**Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups for Thrombophilia Testing**

Ricardo Barini, Joyce Annichino-Bizzache, Egle Couto, Marcelo Luis Nomura, Adriana Goes Soligo and Isabela Nelly Machado

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56819

**1. Introduction**

Normal pregnancy is associated with complex changes of hemostasis, leading to hypercoa‐ gulability states. Such physiological increase of blood coagulation during pregnancy occurs because of changes in the vascular endothelium and blood flow, generating changes from the 10th gestational week on. The changes may create a hypercoagulability state that results in thrombosis. The purpose of the hypercoagulability state during pregnancy is to prevent excessive bleeding by the time of delivery (Moreira, et al. 2008).

Normal hemostasis during pregnancy is the result of a balance between the system that promotes blood coagulation and the one inhibiting excessive coagulation (fibrinolytic system).

Gestational effects on coagulation proteins may be detected after the 3rd month of pregnancy, with significant changes in pro-coagulant proteins in comparison with physiological inhibi‐ tors. So, although changes in hemostatic system are intended for adaptation and protection of the pregnant woman's body, they may cause increased risk of thromboembolic events.

Despite of being physiological, excessive activation of the coagulation mechanism during pregnancy may lead to thromboembolic events, especially in women with hereditary and/or acquired factors who have a known predisposition to thrombi formation.

Thrombophilia is a hereditary or acquired disease related to changes in hemostasis mechanisms that are characterized by an increased trend to blood coagulation and

consequent risk of thromboembolism (Machac S 2006). Hereditary factors that are consid‐ ered potentially responsible for such trend to thrombosis are: protein C deficiency, protein S deficiency, anti-thrombin deficiency, presence of Factor V Leiden, a change in allele prothrombin 20210 G>A gene and a change in the gene of enzyme methylenetetrahydrofo‐ late reductase (D'Amico 2006).

Women are exposed to a great variety of factors that increase their risk of venous throm‐ boembolism (VTE) such as the use of hormonal contraception, pregnancy, puerperium and

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

41

The incidence of VTE is higher in woman during pregnancy and post partum when compared to not pregnant women. There is an increase between five to ten times the risks of VTE, with

Although there are controversies whether the occurrence of VTE is greater during pregnancy or post partum, it seems that this risk is equally distributed during all gestational period (Pomp

Hereditary thrombophilia include Factor V Leiden (FVL), G20210A prothrombin gene

Prevalence of a hereditary thrombophilia is higher in women that had VTE during pregnancy, especially FVL and G20210A gene mutation. In Japanese populations, protein S seems to be

Normal gestation is characterized by hypercoagulation, with increase of coagulation factors II, VII, IX, X, XII, fibrinogen and Von Willebrand factor. There is a reduction in natural anti coagulant factors, such as protein S, protein C and antithrombin III. There is also a reduction in fibrinolysis caused by reduction in tissue plasminogen activating factor (t-PA) and increase in the inhibitor of plasminogen activator (PAI-1). Increase of Factor VIII and reduction of protein S lead to a resistance to activated protein C. Thus, all these changes in pregnancy favor

After a VTE antecedent, thrombophilia is the most important individual risk factor for a new

Besides VTE, recurrent abortions and other complications during pregnancy can be associated

A recent meta analysis has shown that a pregnant woman with thrombophilia has a greater risk than non pregnant woman, particularly in the presence of homozygosis to Leiden Factor V, to G20210A prothrombin gene mutation, in the presence of double heterozygosis for these two mutations and in the presence of antithrombin III deficiency. All of these thrombophilias, except MTHRF 677C>T gene mutation, even in homozygosis, bring about a statistically higher

The predictive value for the risk of VTE relating pregnancy and thrombophilia is described on

Although, the absolut risk of VTE is low due to the low incidence of VTE itself. Even the risk of VTE in more severe thrombophilia, such as antithrombin III deficiency and protein S

to thrombophilia, particularly protein S deficiency and late complications.

an incidence of 0.6 to 1.3 events for 1.000 deliveries (Heit JA 2005) (McColl MD 1997).

ER 2008). VTE is the most important cause of maternal death (Marik PE 2008).

mutation, protein S, protein C and antithrombin III deficiency.

the most common among pregnant women with VTE (Miyata T 2009).

hormonal replacement therapy for menopause.

VTE.

thrombotic episode during gestation.

table 1. (L. B. Pierangeli SS 2011).

deficiency is low.

risk of VTE during pregnancy (Robertson L 2006).

Thrombophilia hereditary causes have been researched since 1956, when Jordan and Nandorff introduced the term thrombophilia. In 1965 anti-thrombin deficiency was identified as the genetic cause of thrombophilia.

Such studies became larger in the 80's, when protein S and protein C deficiencies were described, as well as Factor V Leiden, in 1994. (Reistma PH 2007). Approximately 40% of thrombosis cases showing arterial occlusion or venous occlusion are hereditary. Venous thromboembolism frequently occurs as a result of several factors. Generally, thrombophilia should be seen as a multi-factorial disorder, not as the expression of a single genetic change (Buchholz T 2003).

The importance of angiogenesis for embryo implantation and the presence of thrombophilia leading to micro-thrombosis at the implantation site with subsequent impairment of embryo nidation and placental development should be considered as well (Vaquero E 2005).

The presence of thrombophilia was proven to be related to an increased risk of complica‐ tions during pregnancy, such as pre-eclampsia, intrauterine growth restriction, premature detachment of placenta, preterm delivery, recurrent miscarriage, chronic fetal distress, besides ischemic events during pregnancy (Couto E 2005) (Ren A 2006) (Hoffman E 2012) (Bennet SA 2012).

Events related to thrombophilic changes during pregnancy are shown below.

## **2. Thrombophilia and pregnancy**

One of the most important discussions in clinical practice regards the indication to search for a thrombophilic factor. This is due to elevated testing costs and its relevance to medical management once diagnose is done.

Access to Internet and available information on practically all matters brings up questioning by patients who look for these data regarding their personal risks and ask doctors how would they have to behave. It is an important role for doctors to help patients to discriminate which information are relevant for them, helping patient to pursue adequate options for personal treatment and prophylaxis.

There has been a great deal of research relating thrombophilia to many clinical situations where no scientific data is relevant. On the other side, there are still other clinical situations where there is no consensus, or even there will not be a practical condition to define a medical practice, based on studies performed up until now.

Women are exposed to a great variety of factors that increase their risk of venous throm‐ boembolism (VTE) such as the use of hormonal contraception, pregnancy, puerperium and hormonal replacement therapy for menopause.

consequent risk of thromboembolism (Machac S 2006). Hereditary factors that are consid‐ ered potentially responsible for such trend to thrombosis are: protein C deficiency, protein S deficiency, anti-thrombin deficiency, presence of Factor V Leiden, a change in allele prothrombin 20210 G>A gene and a change in the gene of enzyme methylenetetrahydrofo‐

Thrombophilia hereditary causes have been researched since 1956, when Jordan and Nandorff introduced the term thrombophilia. In 1965 anti-thrombin deficiency was identified as the

Such studies became larger in the 80's, when protein S and protein C deficiencies were described, as well as Factor V Leiden, in 1994. (Reistma PH 2007). Approximately 40% of thrombosis cases showing arterial occlusion or venous occlusion are hereditary. Venous thromboembolism frequently occurs as a result of several factors. Generally, thrombophilia should be seen as a multi-factorial disorder, not as the expression of a single genetic change

The importance of angiogenesis for embryo implantation and the presence of thrombophilia leading to micro-thrombosis at the implantation site with subsequent impairment of embryo

The presence of thrombophilia was proven to be related to an increased risk of complica‐ tions during pregnancy, such as pre-eclampsia, intrauterine growth restriction, premature detachment of placenta, preterm delivery, recurrent miscarriage, chronic fetal distress, besides ischemic events during pregnancy (Couto E 2005) (Ren A 2006) (Hoffman E 2012)

One of the most important discussions in clinical practice regards the indication to search for a thrombophilic factor. This is due to elevated testing costs and its relevance to medical

Access to Internet and available information on practically all matters brings up questioning by patients who look for these data regarding their personal risks and ask doctors how would they have to behave. It is an important role for doctors to help patients to discriminate which information are relevant for them, helping patient to pursue adequate options for personal

There has been a great deal of research relating thrombophilia to many clinical situations where no scientific data is relevant. On the other side, there are still other clinical situations where there is no consensus, or even there will not be a practical condition to define a medical practice,

nidation and placental development should be considered as well (Vaquero E 2005).

Events related to thrombophilic changes during pregnancy are shown below.

late reductase (D'Amico 2006).

40 Pregnancy Thrombophilia - The Unsuspected Risk

genetic cause of thrombophilia.

(Buchholz T 2003).

(Bennet SA 2012).

**2. Thrombophilia and pregnancy**

based on studies performed up until now.

management once diagnose is done.

treatment and prophylaxis.

The incidence of VTE is higher in woman during pregnancy and post partum when compared to not pregnant women. There is an increase between five to ten times the risks of VTE, with an incidence of 0.6 to 1.3 events for 1.000 deliveries (Heit JA 2005) (McColl MD 1997).

Although there are controversies whether the occurrence of VTE is greater during pregnancy or post partum, it seems that this risk is equally distributed during all gestational period (Pomp ER 2008). VTE is the most important cause of maternal death (Marik PE 2008).

Hereditary thrombophilia include Factor V Leiden (FVL), G20210A prothrombin gene mutation, protein S, protein C and antithrombin III deficiency.

Prevalence of a hereditary thrombophilia is higher in women that had VTE during pregnancy, especially FVL and G20210A gene mutation. In Japanese populations, protein S seems to be the most common among pregnant women with VTE (Miyata T 2009).

Normal gestation is characterized by hypercoagulation, with increase of coagulation factors II, VII, IX, X, XII, fibrinogen and Von Willebrand factor. There is a reduction in natural anti coagulant factors, such as protein S, protein C and antithrombin III. There is also a reduction in fibrinolysis caused by reduction in tissue plasminogen activating factor (t-PA) and increase in the inhibitor of plasminogen activator (PAI-1). Increase of Factor VIII and reduction of protein S lead to a resistance to activated protein C. Thus, all these changes in pregnancy favor VTE.

After a VTE antecedent, thrombophilia is the most important individual risk factor for a new thrombotic episode during gestation.

Besides VTE, recurrent abortions and other complications during pregnancy can be associated to thrombophilia, particularly protein S deficiency and late complications.

A recent meta analysis has shown that a pregnant woman with thrombophilia has a greater risk than non pregnant woman, particularly in the presence of homozygosis to Leiden Factor V, to G20210A prothrombin gene mutation, in the presence of double heterozygosis for these two mutations and in the presence of antithrombin III deficiency. All of these thrombophilias, except MTHRF 677C>T gene mutation, even in homozygosis, bring about a statistically higher risk of VTE during pregnancy (Robertson L 2006).

The predictive value for the risk of VTE relating pregnancy and thrombophilia is described on table 1. (L. B. Pierangeli SS 2011).

Although, the absolut risk of VTE is low due to the low incidence of VTE itself. Even the risk of VTE in more severe thrombophilia, such as antithrombin III deficiency and protein S deficiency is low.


