Incidence of hereditary thrombophilia in women with history of obstetrical complications

Introduction

Thrombophilia is a spectrum of coagulation disorders associated with a predisposition to thrombotic events due to abnormalities in blood composition, blood flow, or the vascular wall. Thrombophilia is a pathological condition relatively recently identified and introduced into medical research and practice. The term thrombophilia was coined in 1937 by Nygaard and Brown, and it was used in 1965 by Egeberg to describe a family in Norway with a pronounced tendency to venous thrombosis who were later determined to have an antithrombin deficie­ncy. Research has identified two types of thrombophilia: hereditary and acquired. Hereditary thrombophilia is a genetic condition with heterogeneous etiology, produced by mutations described in almost all coagulation factors that either reduce the level of inhibitors of the coagulation cascade, or increase the level or function of coagulation factors^(1,5,6).

Although substantial progress has been made in understanding many human conditions, there are still large knowledge gaps regarding many physiological and pathological processes, especially in obstetrics, where advances in care have led to significant declines in maternal and perinatal mortality in developed countries. Despite this fact, preeclampsia, intrauterine growth restriction (IUGR), stillbirth, spontaneous abortion, and placental abruption (premature detachment of the normally inserted placenta; PDNIP) remain largely idiopathic. The prediction and prevention of these complications remain limited, with full-term delivery representing the only effective treatment strategy in cases of preeclampsia, IUGR and PDNIP. The consistent findings among patients with such complications as areas of thrombosis on histological examination of the placenta suggested that coagulation disorders may contribute to the etiology of these conditions⁽¹⁰⁾.

In general consensus with the literature, hereditary thrombophilia, in addition to its established role in the pathogenesis of venous thromboembolism (VTE), increases the risk of gestational vascular complications and adverse pregnancy outcomes, and women presenting combinations of different forms of thrombophilia have an additional risk. Pregnancy complications secondary to thrombophilia include maternal (VTE, arterial thrombosis, severe preeclampsia), placental (thrombosis, infarcts, PDNIP), and fetal (recurrent pregnancy loss, severe IUGR, fetal death, stroke) outcomes. All adverse maternal and perinatal outcomes – including fetal death, severe preeclampsia, severe IUGR and PDNIP – account for 75% of fetal morbidity and mortality^{(2-6,9,17-19)}.

The role of hereditary thrombophilia in gestational vascular disorders and adverse pregnancy outcomes has been evaluated in several studies, and the results are heterogeneous and contradictory^{(3,6-10,17,18)}. There are no sufficiently powered studies to adequately determine the effect of hereditary thrombophilia on recurrent pregnancy loss (RPL), preeclampsia, small-for-gestational-age neonates, or other placental-mediated pregnancy complications^(8,9,17). There is little robust evidence of the association of adverse pregnancy outcomes with hereditary thrombophilia during pregnancy^(3,9,17). The evaluation of a large number of studies in the literature on hereditary thrombophilia in pregnancy does not assign a global incidence of risk, indicating only an absolute small risk in pregnant women and the fetus during pregnancy^(4,17). In addition, the clinical consequences of hereditary thrombophilia (cardiovascular and cerebral thrombosis, RPL, preeclampsia, IUGR and PDNIP) correlate, in a still incompletely known manner, with the type and number of mutations^(10,12). It is still difficult to accurately quantify the risk of certain adverse events in the presence of thrombophilic mutations⁽⁴⁾. This may be, in part, due to the relatively rare occurrence of obstetrical complications of pregnancy in combination with the rarity of hereditary thrombophilia diagnosis, which makes it difficult to conduct well-designed studies⁽⁷⁾.

Furthermore, the causes of placental-mediated pregnancy complications remain largely unknown, but an important role is thought to be – at least in part – attributed to the activation of hemostasis at the placental level, which contributes to placental vascular thrombosis and abnormal placental development⁽¹⁰⁾.

