Objective: To study, the impact of thrombophilia caused by Factor V, Factor II G20210A mutations on recurrent abortions, the prevelance of Factor V Leiden and Factor II G20210A mutation in patients with habitual abortions.

Methods: Forty one patients with a diagnosis of habitual abortion were enrolled in the study. Control group consisted of 50 women without a history of poor obstetric outcome. 10 ml. peripheral venous blood was taken from study and control groups and transferred to EDTA tubes, and were tested for Factor V Leiden and Factor II G20210A mutations by PCR in genetics laboratory.

Results: Phenotypes of 91 cases were determined by PCR for Factor V Leiden. It was established that of 41 patients in the study group, 31 (75.6%) had GG genotype, 9 (22%) had GA genotype and 1 (2.4%) had AA genotype. In the control group, 45 (90%)of 50 women had GG genotype and 5 (10%) had GA genotype. A allele carrier status was found to be 24.4% in study group and 10% in the control group. The difference between them was not statistically significant (p=0.06).

Conclusion: The results obtained from patients and control group have no difference in Factor V Leiden and Factor II G20210A mutations.These results suggest that mutations have no role in etiology of 1. and 2. trimester recurrent abortions (JPMA 55:104;2005).

In 68% of recurrent abortions, the etiology is unknown. It may be thought that the alterations in coagulation factors during pregnancy can lead to abortions, based on the fact that hemostatic disorders can cause obstruction in vessels of placental bed. Efficient uteroplacental circulation is mandatory for a successful pregnancy outcome and this circulation may be influenced by hemostatic disorders. Therefore, maternal thrombophilias are important pathologies in obstetrics.1 Factor V Leiden mutation and Active protein C resistance (APCR) are the most frequent subtypes of hereditary thrombophilia. APCR is a risk factor for thromboembolism and accounts for 40% of thrombosis cases. Over 95% of APCR is related to Factor V Leiden mutation and autosomally inherited. Thrombosis risk increases 7 times in heterozygotes and 80 times in homozygotes. The carriage rate is 20-60% in those with personal or familial thrombosis history.2 It has been suggested that hereditary and acquired APCR is among the causes of vascular placental deficiency in some patients with recurrent fetal losses.3 Some investigators have suggested that Factor V Leiden mutation may be a predisposing factor for recurrent abortions but it may not be a frequent cause in such patients.4,5

In some studies, no marked relation has been established between Factor V Leiden mutation and losses in first trimester whereas it has been established with losses in second and third trimesters.5 In addition, the status of Factor V Leiden mutation carrier has been found to be high in foetuses lost. Factor V Leiden is frequently encountered in preeclampsia and foetal losses. Arias et al has determined the presence of Factor V Leiden in 53.8% of patients with poor obstetrics outcome.6

Prothrombin gene is localised on the long arm of 11. chromosome. Recently, G20210A mutation, which is a prothrombin gene mutation increasing the risk of thrombosis, has been identified. This mutation is associated with high plasma concentrations of prothrombin. In carriers of Factor II (prothrombin) G20210A mutation, which occurs at the rate of 2% in healthy individuals, 6% in those with the history of thrombosis and 18% in those with family history, prothrombin levels increase and thrombosis risk is 2.8 times as that in normal population. There are studies supporting the relation of Factor II G20210A mutation with recurrent fetal losses.3,7 It has been proposed that G20210A mutation in prothrombin gene is a genetic risk factor for myocard infarctus. There are studies reporting that this mutation is a risk factor not only for fetal losses but also for thrombosis in young patients with atherosclerotic heart disease.8

The aim of this study was to investigate the role of thrombophilia related to Factor V Leiden and Factor II G20210A mutations in women with recurrent abortions by determining the prevalance of these mutations.