A prospective study with 134 pregnant women heterozygous for FVL failed to show an increase in the incidence of VTE. Thus, although being the most common hereditary throm‐ bophilia, its search is not to be indicated indiscriminately on every pregnant patient, nor

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

43

A recently published meta-analysis including ten prospective studies showed only a small absolute risk for fetal death with demonstration of a small absolut risk with a smaller risk of

It is suggested based on the experience of many studies that generalized search for thrombo‐ philia it is not indicated during pregnancy, except for patients with recent VTE event, or punctual search of a familial or personal in one family member. History of VTE is the most

Although thrombophilia are associated to an increase to the relative risk of complications

Pregnancy by itself is considered a hypercoagulation state. Likewise for diabetes, it is expected to increase venous or arterial thromboembolism during pregnancy. The increase in estrogen levels leads to increase in many of the coagulation factors. Thus, in the presence of a hereditary or acquired thrombophilia a higher incidence of thrombotic events could be expected. Once a thrombophilic state is already identified, than we can expect a higher chance of clinical and obstetrical complications, except if prophylactic or therapeutic treatments are offered by caring

Although this is a disseminated belief, systematic reviews fail to demonstrate such strong relation. As stated above, only patients with a familial history of thrombosis or thrombophilia

However, this is not universally agreed on the literature. For instance, a group of patients with Leiden Factor V were compared to a group of absent mutation. It was observed that the group with the mutation had no thromboembolic events, whereas the group without had 2,7%

It has been also stated that even in relatives of probands who have no history of venous thromboembolism (VTE) should not receive antithrombotic prophylaxis during pregnancy because no difference was seen in the group with and without positive history of VTE prior to pregnancy (Cordoba I 2012). With theses considerations, one must look for evidences

Pregnancy is a clinical situation associated with increased risk of VTE, which increased from twofold to fourfold when these women presented a positive family history of VTE (Bezemer

prophylaxis is to be offered to asymptomatic carriers (Dizon-Townson D 2005).

fetal death on heterozygous FVL (Rodger MA 2010).

during pregnancy, including VTE, the absolut risk is still low.

**3. Clinical interferences of thrombophilia in pregnancy**

indicated event to look for thrombophilia.

physicians and followed by patients.

thromboembolism (Dizon-Townson D 2005).

**3.1. Thrombophilia and venous thromboembolism**

should be investigated.

presented to date.

**Table 1.** Predictive values of hereditary thrombophilias and VTE.

Medical management of pregnant women with thrombophilia will depend on the risk analysis; which may be complicated, because medical actions are based upon retrospective studies, meta-analysis or case control studies.

The evaluation of other risk factors for VTE, personal and familial VTE history (first degree relatives with VTE or arterial disease at age under fifty years old) should be taken into account seriously.

Hereditary thrombophilia can be classified in thee risk categories (Fogerty AE 2009):


As it is observed, personal or familial history of VTE has a strong weight on the VTE risk implication.

A pregnant woman with thrombophilia that presents with VTE during pregnancy has to be treated as a woman without thrombophilia with VTE. Treatment is based on heparin, partic‐ ularly low molecular weight heparin, with doses adjusted by maternal weight. Warfarin can be exceptionally considered after the first trimester up till 34 weeks gestation. Fondaparinux has already been used in pregnant patients who were unable to use other heparin.

Anticoagulation is recommended for six weeks after delivery for all women with hereditary thrombophilia. During gestation, individual risk should be considered. Patients with high risk should receive heparin prophylaxis as a treatment doses or intermediate dose. Patients with intermediate risk should receive prophylactic heparin dose. Low risk patients should be followed up carefully with strict recommendations to look for medical assistance in the event of any symptoms that can be related to VTE.

Thrombophilia should be searched in any patient with familial history or personal history of VTE or with the diagnosis of a hereditary thrombophilia on a first degree relative.

A prospective study with 134 pregnant women heterozygous for FVL failed to show an increase in the incidence of VTE. Thus, although being the most common hereditary throm‐ bophilia, its search is not to be indicated indiscriminately on every pregnant patient, nor prophylaxis is to be offered to asymptomatic carriers (Dizon-Townson D 2005).

A recently published meta-analysis including ten prospective studies showed only a small absolute risk for fetal death with demonstration of a small absolut risk with a smaller risk of fetal death on heterozygous FVL (Rodger MA 2010).

It is suggested based on the experience of many studies that generalized search for thrombo‐ philia it is not indicated during pregnancy, except for patients with recent VTE event, or punctual search of a familial or personal in one family member. History of VTE is the most indicated event to look for thrombophilia.

Although thrombophilia are associated to an increase to the relative risk of complications during pregnancy, including VTE, the absolut risk is still low.

## **3. Clinical interferences of thrombophilia in pregnancy**

**Thrombophilia PPV\*** FVL heterozygous state 1:500 prothrombin 20210 G>A heterozygous state 1:200 Double heterozygous state FVL + G20210A 4.6:100 Protein C deficiency 1:113 Antithrombin III deficiency 1:2.8

Medical management of pregnant women with thrombophilia will depend on the risk analysis; which may be complicated, because medical actions are based upon retrospective studies,

The evaluation of other risk factors for VTE, personal and familial VTE history (first degree relatives with VTE or arterial disease at age under fifty years old) should be taken into account

**•** High risk: FVL homozygosis, prothrombin 20210 G>A gene mutation in homozygosis, double heterozygosis of FVL and prothrombin 20210 G>A, antithrombin III deficiency or

**•** Intermediate risk: thrombophilia not classified as high risk with family history of VTE. **•** Low risk: heterozygosis for FVL, for prothrombin 20210 G>A, protein C and protein S

As it is observed, personal or familial history of VTE has a strong weight on the VTE risk

A pregnant woman with thrombophilia that presents with VTE during pregnancy has to be treated as a woman without thrombophilia with VTE. Treatment is based on heparin, partic‐ ularly low molecular weight heparin, with doses adjusted by maternal weight. Warfarin can be exceptionally considered after the first trimester up till 34 weeks gestation. Fondaparinux

Anticoagulation is recommended for six weeks after delivery for all women with hereditary thrombophilia. During gestation, individual risk should be considered. Patients with high risk should receive heparin prophylaxis as a treatment doses or intermediate dose. Patients with intermediate risk should receive prophylactic heparin dose. Low risk patients should be followed up carefully with strict recommendations to look for medical assistance in the event

Thrombophilia should be searched in any patient with familial history or personal history of

has already been used in pregnant patients who were unable to use other heparin.

VTE or with the diagnosis of a hereditary thrombophilia on a first degree relative.

deficiency, lack of familial history or personal history for VTE.

Hereditary thrombophilia can be classified in thee risk categories (Fogerty AE 2009):

\* PPV- positive predictive values

42 Pregnancy Thrombophilia - The Unsuspected Risk

seriously.

implication.

**Table 1.** Predictive values of hereditary thrombophilias and VTE.

meta-analysis or case control studies.

any thrombophilia with a previous VTE.

of any symptoms that can be related to VTE.

Pregnancy by itself is considered a hypercoagulation state. Likewise for diabetes, it is expected to increase venous or arterial thromboembolism during pregnancy. The increase in estrogen levels leads to increase in many of the coagulation factors. Thus, in the presence of a hereditary or acquired thrombophilia a higher incidence of thrombotic events could be expected. Once a thrombophilic state is already identified, than we can expect a higher chance of clinical and obstetrical complications, except if prophylactic or therapeutic treatments are offered by caring physicians and followed by patients.

Although this is a disseminated belief, systematic reviews fail to demonstrate such strong relation. As stated above, only patients with a familial history of thrombosis or thrombophilia should be investigated.

However, this is not universally agreed on the literature. For instance, a group of patients with Leiden Factor V were compared to a group of absent mutation. It was observed that the group with the mutation had no thromboembolic events, whereas the group without had 2,7% thromboembolism (Dizon-Townson D 2005).

It has been also stated that even in relatives of probands who have no history of venous thromboembolism (VTE) should not receive antithrombotic prophylaxis during pregnancy because no difference was seen in the group with and without positive history of VTE prior to pregnancy (Cordoba I 2012). With theses considerations, one must look for evidences presented to date.

#### **3.1. Thrombophilia and venous thromboembolism**

Pregnancy is a clinical situation associated with increased risk of VTE, which increased from twofold to fourfold when these women presented a positive family history of VTE (Bezemer ID 2009). Hereditary thrombophilia also increases the risk of VTE. However, the most important for prevention of thrombosis in pregnant women with these additive risk factors is the negative or positive history of previous VTE.

Pregnant women with all other thrombophilias and no prior VTE who do not have a positive family history for VTE, antepartum and postpartum clinical vigilance are indicated (Bates SM

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

45

Stroke in pregnancy is one of the main causes of maternal death and pregnancy and puerpe‐ rium are known to increase its occurrence. The incidence of stroke is not precisely known and there is a wide variation among reports across the world, ranging from 1.5 to 69 per 100 thousand pregnancies (Jaigobin C 2000) (Scott CA 2012). Stroke accounts for 2.2% of all deaths in women of reproductive age and most of these deaths occur during pregnancy (WHO, 2004). Puerperium increases the risk of stroke 5-18-fold and cerebral thromboembolism carries a mortality rate 3 times higher in pregnant women. Despite the epidemiological association, stroke is considered a multifactorial disease, and genetic, environmental, vascular and hormonal factors play a complex and integrated role. Multiple genes have been studied, and even in the same individual, more than one polymorphism, acting in inflammatory, vascular

With increasing worldwide efforts and acknowledgement to know about the causes and lessen the consequences of stroke, pregnant women seem to be a special population, not only because

Obstetrical associated conditions are chronic hypertension, preeclampsia and cesarean delivery. However, most cases do not have predisposing factors and occur in apparently healthy subjects. Some physiologic changes during pregnancy and puerperium can be associated with an increased risk of stroke: increased circulating blood volume, increased

Preeclampsia seems to be a risk factor even for non-pregnancy associated ischemic stroke, which means that women who had preeclampsia have an increased risk of, is when nonpregnant (Brown DW 2006). Non-obstetric risk factors include thrombophilia (inherited and acquired) migraine, smoking, advanced maternal age, diabetes, sickle cell disease, autoim‐

Stroke in pregnancy and puerperium has three major clinical syndromes: ischemic stroke (IS), intracranial hemorrhage (ICH) and cerebral venous thrombosis (CVT). It is the most frequent presentation and CVT is rare. Maternal mortality can be as high as 50% for ICH and 20-25%

There are few studies addressing the role of thrombophilia in stroke occurring during pregnancy or puerperium. We will focus mainly in thrombotic and ischemic stroke, since hemorrhagic stroke has different pathogenic mechanisms in pregnancy, mainly related to

rupture of undiagnosed intracranial aneurysms and complications of eclampsia.

**3.2. Thrombophilia and arterial vascular accidents in pregnancy**

of the greater mortality but also because of the perinatal implications.

cardiac output, vascular wall fragility, and high levels of steroid hormones.

mune conditions, and severe hypotension (Scott CA 2012).

overall (Nomura ML 2012) (Scott CA 2012).

and thrombotic pathways can lead to stroke.

and Physicians. 2012).

A meta-analysis and a review demonstrated increased risk of VTE in pregnant women with thrombophilia without a family or a positive history of VTE (Robertson L 2006) (Biron-Andreani C 2006). Heterozygosis for factor V Leiden and prothrombin 20210 G>A variant were fortunately associated with the lower risk, as they were the most common inherited throm‐ bophilia. However, the homozygosis for these mutations was associated with the higher risk. Deficiencies of natural anticoagulants were also associated to increased risk of VTE.