The objective of the study was to study the prevalence of different types of hereditary thrombophilia in women with a history of obstetric complications.

Materials and method

In order to achieve the purpose and objectives of the research, a transversal study was conducted within the Department of Obstetrics and Gynecology of the “Nicolae Testemiţanu” State University of Medicine and Pharmacy, Chişinău, Republic of Moldova, and the Medical-Sanitary Institution of the “Gheorghe Paladi” Municipal Clinical Hospital, Chişinău, between 2020 and 2023. The aim of the study was to determine the prevalence of different forms of hereditary thrombophilia in women with a history of obstetric complications, in order to achieve pathogenetic prophylaxis of severe recurrent complications in pregnant women with thrombophilic mutations.

The study was carried out on a group of 114 women, aged between 21 and 42 years old, with a history of obstetric complications of unknown etiology: preeclampsia, eclampsia, stalled pregnancy, antenatal death, severe IUGR, severe PDNIP, early and late miscarriage.

Because folates may be implicated in thrombotic risk, folic acid administration was assessed in previous pregnancies.

For better research accuracy, a series of inclusion and exclusion criteria were considered, with the study being thus better delimited and centered on a certain representative group.

Women aged 21-42 years old, with recurrent severe obstetric complications of unknown etiology in the anamnesis, were included in the research according to the following criteria: 1) two or more miscarriages and/or stalled pregnancies in the first trimester of pregnancy (up to 11 weeks of gestation plus 6 days); 2) one or more spontaneous abortions and/or stalled pregnancies in the second trimester of pregnancy (from 12 to 21 weeks of gestation plus 6 days); 3) intrauterine fetal death or  stillbirth without identifiable causes; 4) the patient’s consent to participate in the study.

The exclusion criteria included the following: 1) women with recurrent obstetric complications of known etiology: cytogenetic (chromosomal) abnormalities, antiphospholipid syndrome, anatomical defects of the uterus, endocrine disorders, metabolic disorders, alloimmune disorders, endometrial infections, recent maternal cytomegalovirus infection, drug or alcohol abuse during pregnancy; 2) women with severe comorbidities (valvular prostheses, diabetes, severe essential hypertension, polycystic kidney disease, severe renal failure, liver failure – acute hepatitis, chronic active hepatitis, cirrhosis, etc.); 3) women with acquired thrombophilia; 4) women aged above 42 years old and below 21 years old; 5) women with pregnancies obtained through assisted reproduction techniques; 6) women who refused to participate in the study. The examined patients had types of hereditary thrombophilia, combined hereditary thrombophilic defects, a family history of VTE and harmful factors (smoking, work environment, maternal age above 35 years old, voluminous varicose veins, parity >3). The study protocol was approved by the Ethics Committee of the “Nicolae Testemiţanu” State University of Medicine and Pharmacy, Chişinău.

Clinical methods. To determine hereditary thrombophilia in women with a history of obstetric complications, general clinical research methods were used. All women included in the study were evaluated using the survey method. The specially developed standardized questionnaire included 42 questions regarding socio-demographic data, risk factors, types of hereditary thrombophilia, personal and family history of VTE, previous obstetric status, comorbidities, and placenta-mediated pregnancy complications in previous pregnancies.

Special molecular-genetic methods. To determine hereditary thrombophilia, the following polymorphisms were identified:

• the folate cycle – methylenetetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C;

• methionine – methionine synthase (MTR) A2756G, methionine synthase-reductase (MTRR) A66G;

• factor II G20210A – prothrombin;

• factor V Leiden (FVL) G1691A;

• factor XIII G103T;

• platelet activating factor – plasminogen activator inhibitor (PAI-1) 4G/5G.

The types of hereditary thrombophilia were determined based on previously documented medical data or on the analysis of the results of the polymerization chain reaction and the method of restriction fragment length polymorphism with specific primers at a certain thermal regime. The obtained amplicon was subjected to restriction analysis with specific enzymes. The DNA samples were visualized on polyacrylamide gel⁽¹¹⁾.

