Hypofibrinogenemia and the α-Fibrinogen Thr312Ala Polymorphism may be Risk Factors for Early Pregnancy Loss

Abstract

We analyzed a cohort of 36 females with pregnancy loss. In addition to 11 patients with antiphospholipid antibody syndrome and 2 patients with congenital antithrombin (AT) or protein C deficiency, we identified 5 patients with low fibrinogen levels (median 110 mg/dL) prior to 10 weeks of gestation. Four of these 5 patients underwent a fibrinogen gene analysis, and all 4 were found to be heterozygotes for the α-fibrinogen (FGA) Thr321Ala polymorphism. One female without hypofibrinogenemia with a history of 8 pregnancy losses was found to be homozygous for the same polymorphism, and she also showed hypercoagulability without thrombosis. In conclusion, there was a relatively high frequency of pregnancy loss in the setting of hypofibrinogenemia and/or the FGA Thr312Ala polymorphism, and this may be an important risk factor for pregnancy loss and a hypercoagulable state in later pregnancy.

Introduction

Pregnancy is characterized as a proinflammatory and hypercoagulable state, with miscarriage occurring in approximately 15% of pregnancies. In contrast, recurrent miscarriage is a rather rare condition with an estimated incidence of 1% to 3%. ¹ Although genetic factors are the major causes of spontaneous miscarriage, they are less frequently associated with recurrent miscarriage. Although autoimmune-based recurrent miscarriage has been described, including primarily antiphospholipid antibody syndrome (APS), ^2,3 the role of alloimmune mechanisms remains poorly understood. The current treatment of patients with APS during pregnancy includes heparin and aspirin, which prevent clot formation and subsequently improve live birth rates. Nevertheless, other obstetric morbidities remain high, especially in patients with a history of thrombotic events. ² In addition to classical thrombotic placental events, other factors involving inflammation and complement activation appear to play a role in certain complications. However, the influence of parameters associated with congenital thrombophilia, such as abnormalities in antithrombin (AT), protein C (PC), or protein S (PS), ⁴ is controversial. ¹

The α-fibrinogen (FGA) Thr312Ala ⁵ polymorphism occurs in a region important for factor XIII-dependent cross linking ⁶ and may therefore affect the clot structure or the degree of rigidity. Hypofibrinogenemia is caused by a variety of structural abnormalities in the fibrinogen molecule which result in a tendency toward bleeding and thrombosis as well as obstetric complications. The obstetric complications of hypofibrinogenemia include first-trimester pregnancy loss, hemorrhage, placental abruption, and thrombosis. ^7,8 A significant association between FGA Thr312Ala and poststroke mortality was observed in patients with atrial fibrillation, with increased mortality in those possessing Ala312 versus those homozygous for Thr312. ⁹ This fibrinogen polymorphism is also associated with venous thromboembolism (VTE). ¹⁰

In this study, hemostatic abnormalities were examined in 36 pregnant females who experienced pregnancy loss, and gene analyses for fibrinogen were performed in 4 of 5 patients with hypofibrinogenemia.

Materials and Methods

Thirty-six women who had experienced pregnancy loss were examined at Mie University School of Medicine between January 1, 2007, and February 28, 2015. The median age (25%-75% tile) was 32.5 years (30.0-36.0 years), and the median number of abortions was 2.0 (1-3). There were 11 patients with APS, 2 patients with systematic lupus erythematosus, 1 patient with idiopathic thrombocytopenic purpura (ITP), 1 patient with Sjögren’s syndrome, 1 patient with congenital AT deficiency, 1 patient with congenital PC deficiency, and 1 patient with plasminogen deficiency (Table 1). The hemostatic abnormalities were also examined in 20 patients with ITP without pregnancy loss with a median age of 30.0 years (28.0-32.8 years). The diagnosis of APS was made according to the established criteria. ¹¹ The fibrinogen level which was less than 150 mg/dL either before or in the very early phase of pregnancy in the patients without liver dysfunction or disseminated intravascular coagulation was considered to indicate a state of hypofibrinogenemia. The study protocol was approved by the Human Ethics Review Committee of the Mie University School of Medicine, and signed informed consent was obtained from each participant. The study was faithfully carried out in accordance with the principles of the Declaration of Helsinki.

Table 1.

Patients With Pregnancy Loss.