The incidence of VTE in the pregnancy is 1/1,000 deliveries, and the absolute risk of VTE in thrombophilic women without a prior event or family history is in the range of 5-12/1,000 deliveries, except for homozygous carriers of the factor V Leiden or the prothrombin muta‐ tions, in whom the estimated baseline risk is about 4%.

Although the estimated risk of VTE in the presence of a positive family history of VTE and inherited thrombophilia without a previous episode of VTE has been described, it is imprecise, particularly for the rare thrombophilias (Friederich PW 1996).

Previous studies described higher risk of VTE in the presence of deficiencies of the anticoa‐ gulants, particularly antithrombin deficiency. However, methodological limitations could have contributed to these conclusions (Conard J 1990). The recent studies showed similar risks, even in double heterozygous for Leiden FV and prothrombin mutation (Tormene D 2001) (Martinelli I 2008). The homozygosis for MTHFR C>T alone does not lead to an increased risk of VTE in pregnant women (Robertson L 2006).

Women with thrombophilia without a family history presented a low risk of VTE. Because of the absence of high-quality evidence measuring the effectiveness and safety of antithrombotic agents in preventing VTE in patients with thrombophilia and a positive family history the recommendations have limitations.

The most recent guidelines suggest antepartum prophylaxis with prophylactic or intermedi‐ ate-dose LMWH and postpartum prophylaxis for 6 weeks with prophylactic or intermediatedose LMWH or vitamin K antagonists targeted at INR 2.0 to 3.0 for pregnant women with no prior history of VTE who are known to be homozygous for factor V Leiden or the prothrombin 20210 G>A mutation and have a positive family history for VTE (Bates SM and Physicians. 2012).

For patients with other thrombophilias without a previous history of VTE and who have a positive family history for VTE it is indicated antepartum clinical vigilance and postpartum prophylaxis for 6 weeks with prophylactic or intermediate dose LMWH or, in women who are not protein C or S deficient, vitamin K antagonists targeted at INR 2.0 to 3.0 (Bates SM and Physicians. 2012).

The same is indicated for pregnant women with no prior history of VTE who are known to be homozygous for factor V Leiden or the prothrombin 20210 G>A mutation and who do not have a positive family history for VTE (Bates SM and Physicians. 2012).

Pregnant women with all other thrombophilias and no prior VTE who do not have a positive family history for VTE, antepartum and postpartum clinical vigilance are indicated (Bates SM and Physicians. 2012).

#### **3.2. Thrombophilia and arterial vascular accidents in pregnancy**

ID 2009). Hereditary thrombophilia also increases the risk of VTE. However, the most important for prevention of thrombosis in pregnant women with these additive risk factors is

A meta-analysis and a review demonstrated increased risk of VTE in pregnant women with thrombophilia without a family or a positive history of VTE (Robertson L 2006) (Biron-Andreani C 2006). Heterozygosis for factor V Leiden and prothrombin 20210 G>A variant were fortunately associated with the lower risk, as they were the most common inherited throm‐ bophilia. However, the homozygosis for these mutations was associated with the higher risk.

The incidence of VTE in the pregnancy is 1/1,000 deliveries, and the absolute risk of VTE in thrombophilic women without a prior event or family history is in the range of 5-12/1,000 deliveries, except for homozygous carriers of the factor V Leiden or the prothrombin muta‐

Although the estimated risk of VTE in the presence of a positive family history of VTE and inherited thrombophilia without a previous episode of VTE has been described, it is imprecise,

Previous studies described higher risk of VTE in the presence of deficiencies of the anticoa‐ gulants, particularly antithrombin deficiency. However, methodological limitations could have contributed to these conclusions (Conard J 1990). The recent studies showed similar risks, even in double heterozygous for Leiden FV and prothrombin mutation (Tormene D 2001) (Martinelli I 2008). The homozygosis for MTHFR C>T alone does not lead to an increased risk

Women with thrombophilia without a family history presented a low risk of VTE. Because of the absence of high-quality evidence measuring the effectiveness and safety of antithrombotic agents in preventing VTE in patients with thrombophilia and a positive family history the

The most recent guidelines suggest antepartum prophylaxis with prophylactic or intermedi‐ ate-dose LMWH and postpartum prophylaxis for 6 weeks with prophylactic or intermediatedose LMWH or vitamin K antagonists targeted at INR 2.0 to 3.0 for pregnant women with no prior history of VTE who are known to be homozygous for factor V Leiden or the prothrombin 20210 G>A mutation and have a positive family history for VTE (Bates SM and Physicians.

For patients with other thrombophilias without a previous history of VTE and who have a positive family history for VTE it is indicated antepartum clinical vigilance and postpartum prophylaxis for 6 weeks with prophylactic or intermediate dose LMWH or, in women who are not protein C or S deficient, vitamin K antagonists targeted at INR 2.0 to 3.0 (Bates SM and

The same is indicated for pregnant women with no prior history of VTE who are known to be homozygous for factor V Leiden or the prothrombin 20210 G>A mutation and who do not have

a positive family history for VTE (Bates SM and Physicians. 2012).

Deficiencies of natural anticoagulants were also associated to increased risk of VTE.

the negative or positive history of previous VTE.

44 Pregnancy Thrombophilia - The Unsuspected Risk

tions, in whom the estimated baseline risk is about 4%.

of VTE in pregnant women (Robertson L 2006).

recommendations have limitations.

2012).

Physicians. 2012).

particularly for the rare thrombophilias (Friederich PW 1996).

Stroke in pregnancy is one of the main causes of maternal death and pregnancy and puerpe‐ rium are known to increase its occurrence. The incidence of stroke is not precisely known and there is a wide variation among reports across the world, ranging from 1.5 to 69 per 100 thousand pregnancies (Jaigobin C 2000) (Scott CA 2012). Stroke accounts for 2.2% of all deaths in women of reproductive age and most of these deaths occur during pregnancy (WHO, 2004). Puerperium increases the risk of stroke 5-18-fold and cerebral thromboembolism carries a mortality rate 3 times higher in pregnant women. Despite the epidemiological association, stroke is considered a multifactorial disease, and genetic, environmental, vascular and hormonal factors play a complex and integrated role. Multiple genes have been studied, and even in the same individual, more than one polymorphism, acting in inflammatory, vascular and thrombotic pathways can lead to stroke.

With increasing worldwide efforts and acknowledgement to know about the causes and lessen the consequences of stroke, pregnant women seem to be a special population, not only because of the greater mortality but also because of the perinatal implications.

Obstetrical associated conditions are chronic hypertension, preeclampsia and cesarean delivery. However, most cases do not have predisposing factors and occur in apparently healthy subjects. Some physiologic changes during pregnancy and puerperium can be associated with an increased risk of stroke: increased circulating blood volume, increased cardiac output, vascular wall fragility, and high levels of steroid hormones.

Preeclampsia seems to be a risk factor even for non-pregnancy associated ischemic stroke, which means that women who had preeclampsia have an increased risk of, is when nonpregnant (Brown DW 2006). Non-obstetric risk factors include thrombophilia (inherited and acquired) migraine, smoking, advanced maternal age, diabetes, sickle cell disease, autoim‐ mune conditions, and severe hypotension (Scott CA 2012).

Stroke in pregnancy and puerperium has three major clinical syndromes: ischemic stroke (IS), intracranial hemorrhage (ICH) and cerebral venous thrombosis (CVT). It is the most frequent presentation and CVT is rare. Maternal mortality can be as high as 50% for ICH and 20-25% overall (Nomura ML 2012) (Scott CA 2012).

There are few studies addressing the role of thrombophilia in stroke occurring during pregnancy or puerperium. We will focus mainly in thrombotic and ischemic stroke, since hemorrhagic stroke has different pathogenic mechanisms in pregnancy, mainly related to rupture of undiagnosed intracranial aneurysms and complications of eclampsia.

#### *3.2.1. Inherited thrombophilia*

Inherited thrombophilias can be found in up to 11 % of patients with stroke (Bushnell CD 2000). In this systematic review, no association was found between factor V Leiden and IS, but a slight increase in the odds ratio for prothrombin gene mutation was found (1.4; 95% CI 1.03-1.9).

in CVT), preeclampsia/eclampsia, oral contraceptive use and acquired thrombophilias, such

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

47

Acquired thrombophilia is a condition known to be associated with stroke. Transient cerebral ischemia and stroke (including CVT) are clinical manifestations of antiphospholipid syndrome (APS), but in order to establish the diagnosis a laboratory criteria must also be present, which might be detection in the plasma of lupus anticoagulant, or anticardiolipin antibodies (IgG or IgM) or anti-beta2-glycoprotein (IgG or IgM), in at least two occasions, 12 weeks apart, and according to specific standard laboratory guidelines (Miyakis S 2006). The association between IS and APS (primary and lupus-associated) is well established in case-control studies, and even

Patients with previous cerebrovascular events who met criteria for APS should have antithrombosis prophylaxis prescribed when pregnant, with non-fractioned or low-molecular weight heparin. Prophylaxis should be extended into 6 weeks postpartum also. Aspirin can

Patients with APS and previous cerebrovascular events have an increased recurrence risk of

Other acquired thrombophilias or thrombophilic status can potentially increase the risk of stroke in pregnancy and puerperium, including sickle cell disease, nephrotic syndrome, dehydration or severe hypovolemic status, and careful attention must be paid in this situations.

Adverse pregnancy outcome are not infrequent in general population. Pregnancy complica‐ tions include miscarriage, fetal loss, preeclampsia, fetal growth restriction, and placental

The association between inherited thrombophilic disorders and miscarriage, late fetal loss or severe preeclampsia has been described in various studies (Robertson L 2006) (v. d. Coppens

However, there is a high uncertainty about these associations, particularly for the less preva‐ lent thrombophilia (Robertson L 2006). A meta-analysis including only prospective cohort studies showed only association between factor V Leiden and pregnancy loss, but not with

One randomized trial described increased live birth rate in women with factor V Leiden, the prothrombin gene mutation, or protein S deficiency using enoxaparin when compared with

The results of other studies do not provide evidence that LMWH improves pregnancy outcome in women with inherited thrombophilia and recurrent pregnancy loss (F. N. Coppens M 2007).

low-dose aspirin alone (Gris et al s et al), but the methodology was limited.

in unselected populations this association seems strong (Bushnell CD 2000).

IS, and preeclampsia seems to be an additional risk factor (Fischer-Betz R 2012).

also be added, particularly in the acute phase of an IS.

**4. Thrombophilia and pregnancy complications**

as sickle cell disease.

abruption.

M 2006) (Rodger MA 2010).

other thrombophilias (Rodger MA 2010).

*3.2.2. Acquired thrombophilia*

A study by Voetsch (Voetsch B 2000) among 167 patients with ischemic stroke and did not find an association with inherited thrombophilia, except in cerebral venous thrombosis, where prothrombin gene mutation was more prevalent. For the small group of patients of African origin, homozygosis for MTHFR 677C>T might have a potential role. Interestingly, patients with CVT were all in use of oral contraceptives or in the puerperium.

