption, and folate supplementation [
41].
IT is a coagulation disorder related to hypercoagulopathy state and thrombotic events. It has a complex pathophysiology status and is associated with RM by causing placental insufficiency and inhibiting fetal development. Thrombophilia screening is still questionable, so laboratories perform different panels of tests under the name of thrombophilia in unexplained RM cases [
14].
RM is a global health problem that affects all communities worldwide and requires sharing efforts for real effective solutions, but because of the different genetic backgrounds between people of the world, the contribution of IT mutations to RM is expected to differ among ethnic groups. Therefore, it would be helpful to compare the prevalence of IT mutations locally, regionally, and globally to identify similarities and differences between Palestine and other countries. This may facilitate a better understanding and management of unexplained RM.
In our study, we investigated the prevalence of the common eight IT mutations among healthy controls. The most frequent mutations were PAI1 5G/4G (69%), MTHFR C677T (53%), and MTHFR A1298C (47%), while FII G20210A displayed the lowest frequency (3%) (Tables
1 and
5). Our findings exhibit that the most common IT mutations analyzed (FVL, FII G20210A, MTHFR C677T, and MTHFR A1298C) were consistent with the prevalence rates reported by earlier studies from Palestine except for FII G20210A mutation which showed a lower prevalence in our study (3%) compared to earlier Palestinian studies (Table
5) [
6,
32,
33]. The prevalence of PAI1 5G/4G and FV HR2 mutations among the Palestinian population was not reported previously. Also, the prevalence of the main four IT (FVL, FII G20210A, MTHFR C677T and A1298C) among Palestinians was within the range observed in neighboring Arab countries, namely Lebanon [
24], Jordan [
42], Syria [
43] and Egypt [
44]. These neighboring Arab countries and Palestine have similar genetic backgrounds (Table
5). However, the prevalence of the three main inherited thrombophilia mutation (FVL, FII G20210A and MTHFR C677T) among healthy Palestinians and healthy populations in other regions of the world (Europe and America) [
45] revealed that FVL and MTHFR C677T prevalence ranked Palestine among countries with high prevalence of these mutations, but FII G20210A prevalence was consistent with other regions as shown in Table
5 [
45]. Variations in the prevalence of IT mutations among diverse populations can be attributed to the distinct genetic profiles of the populations under investigation. Thus, the determination of the prevalence of these mutations in each population should aid in the development of protocols for thrombophilia screening in the respective population.
Table 5
Comparison of the prevalence of the most common inherited thrombophilia mutations among healthy Palestinian women (healthy controls) observed in this study to that reported by previous Palestinian studies, neighboring Arab countries, and other regions of the world
FVL R506Q | 18 | 18.2 | 23.6 | 20.1 | 9.0 | 30 | 11.5 | 58.1 | 5–9 | 3-5.2 |
FII G20210A | 3 | 4.2 | 5.9 | 9.3 | 2.0 | 8.3 | 2.5 | 4.7 | 2–6 | NA |
MTHFR C677T | 53 | NA | 50.9 | 13.8 | 63.0 | 28.3 | 37.7 | 58.2 | 10–16 | NA |
MTHFR A1298C | 47 | NA | 49.0 | NA | NA | 58.2 | 38.8 | NA | NA | NA |
In addition, the study investigated the association between the main eight inherited thrombophilia mutations and unexplained RM among Palestinian women from the West Bank region of Palestine compared to control women with normal deliveries. Our results indicated a significant association between MTHFR A1298C and unexplained RM in the Palestinian population (p 0.016). In contrary, the other inherited thrombophilia mutations analyzed in this study (FVL, FII G20210A, and MTHFR C677T, BFG − 455G > A, PAI1 5G/4G, FXIIIA V34L, FV HR2) displayed no significant association with unexplained RM. Thrombophilia in general (inherited and acquired) is diagnosed in about 32.5% of miscarriage cases in Palestine [
34]. So far, the six Palestinian previous studies that examined the prevalence of IT and its association with RM showed conflicting results for their association with RM, but they showed consistent findings concerning the prevalence of inherited thrombophilia mutations among healthy women [
6‐
8,
27,
32,
33]. Thus, this study confirmed local previous reports on MTHFR A1298C that reported a significant association of this mutation and RM [
32], as well as no significant association with MTHFR C677T [
6], FVL [
8,
27], FV HR2 [
32], FII G20210A [
6,
8,
32] and BFG − 455G > A [
7,
32]. Our results concerning FVL, FII G20210A and MTHFR C677T were also in contrary to reports from other previous studies [
3,
10,
11,
15,
17,
26].