The detection of thrombophilic polymorphisms included several successive steps: DNA extraction from blood samples according to standard methods, amplification of some DNA sequences by polymerase chain reaction, electrophoresis and visualization with mutation detection⁽¹¹⁾.

Venous blood was collected by venipuncture of the cubital vein in the morning on an empty stomach, stabilized with 3.8% trisodium citrate solution at the blood ratio of 1:9, and immediately centrifuged.

Diagnostic criteria for obstetric complications in pregnancy. Vascular thrombosis represents one or more clinical episodes of arterial, venous or small vessel thrombosis in any tissue or organ, confirmed by objectively validated criteria. There is a histopathological confirmation that thrombosis is present in the absence of significant evidence of vessel wall inflammation⁽⁸⁾.

Spontaneous abortions include the following types: 1) early spontaneous abortion – in the first trimester of pregnancy (up to 11 weeks of gestation plus 6 days); late spontaneous abortion – in the second trimester of pregnancy (from 12 to 21 weeks of gestation plus 6 days), confirmed by ultrasound or histopathological examination.

A perinatal loss is any fetal or neonatal death at term, greater than 22 weeks of gestation, and equal to or less than 28 days postpartum. Neonatal mortality is considered any neonatal death after birth and at least 28 days postpartum. A stillbirth was found after 22 weeks of gestation without identifiable causes⁽¹³⁾.

Preeclampsia was considered the clinical syndrome composed of hypertension and proteinuria (over 300 mg in 24 hours), or the clinical symptoms of target organ damage occurring after 20 weeks of gestation⁽¹⁴⁾.

Eclampsia was found in the case of the development of convulsions and/or coma with underlying preeclampsia, which cannot be attributed to other causes⁽¹⁴⁾.

Severe IUGR is mostly defined as a fetal weight, estimated on ultrasonographic examination for gestational age, of less than the 5^th percentile⁽²⁾.

Methods of statistical processing of the results. To statistically process the material, special files were developed where all the data of the women included in the study were coded.

The primary data were processed using the functions and modules of the Statistical Package for the Social Sciences, version 21.0 for Windows (SPSS, Inc, Chicago, IL, 2012), through descriptive and inferential statistical procedures. The c² method with Yates’ continuity correction, or Fisher’s exact test, was used to compare discrete variables. The univariate analysis of variance with the application of post hoc analysis tests was used to test the difference between the mean values in the study groups, as well as the correlation analysis to determine the relationship between variables, its strength and its direction.

Results and discussion

Hereditary thrombophilia has a special place among thromboembolic risk factors. The study has expanded from common or sophisticated tests of coagulation cascade research to molecular biology research. From a practical point of view, the extent of the involvement of each thrombophilic factor in the thrombotic risk and the means of thromboprophylaxis and treatment constitute the directions of clinical research^(1,17,18).

The study included 114 women, aged between 21 and 42 years old (on average, 31.09±0.5 years old), with a history of obstetric complications of unknown etiology: early pregnancy loss in 73 women (64%), late pregnancy loss in 20 women (17.5%), antenatal death in 10 women (8.8%), stalled pregnancy in eight women (7%), preeclampsia in one woman (0.9%), eclampsia in one woman (0.9%), and severe IUGR in one woman (0.9%).

Hereditary thrombophilia is the result of inheriting an abnormal gene from one parent (heterozygous mutation) or from both parents (homozygous mutation). Hereditary thrombophilic defects are much more widespread than originally thought, and it is not unusual for several types of thrombophilia to occur in individuals or fami­lies^{(10,12,17-19)}. The FVL G1691A gene mutation, prothrombin G20210A gene mutation and MTHFR gene mutation are common, and combinations of these defects with each other or with other types of hereditary thrombophilia are also common. Carriers of combinations of hereditary thrombophilic defects have an additive or synergistic effect, with a much higher risk of thrombosis, at a much earlier age, and with much more frequent recurrent thrombotic episodes^(8,17-19).