Hankey (Hankey GJ 2001) tested 219 patients with ischemic stroke for inherited thrombophilia in a case-control study and did not find a significant association (prevalence of 14.7% in stroke patients and 11.7% in control subjects) between any thrombophilia or combination of throm‐ bophilias and IS, and the authors conclude that routine testing is not recommended in the majority of patients.

Kim & Becker (Kim RJ 2003) performed a meta-analysis of the association between some inherited thrombophilias and ischemic stroke and factor V, prothrombin, and homocysteine metabolism were found to modestly increase the risk in young women.

Weber & Busch (Weber R 2005) performed a cost analysis of screening for inherited thrombo‐ philias in patients with IS of unknown cause and concluded that screening was of questionable value, with the exception of antiphospholipid antibodies in younger patients.

Corod-Artal et al (Carod-Artal FJ 2005) screened 130 young patients with stroke, and only protein S deficiency was found to be associated with stroke of unknown cause in young subjects, but in this subpopulation 31% were oral contraceptive users.

Hamzi et al (Hamzi K 2011) performed the largest meta-analysis to date regarding possible genes associated with ischemic stroke, with more than 150 thousand subjects included. They found that MTHFR 677C>T, factor V Leiden, 20210 G>A prothrombin and ACE I/D polymor‐ phism had significant, although very modest, associations with IS. However, the authors did not specify results in selected populations, such as pregnant women.

Haeusler et al (Haeusler KG 2012) reported an increased prevalence of factor VII polymor‐ phisms and factor V Leiden (although not significant) in patients with cryptogenic (unknown cause) stroke.

All studies reported might have biases, such as selection of high-risk patients, and there is a lack of controlled, prospective studies in pregnant women. Recommendations for routine screening of inherited thrombophilias in the setting of stroke in pregnant or postpartum women cannot be made at present, since pregnancy itself might be the most important risk factor.

Inherited thrombophilia might play a role when associated with other conditions, acting synergistically or increasing the odds of other risk factors, such as puerperium (particularly in CVT), preeclampsia/eclampsia, oral contraceptive use and acquired thrombophilias, such as sickle cell disease.

#### *3.2.2. Acquired thrombophilia*

*3.2.1. Inherited thrombophilia*

46 Pregnancy Thrombophilia - The Unsuspected Risk

1.03-1.9).

majority of patients.

cause) stroke.

factor.

Inherited thrombophilias can be found in up to 11 % of patients with stroke (Bushnell CD 2000). In this systematic review, no association was found between factor V Leiden and IS, but a slight increase in the odds ratio for prothrombin gene mutation was found (1.4; 95% CI

A study by Voetsch (Voetsch B 2000) among 167 patients with ischemic stroke and did not find an association with inherited thrombophilia, except in cerebral venous thrombosis, where prothrombin gene mutation was more prevalent. For the small group of patients of African origin, homozygosis for MTHFR 677C>T might have a potential role. Interestingly, patients

Hankey (Hankey GJ 2001) tested 219 patients with ischemic stroke for inherited thrombophilia in a case-control study and did not find a significant association (prevalence of 14.7% in stroke patients and 11.7% in control subjects) between any thrombophilia or combination of throm‐ bophilias and IS, and the authors conclude that routine testing is not recommended in the

Kim & Becker (Kim RJ 2003) performed a meta-analysis of the association between some inherited thrombophilias and ischemic stroke and factor V, prothrombin, and homocysteine

Weber & Busch (Weber R 2005) performed a cost analysis of screening for inherited thrombo‐ philias in patients with IS of unknown cause and concluded that screening was of questionable

Corod-Artal et al (Carod-Artal FJ 2005) screened 130 young patients with stroke, and only protein S deficiency was found to be associated with stroke of unknown cause in young

Hamzi et al (Hamzi K 2011) performed the largest meta-analysis to date regarding possible genes associated with ischemic stroke, with more than 150 thousand subjects included. They found that MTHFR 677C>T, factor V Leiden, 20210 G>A prothrombin and ACE I/D polymor‐ phism had significant, although very modest, associations with IS. However, the authors did

Haeusler et al (Haeusler KG 2012) reported an increased prevalence of factor VII polymor‐ phisms and factor V Leiden (although not significant) in patients with cryptogenic (unknown

All studies reported might have biases, such as selection of high-risk patients, and there is a lack of controlled, prospective studies in pregnant women. Recommendations for routine screening of inherited thrombophilias in the setting of stroke in pregnant or postpartum women cannot be made at present, since pregnancy itself might be the most important risk

Inherited thrombophilia might play a role when associated with other conditions, acting synergistically or increasing the odds of other risk factors, such as puerperium (particularly

with CVT were all in use of oral contraceptives or in the puerperium.

metabolism were found to modestly increase the risk in young women.

subjects, but in this subpopulation 31% were oral contraceptive users.

not specify results in selected populations, such as pregnant women.

value, with the exception of antiphospholipid antibodies in younger patients.

Acquired thrombophilia is a condition known to be associated with stroke. Transient cerebral ischemia and stroke (including CVT) are clinical manifestations of antiphospholipid syndrome (APS), but in order to establish the diagnosis a laboratory criteria must also be present, which might be detection in the plasma of lupus anticoagulant, or anticardiolipin antibodies (IgG or IgM) or anti-beta2-glycoprotein (IgG or IgM), in at least two occasions, 12 weeks apart, and according to specific standard laboratory guidelines (Miyakis S 2006). The association between IS and APS (primary and lupus-associated) is well established in case-control studies, and even in unselected populations this association seems strong (Bushnell CD 2000).

Patients with previous cerebrovascular events who met criteria for APS should have antithrombosis prophylaxis prescribed when pregnant, with non-fractioned or low-molecular weight heparin. Prophylaxis should be extended into 6 weeks postpartum also. Aspirin can also be added, particularly in the acute phase of an IS.

Patients with APS and previous cerebrovascular events have an increased recurrence risk of IS, and preeclampsia seems to be an additional risk factor (Fischer-Betz R 2012).

Other acquired thrombophilias or thrombophilic status can potentially increase the risk of stroke in pregnancy and puerperium, including sickle cell disease, nephrotic syndrome, dehydration or severe hypovolemic status, and careful attention must be paid in this situations.

## **4. Thrombophilia and pregnancy complications**

Adverse pregnancy outcome are not infrequent in general population. Pregnancy complica‐ tions include miscarriage, fetal loss, preeclampsia, fetal growth restriction, and placental abruption.

The association between inherited thrombophilic disorders and miscarriage, late fetal loss or severe preeclampsia has been described in various studies (Robertson L 2006) (v. d. Coppens M 2006) (Rodger MA 2010).

However, there is a high uncertainty about these associations, particularly for the less preva‐ lent thrombophilia (Robertson L 2006). A meta-analysis including only prospective cohort studies showed only association between factor V Leiden and pregnancy loss, but not with other thrombophilias (Rodger MA 2010).

One randomized trial described increased live birth rate in women with factor V Leiden, the prothrombin gene mutation, or protein S deficiency using enoxaparin when compared with low-dose aspirin alone (Gris et al s et al), but the methodology was limited.

The results of other studies do not provide evidence that LMWH improves pregnancy outcome in women with inherited thrombophilia and recurrent pregnancy loss (F. N. Coppens M 2007). Based on these findings the guidelines do not recommend screening for inherited thrombo‐ philia for women with a history of pregnancy complications. There is no indication of antith‐ rombotic prophylaxis for women with inherited thrombophilia and a history of pregnancy complications.

(ACOG 2005). Clinical criteria are: one or more confirmed episode of vascular thrombosis of any type (venous, arterial, small vessel) and/or pregnancy complications (three or more

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

49

gestation, one or more fetal deaths at greater than 10 weeks of gestation, one or more preterm

preeclampsia or placental insufficiency). Laboratory criteria are: positive plasma levels of anticardiolipin antibodies of the IgG or IgM isotope at medium to high levels and/or positive plasma levels of lupus anticoagulant. Testing must be positive on two or more occasions, 12

The APS is the autoimmune disease most commonly associated with RPL (Rai RS 1995) to as low as 15% (Empson M 2002) and the presence of antiphospholipid antibodies is a major risk

There is still controversy over the timing (early or late) of pregnancy loss more closely related with aPL. A retrospective study in a group of 366 women with recurrent pregnancy losses compared the type of prior pregnancy loss between women with and without APL (Oshiro et al, 1996). A total of 79 women included in the study tested positive for APL, while 290 did not. The rate of prior early pregnancy loss was similar in both groups (>80%). However, those patients with APL had 50% of prior late pregnancy losses compared with <25% late pregnancy loss rate in women without APL. The specificity of late pregnancy loss for the presence of APL was 76% compared with only 6% for two or more early pregnancy losses, thus suggesting that late pregnancy loss is the most frequent type of loss associated with APS. Other studies have found that most of the APL-related pregnancy losses were biochemical pregnancy losses or early pregnancy losses in nature (Parazzini F 1991) (MacLean MA 1994) (Yetman DL 1996). Experimental data using APS animal models further support the evidence that any type of pregnancy loss (including preimplantation embryos), but mainly embryo reabsorption, may

The association of APL with recurrent pregnancy losses in patients with SLE and the APS suggests a causative role but, by no means, it does prove it. The major pregnancy-related target for APL is the placenta and utero-placental insufficiency is often attributed to vasculopathy of the terminal spiral arteries that nourish the placenta intervillous space. These vessels had smaller diameter and showed intimal lawyer thickening, fibrinoid necrosis, and intraluminal thrombosis (De Wolf et al, 1982). In other cases, the infarcted region may show villous congestion and hemorrhage and early trophoblastic necrosis (Bendon RW 1987). In addition to placental infarction and thrombosis, perivillous fibrin deposition and evidence of decidua vascular atherosis, indicative of spiral artery vasculopathy, are seen in some APS cases

The mechanisms by which aPL cause the above described changes are not completely under‐ stood and several hypotheses have been proposed. The earliest one is eicosanoid balance alteration mediated by aPL. Inhibition of endothelial cell production of PGI2 (a potent inhibitor of platelet aggregation and vasodilator) and enhancement of placental TXA2 production by plasma from aPL-positive women have been demonstrated by some investigators (Carreras LO 1981) (Schorer AE 1992). Another possible mechanism for thrombosis in APS is the cross-

consecutive spontaneous pregnancy losses at less than 10 weeks of

births at less than 34 weeks of gestation secondary to severe

factor for an adverse pregnancy outcome (Out HJ 1992).

weeks or more apart (Miyakis S 2006).

be associated with APL (Ziporen L 1998).

(Gharavi AE 2001).

The results of two studies that address this issue, Heparin for Pregnant Women with Throm‐ bophilia [NCT01019655] and TIPPS: Thrombophilia in Pregnancy Prophylaxis Study [NCT00967382] are awaited with interest.

#### **4.1. Thrombophilia and recurrent pregnancy loss**

Pregnancy loss in humans occurs in up to 75% of fertilized ova and 15% of well-confirmed pregnancies (Boklage 1990) and recurrent pregnancy losses (RPL) affect 2–5% of women in reproductive age (Hatasaka 1994). RPL is usually defined as the loss of three or more consec‐ utive pregnancies before 20 weeks of gestation or with fetal weights less than 500 grams. Within this definition is a large and heterogeneous group of patients with many different causes of miscarriage. RPL frequency increases up to 5% when clinicians define RPL as two or more losses of pregnancy (Hogge 2003). In addition, epidemiological investigations have demon‐ strated that the frequency of subsequent pregnancy loss is 24% after two pregnancy losses, 30% after three and 40% after four successive pregnancy losses (Regan 1989). Additionally, recurrent risk for RPL may increase up to 50 percent even after six losses (Poland B 1977).