	Age, yr	Pregnancy Loss	Pregnancy	Family History for Pregnancy Loss	Underlying Diseases	Fibrinogen, mg/dL	UFH Treatment
1	27	2	3	None	-	198	1
2	36	2	3	None	SLE, APS	325	1
3	30	2	3	None	ITP, APS	213	1
4	27	1	2	None	Congenital AT deficiency	200	1
5	40	5	5	None	-	286	1
6	26	1	3	None	Pregnant hypertension	240	1
7	30	2	3	None	Hypofibrinogenemia	147	1
a8	33	3	5	Yes	Hypofibrinogenemia	137	1
9	40	2	2	None	Congenital PC deficiency	449	0
10	32	2	3	None	-	250	1
11	39	3	4	None	-	388	1
12	27	3	4	None	Sjögren syndrome	170	1
13	28	2	2	None	-	261	0
14	35	2	4	None	SLE, APS	405	2
15	31	3	4	None	-	170	0
16	31	2	3	None	-	319	1
17	40	2	3	None	APS	320	1
18	30	1	3	Yes	-	332	1
19	54	3	6	None	Plasminogen deficiency	340	0
20	30	1	3	None	-	367	2
21	23	1	3	None	-	272	1
22	39	4	4	None	Acquired AT deficiency	333	1
23	36	3	4	None	APS	246	1
24	32	2	3	None	Hypofibrinogenemia	110	0
25	36	2	3	None	Hypofibrinogenemia	102	1
26	31	2	4	None	APS	375	3
27	33	3	4	None	APS	245	1
28	30	2	3	None	APS	282	1
29	28	3	5	None	-	230	2
30	34	3	4	None	APS	293	1
31	37	1	2	None	-	340	1
32	33	1	2	None	-	352	1
33	33	1	2	None	APS	221	1
34	33	2	2	None	APS	278	0
a35	29	2	3	Yes	Hypofibrinogenemia	104	１
36	36	8	9	No		260	1

Abbreviations: UFH, unfractionated heparin; APS, antiphospholipid syndrome; SLE, systemic lupus erythematosus; ITP, idiopathic thrombocytopenic purpura; AT, antithrombin; PC, protein C.

^aCases 8 and 35 are sisters.

The level of lupus anticoagulant (LA) was measured using diluted Russell viper venom time based on a LA test, the “Guardipore” (Medical and Biological Laboratories CO, LTD [MBL], Nagoya, Japan). The titers of anticardiolipin immunoglobulin G (IgG) antibodies (aCL-IgG) and anticardiolipin β2-GPI complex antibodies were measured according to an enzyme-linked immunosorbent assay using an MESACUP cardiolipin IgG test (MBL) and anti-CL β2GPI kit Yamasa enzyme immunoassay (Yamasa Shoyu Co, Tyoushi, Japan). ¹² The concentration of fibrinogen was measured by thrombin time method using a datafi fibrinogen kit (Sysmex, Kobe, Japan), a turbidimetric immunoassay (TIA) using a N-assay TIA Fib kit (Nittobo, Koriyama, Japan), and the heat precipitation method. ^11,13

A gene analyses of all 3 fibrinogen genes (FGA, β-, and γ-fibrinogen, respectively) was performed using the cycle sequencing method. Genomic DNA was prepared from peripheral blood leukocytes using a QIAamp DNA Blood Mini Kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. Each exon, including the exon–intron boundary of the gene, was amplified from genomic DNA via polymerase chain reaction (PCR). The PCR products were directly sequenced using a Big-Dye Terminator Cycle Sequencing Kit and a 310 or 3130 Genetic Analyzer (Applied Biosystems, Foster City, California).

Statistical Analysis

The data are expressed as the median and 25th to 75th percentiles. Differences between the groups were examined for significance using the McNemar test. A P value of less than .05 was considered to indicate a significant difference. All statistical analyses were performed using the Stat flex, version 6, software package (Artec Co Ltd, Osaka, Japan).