The study included patients referred to Ankara University Medical School department of Obstretrics and Gynecology, with the complaint of recurrent abortions and diagnosed as primary or secondary habitual abortions. A patient was considered to have habitual abortions if she had 2 or more spontaneous abortions before 20 weeks of pregnancy. Exclusion criteria included the presence of uterine anomaly that may cause recurrent pregnancy losses, chronic systemic disease and the presence of antiphosopholipid and anticardiolipin antibodies. The patients in the study were evaluated for age, pregnancy history, parity, number of abortions, week of recurrent abortions, gynecological history, previous operations, the presence of systemic diseases (Diabetes mellitus, hypertension, chronic liver and kidney disease, autoimmune diseases etc.) bleeding diathesis, or symptoms suggesting thrombosis, and consanginous marriages.

Forty one patients fulfilled the inclusion criteria for habitual abortion and 50 controls matched for age without systemic disease and personal and familial history for thrombosis and had previous healthy pregnancies were included in the study. Informed consent was obtained from patients included in the study.

Ten ml. peripheral venous blood was withdrawn from study and control groups and transferred to EDTA tubes.This was investigated for Factor V Leiden and Factor II G20210A mutations by PCR in genetics laboratory. In order to determine whether genotype distributions reflect actual Mendelian population, namely if they are consistent with Hardy-Weinberg equation9, the frequency of G and A alleles in the groups were determined . The probability of each allele and GG,GA and AA genotypes occurring was calculated according to the computed frequency of alleles. Expected genotype frequencies were compared with those in the study and control groups. It was determined that distribution was in accord with Hardy-Weiberg equation (p>0.05).

To compare the obtained data of the control and habitual abortion groups, student's t test, in the comparison of categorical data chi square, Fischer-Exact and Mann Whitney U tests were used. In addition, odds ratio values and 95% confidence intervals are given to determine whether genotype distribution in two groups produces a risk. This study was approved by the ethics Committee of Ankara University.

The mean age was 29±4.66 years (20-38) in the control group and 32±5.54 (21-41) in the study group, with a statistically significant difference (p<0.05).When two groups were compared with respect to pregnancy, parity, abortus and live births, significant differences were found (p<0.001). The mean abortion week of the study group was 12.9±2.5 (5-24) weeks. When groups were compared in terms of marriage between relatives, it was observed in 6

of 50 women in control group and 12 of 41 women in the study group, with a statistically significant difference. (p<0.05) had consangenous marriages (Table 1).

Table 1. Charecteristics of patients and control groups.
	Control	H. Abortion	P-value
	(Mean±SD)	(Mean±SD)
Number of pregnancies	2.18±1.22	5.07±2.15	<0.001
Parity	1.38±0.75	0.71±1.08	<0.001
Number of abortions	0.24±0.43	3.90±1.92	<0.001
Number of live births	1.30±0.71	0.46±0.71	<0.001
Marriage with relative
Yes	6 (6%)	12 (29.3%)	<0.05
No	44	29	0.05
Age (years)	29+4.66	32+5.54	0.05

When the study and control groups were compared with respect to tendency to thrombosis, deep vein thrombosis history and bleeding diathesis, previous deep vein thrombosis was found in 3 of 41 women in the study group (7.2%), the difference with the control group being insignificant (p>0.05).

Phenotypes of 91 cases were determined by PCR for Factor V Leiden. It was established that of 41 cases in the study group, 31 (75.6%) had GG genotype, 9 (22%) had GA genotype and 1 ((2.4%) had AA genotype. In the control group, 45 (90%)of 50 women had GG Genotype and 5 (10%) had GA genotype. A allele carrier status was found to be 24.4% in study group and 10% in the control group. The difference between two groups was not statistically significant (p=0.06) (Table 2).