Recurrent abortion involves more than 500,000 women in the United States per year (Bick 2000). Within the past ten years interest in correlations between thrombophilia and complica‐ tions of pregnancy has remarkably increased. Thrombotic processes may also be involved in other serious obstetric complications, such as pre-eclampsia, intrauterine growth retardation and placental abruption by impairment of placental perfusion. Pregnancy itself induces a physiological hyper-coagulation state (Bick 2000) (Clark P 1998) (Stirling Y 1984) that might be aggravated by inherited or acquired thrombophilia. Results of studies on pregnancy complications in women with thrombophilia have been conflicting. This heterogeneous group of disorders results in increased venous and arterial thrombosis. Some thrombophilic states in RPL may be acquired such as antiphospholipid syndrome (APS) or heritable.

#### *4.1.1. Acquired thrombophilia*

Several studies have reported the presence of various autoantibodies in patients with RPL (Roussev RG 1996). However, only the antiphospholipid antibodies (APL) have been clearly associated to recurrent pregnancy losses both in patients with a known autoimmune disease, as APS or systemic lupus erythematous (SLE), and in the general population.

APL were thought to be directed against negatively charged phospholipids, but it has been shown that they are often directed against a protein cofactor, called beta 2 glycoprotein 1, that assists antibody association with the phospholipid (McNeil HP 1990). APL has been associated with thrombotic complications: some are systemic and some are pregnancy specific—sponta‐ neous abortion, stillbirth, intrauterine growth retardation, and preeclampsia (Harris 1986). Diagnosis of this syndrome requires at least one of each clinical and laboratory criterion (ACOG 2005). Clinical criteria are: one or more confirmed episode of vascular thrombosis of any type (venous, arterial, small vessel) and/or pregnancy complications (three or more consecutive spontaneous pregnancy losses at less than 10 weeks of

Based on these findings the guidelines do not recommend screening for inherited thrombo‐ philia for women with a history of pregnancy complications. There is no indication of antith‐ rombotic prophylaxis for women with inherited thrombophilia and a history of pregnancy

The results of two studies that address this issue, Heparin for Pregnant Women with Throm‐ bophilia [NCT01019655] and TIPPS: Thrombophilia in Pregnancy Prophylaxis Study

Pregnancy loss in humans occurs in up to 75% of fertilized ova and 15% of well-confirmed pregnancies (Boklage 1990) and recurrent pregnancy losses (RPL) affect 2–5% of women in reproductive age (Hatasaka 1994). RPL is usually defined as the loss of three or more consec‐ utive pregnancies before 20 weeks of gestation or with fetal weights less than 500 grams. Within this definition is a large and heterogeneous group of patients with many different causes of miscarriage. RPL frequency increases up to 5% when clinicians define RPL as two or more losses of pregnancy (Hogge 2003). In addition, epidemiological investigations have demon‐ strated that the frequency of subsequent pregnancy loss is 24% after two pregnancy losses, 30% after three and 40% after four successive pregnancy losses (Regan 1989). Additionally, recurrent risk for RPL may increase up to 50 percent even after six losses (Poland B 1977).

Recurrent abortion involves more than 500,000 women in the United States per year (Bick 2000). Within the past ten years interest in correlations between thrombophilia and complica‐ tions of pregnancy has remarkably increased. Thrombotic processes may also be involved in other serious obstetric complications, such as pre-eclampsia, intrauterine growth retardation and placental abruption by impairment of placental perfusion. Pregnancy itself induces a physiological hyper-coagulation state (Bick 2000) (Clark P 1998) (Stirling Y 1984) that might be aggravated by inherited or acquired thrombophilia. Results of studies on pregnancy complications in women with thrombophilia have been conflicting. This heterogeneous group of disorders results in increased venous and arterial thrombosis. Some thrombophilic states in

Several studies have reported the presence of various autoantibodies in patients with RPL (Roussev RG 1996). However, only the antiphospholipid antibodies (APL) have been clearly associated to recurrent pregnancy losses both in patients with a known autoimmune disease,

APL were thought to be directed against negatively charged phospholipids, but it has been shown that they are often directed against a protein cofactor, called beta 2 glycoprotein 1, that assists antibody association with the phospholipid (McNeil HP 1990). APL has been associated with thrombotic complications: some are systemic and some are pregnancy specific—sponta‐ neous abortion, stillbirth, intrauterine growth retardation, and preeclampsia (Harris 1986). Diagnosis of this syndrome requires at least one of each clinical and laboratory criterion

RPL may be acquired such as antiphospholipid syndrome (APS) or heritable.

as APS or systemic lupus erythematous (SLE), and in the general population.

complications.

[NCT00967382] are awaited with interest.

48 Pregnancy Thrombophilia - The Unsuspected Risk

*4.1.1. Acquired thrombophilia*

**4.1. Thrombophilia and recurrent pregnancy loss**

gestation, one or more fetal deaths at greater than 10 weeks of gestation, one or more preterm births at less than 34 weeks of gestation secondary to severe

preeclampsia or placental insufficiency). Laboratory criteria are: positive plasma levels of anticardiolipin antibodies of the IgG or IgM isotope at medium to high levels and/or positive plasma levels of lupus anticoagulant. Testing must be positive on two or more occasions, 12 weeks or more apart (Miyakis S 2006).

The APS is the autoimmune disease most commonly associated with RPL (Rai RS 1995) to as low as 15% (Empson M 2002) and the presence of antiphospholipid antibodies is a major risk factor for an adverse pregnancy outcome (Out HJ 1992).

There is still controversy over the timing (early or late) of pregnancy loss more closely related with aPL. A retrospective study in a group of 366 women with recurrent pregnancy losses compared the type of prior pregnancy loss between women with and without APL (Oshiro et al, 1996). A total of 79 women included in the study tested positive for APL, while 290 did not. The rate of prior early pregnancy loss was similar in both groups (>80%). However, those patients with APL had 50% of prior late pregnancy losses compared with <25% late pregnancy loss rate in women without APL. The specificity of late pregnancy loss for the presence of APL was 76% compared with only 6% for two or more early pregnancy losses, thus suggesting that late pregnancy loss is the most frequent type of loss associated with APS. Other studies have found that most of the APL-related pregnancy losses were biochemical pregnancy losses or early pregnancy losses in nature (Parazzini F 1991) (MacLean MA 1994) (Yetman DL 1996). Experimental data using APS animal models further support the evidence that any type of pregnancy loss (including preimplantation embryos), but mainly embryo reabsorption, may be associated with APL (Ziporen L 1998).

The association of APL with recurrent pregnancy losses in patients with SLE and the APS suggests a causative role but, by no means, it does prove it. The major pregnancy-related target for APL is the placenta and utero-placental insufficiency is often attributed to vasculopathy of the terminal spiral arteries that nourish the placenta intervillous space. These vessels had smaller diameter and showed intimal lawyer thickening, fibrinoid necrosis, and intraluminal thrombosis (De Wolf et al, 1982). In other cases, the infarcted region may show villous congestion and hemorrhage and early trophoblastic necrosis (Bendon RW 1987). In addition to placental infarction and thrombosis, perivillous fibrin deposition and evidence of decidua vascular atherosis, indicative of spiral artery vasculopathy, are seen in some APS cases (Gharavi AE 2001).

The mechanisms by which aPL cause the above described changes are not completely under‐ stood and several hypotheses have been proposed. The earliest one is eicosanoid balance alteration mediated by aPL. Inhibition of endothelial cell production of PGI2 (a potent inhibitor of platelet aggregation and vasodilator) and enhancement of placental TXA2 production by plasma from aPL-positive women have been demonstrated by some investigators (Carreras LO 1981) (Schorer AE 1992). Another possible mechanism for thrombosis in APS is the crossreactivity between APL and glycosaminoglycans, a family of heparin-like substances related with the non-thrombotic properties of the vascular endothelium. The inhibition of this function by APL may in part explain the thrombosis associated with them (Chamley LW 1993). Additionally, APL may interfere with the function of natural inhibitors of coagulation such as placental anticoagulant proteins (PAP) and others. PAP is a group of four calcium-dependent phospholipid-binding proteins that inhibit phospholipid- dependent steps of coagulation by making phospholipid inaccessible to clothing factors (Walker JH 1992). The major component of the PAP family is the PAP-1, also called annexin V, which is most abundant in the placenta. Annexin V and aPL compete for phospholipids in coagulation assays (Sammaritano LR 1992). It has been shown that distribution of annexin V over the intervillous surface was significantly lower in patients with APS that in women with recurrent pregnancy losses (Rand JH 1994). These findings suggest that reduced annexin V production and inhibition of its anticoagulant function by aPL may play a role in pregnancy loss in APS patients.

V Leiden (Factor V 1691 G>A), prothrombin 20210 G>A and the methylene tetrahydrofolate gene 677 C>T variations were determined. Some studies also included other classical markers of thrombophilia, such as antithrombin III, protein C and protein S deficiency and APL

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

51

The normal coagulation pathway is pivotal for the pregnancy outcomes. Also any kind of disorder in coagulation pathway may cause thrombophilia that may be the reason of placental insufficiency and pregnancy loss (Reznikoff-Etievan MF, 2001). It has become clear that prothrombotic changes are associated with a substantial proportion of these fetal losses. Throm‐ bophilic defects, including mutations in factor V Leiden and prothrombin 20210 G>A, and deficiencies in protein C, protein S, and antithrombin III, have been reported in 49–65% of women with pregnancy complications and in 18–22% of women with normal pregnancies (B.

Therefore, the role of thrombophilias in RPL has generated a great deal of interest. This heterogeneous group of disorders results in increased venous and arterial thrombosis. Although some thrombophilic states in RPL may be acquired such as APS, most are heritable such as hyperhomocyteinemia, activated protein C resistance, deficiencies in proteins C and S, mutations in prothrombin, and mutations in antithrombin III. The three most known common genetic markers for thrombophilia to predispose to venous thrombosis are; factor V Leiden (FVL), methylenetetrahydrofolate reductase mutation (MTHFR 677 C>T) and pro‐ thrombin gene mutation. Thrombophilic disorders have generated considerable interest in the field of RPL. Thrombophilia is an important predisposition to thrombosis due to a procoagulant state. Several blood-clotting disorders are grouped under the term of thrombophilia. Clinical studies suggest that the underlying pathophysiological mechanism is mediated via hypercoagulation, leading to utero-placental insufficiency with resultant pregnancy loss. The basis for the association between adverse fetal outcomes and heritable thrombophilias has focused on the mechanisms of impaired placental development and function secondary to venous or arterial thrombosis at the maternal–fetal interface (Aubard Y, 2000) (Cotter AM 2001)

Mutation in the gene-encoding factor V results in a protein that is resistant to the effects of activated protein C (aPC). The most common of a variety of mutations is at position 506 with a glutamine substitution for arginine; this FV: R506Q mutation is called the factor V Leiden mutation. The mutation results in a protein resistant to the effects of activated protein C (aPC). The net result is increased the cleavage of prothrombin to thrombin, which causes excessive

The resistance to aPC has emerged as the commonest genetic cause of thromboembolism. It is caused by FVL in 95% of cases. The risk of thrombosis is increased 5- to 10-fold in heterozygous

Inherited decreased or absent antithrombin III activity will lead to increased thrombin formation and clotting. Prothrombin gene mutation is signaled by a defect in clotting factor II at position G20210A. The relative risk for thrombosis in patients with this mutation is two-fold

carriers of FVL, and 100-fold in homozygosis (Kovalevsky G, 2004).