For further investigation of the association between unexplained RM and inherited thrombophilia mutations, we analyzed the frequency of mutant alleles for the common eight mutations. Our findings revealed that the frequencies of mutant alleles of MTHFR A1298C (
p < 0.001) and FXIIIA V34L (p 0.009) were significantly higher in patients compared to controls (Table
2). Additionally, the mutant allele MTHFR A1298C was associated with a 2-fold increased risk (odds ratio 2.005; 95% CI 1.138–3.533) of RM, while such an effect was not observed with FXIIIA V34l (odds ratio 1.942; 95% CI 0.971–3.884). Most subjects in the patient (75%) and control groups (75%) have 2–4 mutant alleles out of the eight mutant alleles analyzed here; and only 1% of patients and 2% of controls have zero mutant alleles out of the eight alleles studied Table
3). In addition, we have analyzed the combination of the most frequent mutant alleles of FVL, FII G20210A, MTHFR C677T and A1298C (Table
4), and no significant association was observed either between the number of mutations nor the number of their alleles with unexplained RM. The most frequent combination of alleles was 1–2 alleles and it was observed in 82% of patients and 78% of controls (Table
4).
A recent bibliometric analysis study by Deng et al. [
23] analyzed 725 articles performed in the last 30 years and published by 3205 authors from 1139 organizations and 68 countries; and concluded that there is a clear causal relationship between thrombophilia and RM and tests evaluating thrombophilia should always be considered. In addition, the authors advise investigation of the unknown thrombophilia status of controls [
23]. A systematic review of the literature that included only case-control studies, 16 articles were selected for the FVL and 7 for the FII G20210A analysis, and the authors concluded that women with either one of these mutations have a 2-fold risk of RM and recommended testing for these mutations in RM cases [
26]. On the other side, a retrospective cohort study of 1155 women with RM in the UK reported that the prevalence of inherited thrombophilia in women with RM and the general population are similar and [
19].
Controversial findings are commonly encountered with association studies and especially if conducted on different populations and ethnic groups [
7]. These variations can be attributed to different genetic backgrounds of the different populations or ethnic groups studied, as well as environmental factors and genetic modifiers, especially with a multifactorial condition like RM. Therefore, the findings of inherited thrombophilia mutations in patients should be interpreted within the context of the clinical manifestation of each patient. Also, most studies that tackled the role of inherited thrombophilia in RM have focused on a small group of mutations [
3,
10,
11,
17,
24,
26,
29], namely FVL, FII G20210A, MTHFR C677T and A1298C, while studies that investigated a larger panel of thrombogenic mutations are rare [
15]. Consequently, the later four main mutations have been extensively studied and several studies have reported risk values for them [
3,
10,
15,
17,
24,
26,
29], albeit with wide variations, while such a risk value is not available for other mutations. In clinical practice, thrombophilia screening of RM cases is still used, and the interpretation of the results is somehow arbitrary due to the lack of consensus risk values for the mutations analyzed or a consensus list of mutations that should be tested. Although, there are probably differences in the clinical relevance of the different types of thrombophilia as well as single or multiple IT mutations [
23] and this may influence their interpretation. Additionally, most studies concerning IT as a hypercoagulable state are based on scientific facts and theories. While most gynecologists tend to manage RM cases with aspirin and heparin whether inherited thrombophilia mutations are detected or not [
46‐
48]. Many studies do not support the use of anticoagulants or intravenous immunoglobulin in women with unexplained RM either with or without IT [
46‐
54]. In exploring the possible causes of RM, it is interesting to note that several studies advise psychological supportive care alone for RM cases [
46,
55].
A limitation of this study is the inability to analyze markers of hypercoagulopathy at different time points of pregnancy and link it to the findings of thrombophilia mutations, because most patients were not pregnant at the time of sample collection. Another limitation is that the low number of patients and controls that have FII G20210A mutation and thus the statistical analysis of this mutation is limited.
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