Table 2. Frequency of genotypes Factor V Leiden.
Factor V	Frequency of	Frequency of	Frequency of	Frequency
Leiden	GG genotype	GA genotype	AA genotype	of A Alelic
				genotype
Case (n=41)	31	9	1	10
	(75.6%)	(22%)	(2.4%)	(22.4%)
Control (n=50)	45	5	0	5
	(90%)	(10%)	(0%)	(10%)

Study and control groups were compared for Factor II G201210A mutation as well. While no patient in the study group was a carrier of this mutation, 2 patients (45) in the control group were found to have GA genotype, but this difference was not statistically sitgnificant (p>0.05) (Table 3).

Table 3. Frequency of genotypes Factor II G210A mutation.
Mutation of Factor II	Frequency of	Frequency of
G20210A	GG genotype	GA genotype
Case (n=41)	41 (100%)	0 (0%)
Contro (n=50)	48 (96%)	2 (4%)

When 41 patients in the study group were compared for reccurrence of abortion and whether their abortions were (primary or secondary, it was established that 25 (61%) had primary and 16 (39) secondary abortions. When 41 patients were evaluated for Factor V Leiden status, 7 (28%) with primary abortion and 3 (18.8%) with secondary abortions, they were found to carry A allele which is not meaningful (p>0.05).

The 41 patients of the study group were investigated for the relation between the Factor V Leiden carrier status and the number of abortions. The 31 patients with GG genotype had mean 3.87±1.92 abortions, 9 patients with GA genotype had mean 4.0±2.17 abortions and 1 patient with AA genotype had 3 abortions. Accordingly, no statistically significant relation was found between Factor V Leiden carrier status and the number of abortions (p>0.05). The 41 patients of the study group were compared with each other in terms of mean pregnancy week of abortions and A allele carrier status for Factor V Leiden. Twenty three patients had abortions in first trimester and five of them were carriers of Factor V Leiden mutation (21.7%). Five of 18 patients had Factor V Leiden mutations, whose losses occured in second trimester (27.8%). No statistically significant relation was found between the losses in 1 and 2 trimesters and Factor V Leiden mutation (p>0.05).

Uteroplacental circulation which is vital for succesful pregnancy outcome may be influenced by hemostatic disorders. Therefore, maternal thrombophilias are important for obstetrics.1

Impairment in uteroplacental circulation is an important factor in fetal losses.10 In patients with recurrent fetal losses, it has been demonstrated by many studies that there is an intimate relation between Factor V Leiden mutation and vascular placental deficiency.1,4,11 Factor V Leiden and Factor II G20210A mutations are more frequent in patients with habitual abortions compared to controls. These findings support the role of two mutations in the increasing risk of recurrent abortions and the idea that thrombophilia plays an important part in clinical picture.10 Forty one patients with two or more than two pregnancy losses were named idiopathic habitual abortion cases and 50 controls were included in the present study. The mean age of the patients in the control group was 29±4.66 years and that of the study group was 32±5.54 years. The statistically significant difference in age between groups ( habitual abortion group being older) may be related to late diagnosis of habitual abortion. However, as study material was a genetic product, this difference can be ignored. When case and control groups were compared in terms of Factor V Leiden and factor II G20210A mutations, it was seen that 9 patients with recurrent abortions were heterozygote for Factor V Leiden (22%) and one was homozygote (2.4%). Overall rate of A allele carriers was 24.4%. In the control group, heterozygosity for Factor V Leiden was determined in 5 patients (10%). This difference was not significantly different. Studies which were carried out to determine the prevalance of Factor V Leiden showed that it is between 7.1-9.8%.12,13

Zoka et al have reported that in a study on 80 habitual abortion patients,Factor V Leiden and Factor II G20210A mutation was seen in one out of four patients with fetal losses and this prevalance was higher in the abortions in second trimester.5 Factor IIG20210A mutation was more frequent in patients with fetal losses than in the control group. It has been reported that these patients have four times the risk for fetal loss. In the prevalance of this polymorphism, no difference has been obtained between patients with 2 abortions and 3 or more abortions.