(Kupferminc MJ 1999)(Kupferminc MJ, 1999) (Gris JC Q. I., 2000).

1999) (Kupferminc MJ, 1999).

(Jeanine F, 2010).

coagulation.

in heterozygotes.

However, other non-thrombotic mechanisms have been implicated, being interference with the embryonic implantation the one that has received more attention. The APL have been found to react directly with third trimester villous trophoblastic cells (Lyden TW 1992) (Di Simone N 2000), prevent proliferation of trophoblast derived from choriocarcinoma cells (Chamley LW 1993), inhibit in vitro chemotaxis and differentiation of villous trophoblast isolated from third trimester placentae (Di Simone N 2000), decrease trophoblast invasion (Sebire NJ 2002) (Bose P 2005), and inhibit extra-villous trophoblast differentiation (Quenby S 2005). Furthermore, APL can induce pregnancy loss in mice by impairing the embryonic implantation capacity, likely because a direct interaction with the throphoectoderm cells (Sthoeger ZM 1993).

Additionally, aPL may impair the placenta production of chorionic gonadotropin during the early phases of pregnancy, thus determining the embryonic evolution (Shurtz-Swirski R 1993) and, in the mice model, APS is associated with a diminished secretion of interleukin-3, positively related with pregnancy the pregnancy loss is prevented by in vitro administration of recombinant interleukin-3 (Fishman P 1993).

Furthermore, the role of complement activation by the aPL has also received a great deal of attention. Several studies have suggested that activation of the complement cascade is necessary for aPL-mediated thrombophilia and fetal loss (G. G.-O. Pierangeli SS n.d.) (Holers VM 2002). It was found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1-related gene protein y (Crry)-Ig, blocks aPLinduced fetal loss and growth retardation, and reversed aPL-mediated thrombosis (Holers VM 2002).

#### *4.1.2. Inherited thrombophilia*

In 1996 the first reports on an association between other forms of thrombophilia and recurrent pregnancy loss were published (Preston FE 1996) (Rai RS 1995) (Sanson BJ, 1996). Since then numerous case control studies investigating the impact of thrombophilia on pregnancy loss have been conducted (Kupferminc MJ 1999) (Gris JC R.-N. S., 1997) (Grandone E 1997) (Younis JS, 2000) (Pihusch R 2001) (Alonso A, 2002) (Rasmussen A, 2004). In most of these studies factor V Leiden (Factor V 1691 G>A), prothrombin 20210 G>A and the methylene tetrahydrofolate gene 677 C>T variations were determined. Some studies also included other classical markers of thrombophilia, such as antithrombin III, protein C and protein S deficiency and APL (Kupferminc MJ 1999)(Kupferminc MJ, 1999) (Gris JC Q. I., 2000).

reactivity between APL and glycosaminoglycans, a family of heparin-like substances related with the non-thrombotic properties of the vascular endothelium. The inhibition of this function by APL may in part explain the thrombosis associated with them (Chamley LW 1993). Additionally, APL may interfere with the function of natural inhibitors of coagulation such as placental anticoagulant proteins (PAP) and others. PAP is a group of four calcium-dependent phospholipid-binding proteins that inhibit phospholipid- dependent steps of coagulation by making phospholipid inaccessible to clothing factors (Walker JH 1992). The major component of the PAP family is the PAP-1, also called annexin V, which is most abundant in the placenta. Annexin V and aPL compete for phospholipids in coagulation assays (Sammaritano LR 1992). It has been shown that distribution of annexin V over the intervillous surface was significantly lower in patients with APS that in women with recurrent pregnancy losses (Rand JH 1994). These findings suggest that reduced annexin V production and inhibition of its anticoagulant

However, other non-thrombotic mechanisms have been implicated, being interference with the embryonic implantation the one that has received more attention. The APL have been found to react directly with third trimester villous trophoblastic cells (Lyden TW 1992) (Di Simone N 2000), prevent proliferation of trophoblast derived from choriocarcinoma cells (Chamley LW 1993), inhibit in vitro chemotaxis and differentiation of villous trophoblast isolated from third trimester placentae (Di Simone N 2000), decrease trophoblast invasion (Sebire NJ 2002) (Bose P 2005), and inhibit extra-villous trophoblast differentiation (Quenby S 2005). Furthermore, APL can induce pregnancy loss in mice by impairing the embryonic implantation capacity, likely because a direct interaction with the throphoectoderm cells

Additionally, aPL may impair the placenta production of chorionic gonadotropin during the early phases of pregnancy, thus determining the embryonic evolution (Shurtz-Swirski R 1993) and, in the mice model, APS is associated with a diminished secretion of interleukin-3, positively related with pregnancy the pregnancy loss is prevented by in vitro administration

Furthermore, the role of complement activation by the aPL has also received a great deal of attention. Several studies have suggested that activation of the complement cascade is necessary for aPL-mediated thrombophilia and fetal loss (G. G.-O. Pierangeli SS n.d.) (Holers VM 2002). It was found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1-related gene protein y (Crry)-Ig, blocks aPLinduced fetal loss and growth retardation, and reversed aPL-mediated thrombosis (Holers VM

In 1996 the first reports on an association between other forms of thrombophilia and recurrent pregnancy loss were published (Preston FE 1996) (Rai RS 1995) (Sanson BJ, 1996). Since then numerous case control studies investigating the impact of thrombophilia on pregnancy loss have been conducted (Kupferminc MJ 1999) (Gris JC R.-N. S., 1997) (Grandone E 1997) (Younis JS, 2000) (Pihusch R 2001) (Alonso A, 2002) (Rasmussen A, 2004). In most of these studies factor

function by aPL may play a role in pregnancy loss in APS patients.

(Sthoeger ZM 1993).

50 Pregnancy Thrombophilia - The Unsuspected Risk

2002).

*4.1.2. Inherited thrombophilia*

of recombinant interleukin-3 (Fishman P 1993).

The normal coagulation pathway is pivotal for the pregnancy outcomes. Also any kind of disorder in coagulation pathway may cause thrombophilia that may be the reason of placental insufficiency and pregnancy loss (Reznikoff-Etievan MF, 2001). It has become clear that prothrombotic changes are associated with a substantial proportion of these fetal losses. Throm‐ bophilic defects, including mutations in factor V Leiden and prothrombin 20210 G>A, and deficiencies in protein C, protein S, and antithrombin III, have been reported in 49–65% of women with pregnancy complications and in 18–22% of women with normal pregnancies (B. 1999) (Kupferminc MJ, 1999).

Therefore, the role of thrombophilias in RPL has generated a great deal of interest. This heterogeneous group of disorders results in increased venous and arterial thrombosis. Although some thrombophilic states in RPL may be acquired such as APS, most are heritable such as hyperhomocyteinemia, activated protein C resistance, deficiencies in proteins C and S, mutations in prothrombin, and mutations in antithrombin III. The three most known common genetic markers for thrombophilia to predispose to venous thrombosis are; factor V Leiden (FVL), methylenetetrahydrofolate reductase mutation (MTHFR 677 C>T) and pro‐ thrombin gene mutation. Thrombophilic disorders have generated considerable interest in the field of RPL. Thrombophilia is an important predisposition to thrombosis due to a procoagulant state. Several blood-clotting disorders are grouped under the term of thrombophilia. Clinical studies suggest that the underlying pathophysiological mechanism is mediated via hypercoagulation, leading to utero-placental insufficiency with resultant pregnancy loss. The basis for the association between adverse fetal outcomes and heritable thrombophilias has focused on the mechanisms of impaired placental development and function secondary to venous or arterial thrombosis at the maternal–fetal interface (Aubard Y, 2000) (Cotter AM 2001) (Jeanine F, 2010).

Mutation in the gene-encoding factor V results in a protein that is resistant to the effects of activated protein C (aPC). The most common of a variety of mutations is at position 506 with a glutamine substitution for arginine; this FV: R506Q mutation is called the factor V Leiden mutation. The mutation results in a protein resistant to the effects of activated protein C (aPC). The net result is increased the cleavage of prothrombin to thrombin, which causes excessive coagulation.

The resistance to aPC has emerged as the commonest genetic cause of thromboembolism. It is caused by FVL in 95% of cases. The risk of thrombosis is increased 5- to 10-fold in heterozygous carriers of FVL, and 100-fold in homozygosis (Kovalevsky G, 2004).

Inherited decreased or absent antithrombin III activity will lead to increased thrombin formation and clotting. Prothrombin gene mutation is signaled by a defect in clotting factor II at position G20210A. The relative risk for thrombosis in patients with this mutation is two-fold in heterozygotes.

Individuals with hiperhomocisteinemia exhibit a deficiency of folate due to the presence of the methylene tetrahydrofolate reductase mutation (MTHRF 677C>T C677 T). The thrombotic risk is increased two-fold in homozygosis; and in the heterozygous state for Antithrombin III deficiency, the risk is 20- to 50-fold.

associated to preeclampsia or arterial hypertension, colagenosis as Systemic Lupus or any other maternal identified disease. This has been the casa once many patients that would have the profile for antiphospholipid syndrome showed no abnormal antibodies on their laboratory workup. So, there would be another group of women that would have another type of

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

53

Considering the possibility that fibrin deposition on the maternal surface of the placenta may impair gas exchanges and nutritional elements between mother and fetus, the model of a

It has to be emphasized that most studies focus on the maternal aspect of maternal thrombosis in thrombophilia and less is regarded on the obstetrical and fetal complications of thrombo‐

It has been shown by some authors that this association is found in case control study (Monari F, 2012), where all thrombophilias evaluated had a greater incidence within patients with history of fetal death and that Factor II 20210 G>A gene mutation had also a predictive value

Others contest this association when placental infarction is evaluated in comparison to abnormal placentation (Franco C 2011). In this study, no association was found between the

One of us (R.B., personal communication not published) have observed instances where patients develop placental ultrasonography abnormal image - Grade II or III during second trimester – (Grannum PA, 1979) together with fetal growth restriction that demonstrated true catch up in fetal growth and sustained or regression in placental grade upon prophylactic lowdose heparin and aspirin regimen. In two occasions patients showed to be Leiden Factor

One interesting study showed an increase in the frequency of patients with prothrombin gene mutation associated to IUGR and abruptio placentae (Kupferminc MJ, Peri H, Zwang E, Yaron

Although randomized trial are still necessary to address the question whether treatment should be advised for women with diagnosed hereditary thrombophilia to prevent adverse pregnancy results, many authors agree that treatment ensures good results, like the one reported by results Kosar (Kosar A 2011). The live birth rate for treated patients was only 62%, even with treatment, which brings up de the consideration that this is really a high-risk

One group showed that the institution of low-molecular-weight heparin to women with previous fetal growth restriction or pre eclampsia had a dramatic reduction on the recurrence

When reviewing the literature one must be careful with the conclusions offered because most

of the studies do not have a good enough large sample size to draw final conclusions.