All the women in the study were found to have from one to six types of hereditary thrombophilia. A thrombophilic mutation was determined in three women (2.6%), two thrombophilic mutations in 13 women (11.4%), and three or more thrombophilic mutations were determined in 98 women (86%).

The frequency of hereditary thrombophilia types in women with obstetric complications in pregnancy is presented in descending order. PAI-1 4G/5G was determined in 86 women (75.4%), including heterozygous type in 52 women (45.6%) and homozygous type in 34 women (29.8%). MTRR A66G was determined in 84 women (73.6%), including heterozygous type in 73 women (64%) and homozygous type in 11 women (9.6%). MTHFR A1298C was determined in 74 women (64.9%), including heterozygous type in 62 women (54.4%) and homozygous type in 12 women (10.5%). MTHFR C677T was determined in 56 women (49.1%), including heterozygous type in 52 women (45.6%) and homozygous type in four women (3.5%). MTR A2756G was determined in 53 women (46.5%), including heterozygous type in 43 women (37.7%) and homozygous type in 10 women (8.8%). Factor XIII G103T was determined in 50 women (43.9%), including heterozygous type in 41 women (36%) and homozygous type in nine women (7.9%). FVL G1691A was determined in 13 women (11.4%; the heterozygous type), and factor II G20210A wad determined in two women (1.8%; the heterozygous type).

Although, according to literature data, the most widespread genetic abnormalities that are known to predispose to venous thrombosis are the FVL G1691A, MTHFR C677T and factor II G20210A gene mutations^(5,6,10), in this study, the most frequent  mutations were those of the following genes: PAI-1 4G/5G (75.4%), MTRR A66G (73.6%), MTHFR A1298C (64.9%), MTHFR C677T (49.1%), MTR A2756G (46.5%), and factor XIII G103T. The prevalence of genetic abnormalities FVL G1691A (11.4%) and factor II G20210A (1.8%) was significantly lower.

The homozygous mutation rate of different types of hereditary thrombophilia varies significantly and results in a more severe phenotype. There is the homozygous type of the FVL G1691A gene mutation in about 0-1% of cases. The MTHFR C677T gene mutation is present in 10-16% of cases, and the MTHFR A1298C gene mutation is present in 4-6% of cases^(6,7).

The assessment of personal history in the study group found harmful factors in 37 women (32.5%; smoking was reported by two women, representing 1.8%) and harmful work environment factors in 35 women (30.7%), while comorbidities (endocrine, vascular) were found in three women (2.7%) and voluminous varicose veins were found in five (4.4%).

The women with hereditary thrombophilia usually have risk factors, including a family history of thrombosis, a family history of sudden death from pulmonary embolism, or multiple family members on long-term anticoagulant therapy. Another personal risk factor is venous thromboembolism (VTE) by the age of 45⁽⁴⁾.

A family history of venous thromboembolism in a first-degree relative increases the risk of VTE twofold, regardless of the presence of hereditary thrombophilia. In pregnant women, the risk of venous thromboembolism is six times higher compared to non-pregnant women of the same age⁽⁵⁾. About 50% of women with thrombosis during pregnancy have an underlying thrombophilia⁽¹⁰⁾. However, in this study, a personal history of thromboembolism was not found, and family history revealed a history of thromboembolism in first-degree relatives in 24 cases (21.1%).

The obstetric history revealed that, from zero to ten previous pregnancies (mean: 2.4±0.2 pregnancies), 28 women (24.6%) had live births, including one live child in 24 cases (21.1%), two live children in four cases (3.5%), and three live children in one case (0.9%). Full-term births occurred in 26 women (22.8%), and premature birth occurred in three women (2.6%).