Results

There were 11 patients with APS who had experienced pregnancy loss. Ten of the 11 patients with APS were pregnant and received treatment with aspirin and unfractionated heparin (UFH), and all patients either had a healthy baby or maintained a normal pregnancy (Table 1). Although no relationships between positive findings for antiphospholipid antibody (aPL) and the number of pregnancy losses were observed, the patients triple positive results for aPL-developed thrombosis (Table 2). In 35 cases, the pregnant females were treated with UFH because of elevation in d-dimer levels, 32 of whom had a healthy baby, while 1 patient with acquired AT deficiency experienced early delivery. Two females are still pregnant and are being treated with UFH. The patient with congenital AT deficiency presented with cerebral venous sinus thrombosis and deep vein thrombosis during her first pregnancy and consequently experienced pregnancy loss (case 4). One patient with congenital PC deficiency experienced abortion twice and associated cerebral venous sinus thrombosis without pregnancy (case 9). In case 22, the AT concentration was within the normal range prior to pregnancy and decreased after pregnancy. That patient displayed recurrent deep vein thrombosis and experienced 4 pregnancy losses after 20 weeks of gestation due to thrombosis. In addition, there were 5 females with hypofibrinogenemia (cases 7, 8, 24, 25, and 35), and all 5 patients in 4 families (cases 8 and 35 are sisters) experienced pregnancy loss before 10 weeks of gestation (Table 3). Five females showed hypofibrinogenemia in the early pregnancy phase and 1 month after delivery, suggesting to be a heterozygous of fibrinogen deficiency. In case 24 and her sister, although no gene analysis of fibrinogen was performed due to the request of the sister, the ratio of activity to antigen was 2:1, thus suggesting that the patient most likely had dysfibrinogenemia. In the gene analyses of 4 patients with hypofibrinogenemia, all patients were found to have a heterozygous of FGA Thr312Ala. In case 25, the patient had 4 other mutations in fibrinogen; however the functions of these mutations were not clear (Table 4). The patients in cases 8 and 35 were sisters, both of whom had a hypofibrinogenemia and experienced pregnancy loss before 10 weeks of gestation. They were treated with UFH due to hypercoagulability during their second pregnancies. Meanwhile, the plasma level of soluble fibrin (SF) in case 8 was significantly increased at 27 weeks of gestation and then decreased after treatment with UFH. Both patients had healthy babies after receiving UFH (Figure 1). Finally, 4 recent pregnant females who had hypofibrinogenemia were treated with UFH after 10 weeks of gestation and a favorable pregnancy outcome was thus obtained. A gene analysis of AT and fibrinogen was carried out to investigate the cause of pregnancy loss in case 36 without hypofibrinogenemia who experienced pregnancy loss 8 times. The patient had no abnormality for AT gene, but she was found to be homogyzous for FGA Thr312Ala, and therefore she was treated with UFH due to hypercoagulability.

Figure 1.

Plasma levels of SF, d-dimer, and fibrinogen in case 8. UFH indicates unfractionated heparin.

Table 2.

Antiphospholipid Antibodies.

Case	LA	aCL-β2GPI-IgG	aCL-IgG	Abortion	Thrombosis
2	0.9	5.7	17	2	None
3	0.98	2.3	8	2	None
14	1	1.5	5	2	None
17	1	<1.3	10	2	None
23	0.91	<1.3	16	3	None
26	0.9	4.3	28	2	None
27	0.9	<1.3	19	3	None
28	0.99	<1.3	<8	2	None
30	1.75	75.9	22	2	Cerebral thrombosis
33	1.85	117	67	1	Skin ulcer
34	0.9	<1.3	13	2	None

Abbreviations: aCL-IgG, anticardiolipin immunoglobulin G antibody; LA, lupus anticoagulant.

Table 3.

Period of Pregnancy Loss.

	Pregnancy Loss	Abortion Period
		First	Second	Third	Fourth	Fifth	Sixth	Seventh	Eighth
1	2	12	6
2	2	28	6
3	2	10	8
4	1	8
5	5	8	6	8	8	8
6	1	34
7	1	10
8	3	6	8	6
9	2	9	10
10	2	7	9
11	3	6	6	6
12	3	6	6	6
13	2	8	6
14	2	8	12
15	3	16	16	8
16	2	9	10
17	2	8	8
18	1	32
19	3	8	10	11
20	1	9
21	1	37
22	4	18	26	20
23	3	8	7	9
24	2	9	10
25	2	8	8
26	2	7	8
27	3	5	7	7
28	2	9	6
29	3	6	8	7
30	3	19	8	9
31	1	10
32	1	9
33	1	8
34	2	8	8
35	2	8	7
36	8	5	9	20	4	4	12	5	16

Table 4.

Patients With Hypofibrinogenemia and Case 36.