"coagulation disorder" that would have to be sorted out.

for previous fetal deaths in this study population.

heterozygous and Protein S deficiency.

philia in pregnancy.

Y, Wolman I, 2000).

obstetrical population.

thrombophilic induced placental insufficiency seems very attractive.

presence of thrombophilia and histological findings of infarction.

of these events on subsequent pregnancies (Kupferminc, 2011).

Consistent with general thrombotic risk, carriage of combinations of two or more inherited thrombophilic defects has particularly strong association with adverse pregnancy outcomes (Lockwood C. J., 2002) (Preston FE, 1996). Considerable attention has been directed recently toward a possible relationship between thrombophilias and certain pregnancy complications other than venous thrombosis (De Santis M, 2006).

#### *4.1.3. In vitro fertilization failures and thrombophilia*

The known or purported causality of phospholipid antibodies and coagulation factors on recurrent pregnancy loss long ago spilled over into the arena of conception with IVF or more precisely, the lack of it. Some have argued that without implantation to signal the arrival of an embryo, it would be improbable for serum or tissue-based response ele‐ ments to prevent implantation. Others have argued that the effect is unrelated to the embryo, but rather the negative impact is at the level of the endometrium. The Practice Committee of the American Society for Reproductive Medicine released a Committee Opinion in 1999, which it reviewed again in 2008, ''Anti-phospholipid antibodies (APA) do not affect IVF success'' (Practice Committee of American Society for Reproductive Medi‐ cine. 2012). The review culled 16 peer-reviewed papers, of which 7 included appropriate endpoints and controls. There was no statistically significant impact of the presence of phospholipid antibodies on IVF outcomes neither when studies were examined individual‐ ly nor when the data were aggregated in the 2,053 patients studied. The authors conclud‐ ed that ''assessment of APA is not indicated among couples undergoing IVF. Therapy is not justified on the basis of existing data.''

A review was recently published on the topic of thrombophilias and IVF outcome (Di Nisio M, 2011). The authors' initial search yielded 694 studies. Case reports, editorials, reviews, metaanalyses, studies with inadequate outcomes, absence of thrombophilia/anti-phospholipid antibodies, and more than one of the above was excluded and 33 (6,092 patients) were ultimately analyzed. They report that twenty-nine studies (5,270 patients) assessed antiphospholipid antibodies in women treated with assisted reproductive techniques (ART). The prevalence of antibodies in infertile patients varied from 0%–45%. When examining casecontrol studies, the authors write ''overall, the presence of one or more anti-phospholipid antibodies was associated with a 3-fold higher risk of ART failure.'' There was a significant degree of heterogeneity across these case-control studies.

#### **4.2. Thrombophilia and fetal growth restriction**

Obstetricians have been very interested on the possible consequences of thrombophilia because in face of an unexpected pregnancy event, such as fetal growth restriction not associated to preeclampsia or arterial hypertension, colagenosis as Systemic Lupus or any other maternal identified disease. This has been the casa once many patients that would have the profile for antiphospholipid syndrome showed no abnormal antibodies on their laboratory workup. So, there would be another group of women that would have another type of "coagulation disorder" that would have to be sorted out.

Individuals with hiperhomocisteinemia exhibit a deficiency of folate due to the presence of the methylene tetrahydrofolate reductase mutation (MTHRF 677C>T C677 T). The thrombotic risk is increased two-fold in homozygosis; and in the heterozygous state for Antithrombin III

Consistent with general thrombotic risk, carriage of combinations of two or more inherited thrombophilic defects has particularly strong association with adverse pregnancy outcomes (Lockwood C. J., 2002) (Preston FE, 1996). Considerable attention has been directed recently toward a possible relationship between thrombophilias and certain pregnancy complications

The known or purported causality of phospholipid antibodies and coagulation factors on recurrent pregnancy loss long ago spilled over into the arena of conception with IVF or more precisely, the lack of it. Some have argued that without implantation to signal the arrival of an embryo, it would be improbable for serum or tissue-based response ele‐ ments to prevent implantation. Others have argued that the effect is unrelated to the embryo, but rather the negative impact is at the level of the endometrium. The Practice Committee of the American Society for Reproductive Medicine released a Committee Opinion in 1999, which it reviewed again in 2008, ''Anti-phospholipid antibodies (APA) do not affect IVF success'' (Practice Committee of American Society for Reproductive Medi‐ cine. 2012). The review culled 16 peer-reviewed papers, of which 7 included appropriate endpoints and controls. There was no statistically significant impact of the presence of phospholipid antibodies on IVF outcomes neither when studies were examined individual‐ ly nor when the data were aggregated in the 2,053 patients studied. The authors conclud‐ ed that ''assessment of APA is not indicated among couples undergoing IVF. Therapy is

A review was recently published on the topic of thrombophilias and IVF outcome (Di Nisio M, 2011). The authors' initial search yielded 694 studies. Case reports, editorials, reviews, metaanalyses, studies with inadequate outcomes, absence of thrombophilia/anti-phospholipid antibodies, and more than one of the above was excluded and 33 (6,092 patients) were ultimately analyzed. They report that twenty-nine studies (5,270 patients) assessed antiphospholipid antibodies in women treated with assisted reproductive techniques (ART). The prevalence of antibodies in infertile patients varied from 0%–45%. When examining casecontrol studies, the authors write ''overall, the presence of one or more anti-phospholipid antibodies was associated with a 3-fold higher risk of ART failure.'' There was a significant

Obstetricians have been very interested on the possible consequences of thrombophilia because in face of an unexpected pregnancy event, such as fetal growth restriction not

deficiency, the risk is 20- to 50-fold.

52 Pregnancy Thrombophilia - The Unsuspected Risk

other than venous thrombosis (De Santis M, 2006).

*4.1.3. In vitro fertilization failures and thrombophilia*

not justified on the basis of existing data.''

degree of heterogeneity across these case-control studies.

**4.2. Thrombophilia and fetal growth restriction**

Considering the possibility that fibrin deposition on the maternal surface of the placenta may impair gas exchanges and nutritional elements between mother and fetus, the model of a thrombophilic induced placental insufficiency seems very attractive.

It has to be emphasized that most studies focus on the maternal aspect of maternal thrombosis in thrombophilia and less is regarded on the obstetrical and fetal complications of thrombo‐ philia in pregnancy.

It has been shown by some authors that this association is found in case control study (Monari F, 2012), where all thrombophilias evaluated had a greater incidence within patients with history of fetal death and that Factor II 20210 G>A gene mutation had also a predictive value for previous fetal deaths in this study population.

Others contest this association when placental infarction is evaluated in comparison to abnormal placentation (Franco C 2011). In this study, no association was found between the presence of thrombophilia and histological findings of infarction.

One of us (R.B., personal communication not published) have observed instances where patients develop placental ultrasonography abnormal image - Grade II or III during second trimester – (Grannum PA, 1979) together with fetal growth restriction that demonstrated true catch up in fetal growth and sustained or regression in placental grade upon prophylactic lowdose heparin and aspirin regimen. In two occasions patients showed to be Leiden Factor heterozygous and Protein S deficiency.

One interesting study showed an increase in the frequency of patients with prothrombin gene mutation associated to IUGR and abruptio placentae (Kupferminc MJ, Peri H, Zwang E, Yaron Y, Wolman I, 2000).

Although randomized trial are still necessary to address the question whether treatment should be advised for women with diagnosed hereditary thrombophilia to prevent adverse pregnancy results, many authors agree that treatment ensures good results, like the one reported by results Kosar (Kosar A 2011). The live birth rate for treated patients was only 62%, even with treatment, which brings up de the consideration that this is really a high-risk obstetrical population.

One group showed that the institution of low-molecular-weight heparin to women with previous fetal growth restriction or pre eclampsia had a dramatic reduction on the recurrence of these events on subsequent pregnancies (Kupferminc, 2011).

When reviewing the literature one must be careful with the conclusions offered because most of the studies do not have a good enough large sample size to draw final conclusions.

This is the case of the Australian study comparing pregnancy results of women with and without inherited thrombophilia and positive pregnancy with pre eclampsia, fetal restriction, fetal death or placental abruption. Groups comprised 115 women on each arm and no difference on the frequency of thrombophilia in the group with and without adverse history. Now we have to wonder that for instance, Leiden Factor V is expected in less than 2% of any Caucasian population and 20210 G>A prothrombin gene mutation is even less frequent. A case control study would have to add up at least 600 hundred woman on each arm to be able to draw final conclusions.

Initial reports showed a marked increase in the prevalence of thrombophilic mutations in women with preeclampsia compared to women with uneventful pregnancies, with figures of 40% to 72% for at least one mutation (Stella CL, 2006) (Kupferminc MJ, 1999). These studies were in most part case-control and included women with late and early-onset, mild and severe

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

55

Despite these convincing evidences, several studies further did not confirm such a strong

Kosmas et al (Kosmas, 2003) reported a meta-analysis of more than 5,000 women among 19 studies. For the studies published until 2000, an association was found between factor V Leiden and preeclampsia, however, for the studies published in 2001-2002 this association was not

Dizon-Towson et al (Dizon-Townson D, 2005) also found no association between factor V Leiden and prothrombin mutations with preeclampsia, and this was a multi-center, prospec‐

Another prospective study (Said, 2010) involving 2034 women did not find significant associations between any inherited thrombophilia and preeclampsia and the authors conclud‐ ed that the majority of women with inherited thrombophilia have normal pregnancy outcomes.

Alfirevic et al. (Alfirevic, 2002) reported the first systematic review of the association between maternal thrombophilia and preeclampsia, and factor V Leiden mutation (heterozygous), 20210 G>A prothrombin mutation (heterozygous), MTHFR 677C>T (homozygous), protein C deficiency, protein S deficiency and activated protein C resistance were more prevalent among

In a systematic review performed by Robertson et al. (Robertson L, 2006), the odds ratios for several genetic mutations (factor V Leiden, prothrombin mutation, MTHFR 677C>T and protein S) ranged from 1.37 to 3.49, with evidence of heterogeneity among some studies.

In a systematic review and meta-analysis, Rodger et al. (Rodger MA, 2010) failed to show a significant association between factor V Leiden and prothrombin gene mutation. This review

Combined thrombophilias (being carrier of more than one mutation) might have a stronger association with early-onset, severe preeclampsia and HELLP syndrome, however conclusive

The American College of Obstetricians and Gynecologists (Lockwood C, Wendel G; Committee on Practice Bulletins— Obstetrics., 2011) stated that the evidence is insuffi‐ cient to conclude that inherited thrombophilia increases the occurrence of preeclampsia and therefore do not recommend screening and treatment for thrombophilia in women with

Lockwood (Lockwood C., 2010) also cautioned against screening and treatment for inherited thrombophilias, unless in the setting of a clinical trial. The author argues that methodological

included ten prospective cohort studies and more than twenty thousand patients.

preeclampsia, as also HELLP syndrome and eclampsia.

association (Rodger, 2007).

women with preeclampsia.

and solid evidence is also lacking.

previous preeclampsia.

confirmed.

tive study.