Among women who gave birth to live children, MTRR A66G was present in 22 women (88.6%), including heterozygous type in 19 women (67.9%) and homozygous type in three women (10.7%). PAI-1 4G /5G was present in 24 women (85.7%), including heterozygous type in 14 women (5%) and homozygous type in 10 women (35.7%). Factor XIII G103T was present in 16 women (57.2%), including heterozygous type in 12 women (42.9%) and homozygous type in four women (14.3%). MTHFR A1298C was present in 14 women (50%), including heterozygous type in 11 women (39.3%) and homozygous type in three women (10.7%). MTR A2756G was present in 13 women (46.4%), including heterozygous type in 11 women (39.3%) and homozygous type in two women (7.1%). MTHFR C677T was present in 12 women (42.8%), including heterozygous type in 10 women (35.7%) and homozygous type in two women (7.1%). FVL G1691A was present in two women (7.1%; the heterozygous type). Factor II G20210A in this group of women was not determined.

Although the frequency of different forms of hereditary thrombophilia varies among women who have given birth to live children and among women who have not given birth to live children, these differences did not reach statistical certainty, a fact probably accounted for by the small groups of women.

Previous fetal deaths after 10 weeks of gestation were found in 33 women (28.9%), including one death in 24 cases (21.1%), two deaths in five cases (4.4%), and three deaths in one case (0.9%). Three women (2.6%) had a medical abortion.

The etiopathogenesis of recurrent pregnancy loss (RPL) is multifactorial, but largely begins with determining who meets the diagnostic criteria for RPL, as definitions vary and change frequently. The commonly used definitions include: 1) two or more failed clinical pregnancies documented by ultrasonography or histopathological examination; 2) three consecutive pregnancy losses that must not be intrauterine⁽⁷⁾.

About 15% of pregnant women experience a clinically recognized pregnancy loss, 2-5% report two or more consecutive pregnancy losses, and 1-2% present three or more consecutive pregnancy losses^(7,9). Although the results are not conclusive, several studies have suggested a higher frequency and a positive association of different forms of hereditary thrombophilia with early pregnancy loss and RPL^(10,15-20). In addition, the rate of fetal loss was higher in women who had two or more forms of hereditary thrombophilia^(10,15). In the study, pregnant women with severe obstetric complications reported significantly more recurrent pregnancy loss: a total of 108 women (94.7%), including one pregnancy loss in 40 cases (35.1%), two pregnancy losses in 44 cases (38.6%), and three or more pregnancy losses in 24 cases (26.6%).

The average value of the gestational age of the first recurrent pregnancy loss was 8.7±0.6 weeks, for the second RPL, the average gestational age was 8.72±0.7 weeks, for the third RPL – 8.64±1.3 weeks, and for the fourth RPL – 8±1.9 weeks.

The association of hereditary thrombophilia with recurrent pregnancy loss is manifested more by the total number of mutations than by the specific genes involved. Some scientists have found an overall higher risk for late pregnancy loss than for early pregnancy loss in women with hereditary thrombophilia. The odds ratio for late pregnancy loss in women with combined hereditary thrombophilia is 14.3⁽⁸⁾. However, other studies found that the combination of two or more forms of hereditary thrombophilia (FVL G1691A gene mutation, factor V A5279G gene mutation, prothrombin G20210A gene mutation, MTHFR C677T gene mutation, MTHFR A1298C gene mutation, factor XIII A614T gene muta­tion, b-fibrinogen G-455A gene mutation), especially the combination of FVL G1691A gene mutation with MTHFR (C677T or A1298C) gene mutation, revealed a significant correlation between them and with early RPL^(15,16).

In the study, women with two (66.7%) and three (61.3%) types of hereditary thrombophilia had a history of three or more RPL more frequently. However, significant statistical differences were not determined, probably due to the small study group.  

Corresponding author: Angela Marian-Pavlenco, e-mail: angela.marian@usmf.md

Conflict of interests: none declared.

Financial support: none declared.

This work is permanently accessible online free of charge and published under the CC-BY.