Case	Age, yr	TIA Method, mg/dL	Thrombin Time, mg/dL	Heat Coagulation, mg/dL	Pregnancy Loss	Amino Acid Mutated
7	30	–	147	148	1	FGA Thr312Ala (heterozygous)
8	33	139	137	145	3	FGA Thr312Ala (heterozygous)
24	32	–	110	220	2	Not done
25	36	110	102	118	2	FGA Ile6-14Val (heterozygous); FGA Thr312Ala (heterozygous); FGBPro235Leu (heterozygous); FGB448Lys (heterozygous); and FGGArg275His (heterozygous)
35	29	120	104	110	2	FGA Thr312Ala (heterozygous)
36	36	–	260	–	7	FGA Thr312Ala (homozygous)

Abbreviations: FGA, α-fibrinogen; FGB, β-fibrinogen; FGG, γ-fibrinogen; TIA, turbidimetric immunoassay.

There were no cases of hypofibrinogenemia, APS, AT deficiency, or PC deficiency among the 20 pregnant females with ITP without pregnancy loss, and the frequency of combination treatment with hypofibrinogenemia and FGA Thr312Ala was significantly higher (P < .001) in the females with pregnancy loss than in the pregnant females with ITP without pregnancy loss.

According to an analysis of the frequency of FGA Thr312Ala, 13 (86.7%) of 15 patients had pregnancy loss, 20 (83.3%) of 24 patients had deep vein thrombosis (DVT), and 40 (81.6%) of 49 patients had prosthetic valves.

Discussion

In this study, 11 patients exhibited APS, suggesting that APS is one of the most important causes of recurrent miscarriage. A causative relationship between aPLs and recurrent early miscarriage (prior to 10 weeks of gestation) is supported by many studies, ¹⁴ and treatment with heparin and low-dose aspirin is associated with favorable pregnancy outcomes. ¹⁵ In the current study, UFH therapy was associated with a favorable pregnancy outcome in 34 of 35 cases, including 2 cases who have successfully maintained their pregnancy after UFH treatment. These findings suggest that the status of aPL is not related to the frequency of abortion, but triple positive finding for aPL is associated with thrombosis. Abnormal AT or PC levels have also been suggested to correlate with thrombosis but not directly with pregnancy loss. The findings of 5 pregnant females in 4 families with hypofibrinogenemia among 36 pregnant females who experienced pregnancy loss indicate a markedly high frequency. All 5 patients had pregnancy loss before 10 weeks of gestation. Although these patients were not treated with fibrinogen concentrate, fibrinogen concentrate may be recommended for females with a fibrinogen level of less than 150 mg in very early phase of pregnancy. In addition, UFH should not be administrated to pregnant women with a fibrinogen level of less than 150 mg/dL. Although the relationship between a heterozygous status for afibrinogenemia and pregnancy loss has not been reported, the relationship combination of hypofibrinogenemia and heterozygous of FGA Thr312Ala with pregnancy loss has not been clarified. Because the plasma fibrinogen level increases in pregnancy, it is difficult to diagnose hypofibrinogenemia in pregnant females.

In the gene analyses of 4 patients with hypofibrinogenemia, all patients were found to have a heterozygous of FGA Thr312Ala. One female with homozygous FGA Thr312Ala experienced pregnancy loss 7 times. These findings suggest that more than 2 abnormalities of fibrinogen may make it difficult to maintain the placenta in a good condition.

This fibrinogen polymorphism has been reported to be a strong risk factor, such as intracerebral hemorrhage, ¹⁶ chronic thromboembolic pulmonary hypertension, ¹⁷ and VTE ¹⁸ as well as myocardial infarction and ischemic stroke. ¹⁹ In Japan, FGA Thr312Ala in cases involving pregnancy loss is rarely reported. The frequency of FGA Thr312Ala polymorphism was reported to be 46.5% in Japanese ²⁰ and 29.9% in Europeans. ²¹ According to the findings of our study, this frequency was high in patients with pregnancy loss, those with DVT, and those with prosthetic valves. Complications with FGA Thr312Ala and hypofibrinogenemia were observed in all 4 patients who had pregnancy loss, thus suggesting that such complications may be closely associated with pregnancy loss.

The patients in cases 8 and 35 were sisters, both of whom experienced pregnancy loss in the early period of pregnancy and consequently received UFH treatment for hypercoagulability in later pregnancies. In addition, both patients experienced a normal delivery after receiving treatment with UFH, suggesting that this fibrinogen abnormality may cause both bleeding tendency and thrombosis.

In conclusion, the frequency of complications with FGA Thr312Ala and hypofibrinogenemia is relatively high, and such complications may be associated with pregnancy loss in early pregnancy and hypercoagulability in later pregnancy.