Dizon-Towson et al have reported that no patient carried Factor V Leiden mutation in a study they carried out on 40 patients with three or more pregnancy losses ( 22 in the 1. trimester and 18 in the 2. trimester) and 25 controls, but one male partner carried it in each group. They have suggested that the status of male partner does not influence pregnancy outcome and larger studies should be performed.1,4 Drawing upon their study, we did not include male partners in this study.

In the present study, 23 of 41 patients were the ones with first trimester losses (56.1%), whereas 18 (43.9%) patients had fetal losses in the second trimester. Factor V Leiden carrier rate was 21.7% (5/23) in patients with fetal losses in the first trimester, whilst it was 27.8% (5/18) in those with losses in the second trimester. The majority of women with recurrent fetal losses were those with first trimester losses while Factor V Leiden carrrier status was more frequent in women with 2. trimester losses, but this difference was not statistically significant. Our control patients did not have any thrombosis history. Nevertheless, the prevalance of carrier status was determined to be 10%. In 2 of 41 patients in the study group, suspicious thrombosis history was elicited, yet their genetic analysis revealed that they were negative for Factor V Leiden mutation. In the study period, one patient was diagnosed with active deep vein thrombosis and Factor V genotype was homozygous for mutation, namely it was AA genotype.

Wramsky attempted to determine the prevalence of G1691 mutation in Factor V gene, C677T mutation in MTHFR gene and to analyse G20210A polymorphism in Factor II gene and found that Factor V Leiden mutation prevalence was higher in patients with primary habitual abortion and that the prevalance of Factor II G20210A mutation and MTHFR gene C677T mutation carriers was not significantly different between patients with fetal losses and control group. As to Factor V Leiden, they have reported that patients with primary abortion carry a higher risk than those with secondary abortions.14

Of 41 patients in the study, 25 (61%) had primary abortions whereas 16 (395) had secondary abortions. The patients with primary abortions constituted 28% of Factor V Leiden mutation carriers (7/10). The prevalance of Factor V Leiden was 18.8% (3/10) in women with secondary abortions. Although, the rate was higher in those with primary abortions, the difference was not statistically significant. These findings are consistent with those of Kutteh et al and discordant with those of Wramsky et al.14

Factor V Leiden mutation carrier status was more frequent in women with second trimester abortions. Although there were more abortions in the first trimester may be these are loss occurred in 7 of 12 patients married with relatives and abortions may be related to other genetic anomalies. Fetal loss is related to placental thrombosis in carriers of Factor V Leiden. This view is supported by the observation of placental infarct in abortion material in various studies.1,4 Dizon Townson et al have demonstarted that in the abortion material of women with recurrent abortions, the rate of placental infarct increases in fetuses with Factor V Leiden. In addition, it has been reported that the rate of spontaneous abortion increases in fetuses heterozygote for factor V Leiden.4

In conclusion, it has been observed that in Turkish women with recurrent abortions at the first and second trimester of pregnancy, although Factor V Leiden was more frequent than control group, the difference was not statistically significant. Factor II G20210A mutation was not observed in patients with fetal losses while it was observed in control group. Our findings demonstrate that the presence of Factor V Leiden mutation, and Factor II G20210A mutations can not account for the etiology of abortions in patients with no explicable reason for recurrent fetal losses. Larger prospective studies are required to elucidate this issue.

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3. Brenner B, Mandel H, Lanir N, Younis J, Rothbart H, Ohel G, et al. Activated protein C resistance can be associated with recurrent fetal loss. Br J Haematol 1997;97:551-4 .

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5. Foka ZJ, Lambropoulos AF, Saravelos H, Karas GB, Karavida A, Agorastos T. Factor V Leiden and prothrombin G20210A mutations, but not methylenetetrahydrofolate reductase C677T are associated with recurrent miscarriages. Hum Reprod 2000;15:458-62 .

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10. Brigden ML. The hypercoagulable state who how and when to test and treat. Postgrad Medicine 1997;101:249-52, 254-6, 259-62 passim

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