A large cohort study of nulliparous women as performed where the results of inherited thrombophilia was blind to caring physicians and only a strong association of 20210 G>A prothrombin gene mutation was found to adverse results of their pregnancies (pre eclampsia, fetal death, fetal restriction, placenta abruption). None of the other thrombophilia showed association to any of these events in this asymptomatic population (Said, 2010).

When we look in the literature on acquired thrombophilia and fetal growth restriction, there is greater agreement that there is a relation between them.

#### **4.3. Thrombophilia and preeclampsia**

Preeclampsia is a leading cause of maternal-fetal morbidity and mortality. It accounts for a significant fraction of maternal deaths in developed and underdeveloped countries. Pree‐ clampsia is one of the most researched diseases in medicine, and so far several aspects of its pathophysiology have been elucidated. However, a major concept that prevails among the most important studies is that preeclampsia is a multifactorial disease.

One of the most controversial aspects is the role of acquired and inherited thrombophilia in the development of preeclampsia. In this chapter, we present a review on the studies about thrombophilia and preeclampsia, with a critical standing point and future perspectives on this issue.

How thrombophilia might act as cause or contributor in preeclampsia? Thrombophilias may act as co-factor in decreasing placental function through vascular thrombosis and also may regulate inflammatory pathways and increase intravascular coagulation. Alltogether with other contributors these features may trigger endothelial dysfunction and lead to the clinical and laboratorial picture of preeclampsia (Kupferminc., 2003).

#### *4.3.1. Inherited thrombophilias*

Among inherited thrombophilias, the most studied are factor V Leiden mutation, 20210 G>A prothrombin mutation, MTHRF 677C>T, protein C deficiency, protein S deficiency and activated protein C resistance.

There are conflicting results regarding the association between inherited thrombophilias and preeclampsia. This might be due to several factors: small sample sizes, poor methodological quality, retrospective nature of most studies and heterogeneity in the prevalence of thrombo‐ philia in different populations.

Initial reports showed a marked increase in the prevalence of thrombophilic mutations in women with preeclampsia compared to women with uneventful pregnancies, with figures of 40% to 72% for at least one mutation (Stella CL, 2006) (Kupferminc MJ, 1999). These studies were in most part case-control and included women with late and early-onset, mild and severe preeclampsia, as also HELLP syndrome and eclampsia.

This is the case of the Australian study comparing pregnancy results of women with and without inherited thrombophilia and positive pregnancy with pre eclampsia, fetal restriction, fetal death or placental abruption. Groups comprised 115 women on each arm and no difference on the frequency of thrombophilia in the group with and without adverse history. Now we have to wonder that for instance, Leiden Factor V is expected in less than 2% of any Caucasian population and 20210 G>A prothrombin gene mutation is even less frequent. A case control study would have to add up at least 600 hundred woman on each arm to be able to

A large cohort study of nulliparous women as performed where the results of inherited thrombophilia was blind to caring physicians and only a strong association of 20210 G>A prothrombin gene mutation was found to adverse results of their pregnancies (pre eclampsia, fetal death, fetal restriction, placenta abruption). None of the other thrombophilia showed

When we look in the literature on acquired thrombophilia and fetal growth restriction, there

Preeclampsia is a leading cause of maternal-fetal morbidity and mortality. It accounts for a significant fraction of maternal deaths in developed and underdeveloped countries. Pree‐ clampsia is one of the most researched diseases in medicine, and so far several aspects of its pathophysiology have been elucidated. However, a major concept that prevails among the

One of the most controversial aspects is the role of acquired and inherited thrombophilia in the development of preeclampsia. In this chapter, we present a review on the studies about thrombophilia and preeclampsia, with a critical standing point and future perspectives on this

How thrombophilia might act as cause or contributor in preeclampsia? Thrombophilias may act as co-factor in decreasing placental function through vascular thrombosis and also may regulate inflammatory pathways and increase intravascular coagulation. Alltogether with other contributors these features may trigger endothelial dysfunction and lead to the clinical

Among inherited thrombophilias, the most studied are factor V Leiden mutation, 20210 G>A prothrombin mutation, MTHRF 677C>T, protein C deficiency, protein S deficiency and

There are conflicting results regarding the association between inherited thrombophilias and preeclampsia. This might be due to several factors: small sample sizes, poor methodological quality, retrospective nature of most studies and heterogeneity in the prevalence of thrombo‐

association to any of these events in this asymptomatic population (Said, 2010).

most important studies is that preeclampsia is a multifactorial disease.

and laboratorial picture of preeclampsia (Kupferminc., 2003).

is greater agreement that there is a relation between them.

**4.3. Thrombophilia and preeclampsia**

draw final conclusions.

54 Pregnancy Thrombophilia - The Unsuspected Risk

issue.

*4.3.1. Inherited thrombophilias*

activated protein C resistance.

philia in different populations.

Despite these convincing evidences, several studies further did not confirm such a strong association (Rodger, 2007).

Kosmas et al (Kosmas, 2003) reported a meta-analysis of more than 5,000 women among 19 studies. For the studies published until 2000, an association was found between factor V Leiden and preeclampsia, however, for the studies published in 2001-2002 this association was not confirmed.

Dizon-Towson et al (Dizon-Townson D, 2005) also found no association between factor V Leiden and prothrombin mutations with preeclampsia, and this was a multi-center, prospec‐ tive study.

Another prospective study (Said, 2010) involving 2034 women did not find significant associations between any inherited thrombophilia and preeclampsia and the authors conclud‐ ed that the majority of women with inherited thrombophilia have normal pregnancy outcomes.

Alfirevic et al. (Alfirevic, 2002) reported the first systematic review of the association between maternal thrombophilia and preeclampsia, and factor V Leiden mutation (heterozygous), 20210 G>A prothrombin mutation (heterozygous), MTHFR 677C>T (homozygous), protein C deficiency, protein S deficiency and activated protein C resistance were more prevalent among women with preeclampsia.

In a systematic review performed by Robertson et al. (Robertson L, 2006), the odds ratios for several genetic mutations (factor V Leiden, prothrombin mutation, MTHFR 677C>T and protein S) ranged from 1.37 to 3.49, with evidence of heterogeneity among some studies.

In a systematic review and meta-analysis, Rodger et al. (Rodger MA, 2010) failed to show a significant association between factor V Leiden and prothrombin gene mutation. This review included ten prospective cohort studies and more than twenty thousand patients.

Combined thrombophilias (being carrier of more than one mutation) might have a stronger association with early-onset, severe preeclampsia and HELLP syndrome, however conclusive and solid evidence is also lacking.

The American College of Obstetricians and Gynecologists (Lockwood C, Wendel G; Committee on Practice Bulletins— Obstetrics., 2011) stated that the evidence is insuffi‐ cient to conclude that inherited thrombophilia increases the occurrence of preeclampsia and therefore do not recommend screening and treatment for thrombophilia in women with previous preeclampsia.

Lockwood (Lockwood C., 2010) also cautioned against screening and treatment for inherited thrombophilias, unless in the setting of a clinical trial. The author argues that methodological quality of the positive associations is questionable, that these associations are modest (3-fold increase) and that large prospective cohort studies did not show a consistent association.

**References**

[1] Abou-Nassar K, Carrier M, Ramsay T, Rodger MA. "The association between anti‐ phospholipid antibodies and placenta mediated complications: a systematic review

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

57

[3] Alfirevic, Z., Roberts, D., Martlew, V. "How strong is the association between mater‐ nal thrombophilia and adverse pregnancy outcome? A systematic review." Eur J Ob‐

[4] Alonso A, Soto I, Urgelles MF, Corte JR, Rodriguez MJ, Pinto CR. "Acquired and in‐ herited thrombophilia in women with unexplained fetal losses." Am J Obstet Gyne‐

[5] Aubard Y, et al. "Hyperhomocysteinema and pregnancy review of our present un‐ derstanding and therapeutic implications." Eur J Obstet Gynecol, 2000: 157–65.

[6] B., Brenner. "Inherited thrombophilia and pregnancy loss." Thromb Haemost, 1999:

[7] Bates SM, Greer IA, Middeldorp S, Veenstra DL, Prabulos AM, Vandvik PO, and American College of Chest Physicians. "VTE, thrombophilia, antithrombotic therapy, and pregnancy: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines."

[8] Bendon RW, Wilson J, Getahun B, van der Bel-Kahn J. "A maternal death due to thrombotic disease associated with anticardiolipin antibody." Arch Pathol Lab, 1987:

[9] Bennet SA, Bagot CN, Arya R. "Pregnancy loss and thrombophilia: the elusive link."

[10] Bezemer ID, van der Meer FJ, Eikenboom JC, Rosendaal FR, Doggen CJ. "The value of family history as a risk indicator for venous thrombosis." Arch Intern Med 169

[11] Bick, R L. "Recurrent miscarriage syndrome due to blood coagulation protein/platelet defects: prevalence, treatment and outcome results. DRW Metroplex Recurrent Mis‐ carriage Syndrome Cooperative Group." Clin Appl Thromb Hemost 6 (2000): 115-25.

[12] Biron-Andreani C, Schved JF, Daures JP. "Factor V Leiden mutation and pregnancyrelated venous thromboembolism:what is the exact risk? Results from a meta-analy‐

[13] Boklage, CE. "Survival probability of human conceptions from fertilization to term."

and meta-analysis." Thromb Res 128, no. 1 (Jul 2011): 77-85.

stet Gynecol Reprod Biol, Feb 2002: 6-14.

Chest. 141, no. 2 Suppl (Feb 2012): e691S-736S.

sis." Thromb Haemost 18, no. 96 (2006): 14-18.

Int J Fertil 35, no. 2 (Mar-Apr 1990): 75, 79-80, 81-94.

Br J Haematol 157 (2012): 529-42.

col, 2002: 1337 - 42.

634–640.

370–372.

(2009): 610 - 615.

[2] ACOG. "Antiphospholipid syndrome." Practice Bulletin No. 68, Nov 2005.

In summary, the most recent evidence points to a weak association between preeclampsia and inherited thrombophilias. Even this evidence derives from small studies, with possible selection and report biases. The recent evidence also discourages treatment with heparin based on a diagnosis of inherited thrombophilia.

#### *4.3.2. Acquired thrombophilia*

Antiphospholipid antibodies are more frequently encountered in patients with preeclampsia, and in this setting they might be a modulator of the severity of the disease rather than a direct cause.

The Sydney Consensus Statement on Investigational Classification Criteria for the Antiphos‐ pholipid Syndrome (Miyakis S, 2006) included eclampsia or severe preeclampsia leading to preterm delivery prior to the 34th week as clinical criteria. To establish the diagnosis of antiphospholipid syndrome (APS) a laboratory criteria must also be present, which might be detection in the plasma of lupus anticoagulant, or anticardiolipin antibodies (IgG or IgM) or anti-beta2-glycoprotein (IgG or IgM), in at least two occasions, 12 weeks apart, and according to specific standard laboratory guidelines.

Do Prado et al (do Prado AD, 2010) performed a systematic review on the association between preeclampsia and anticardiolipin antibodies. The authors found a significant association with an odds ratio of 11.15 for severe preeclampsia and 2.86 for preeclampsia.

Abou-Nassar et al. (Abou-Nassar K, 2011) Published a systematic review of antiphospholipid antibodies and preeclampsia and found an inconsistent association, detected only in casecontrol studies but not in cohort studies, and of lower magnitude.

Although controversial, recent evidence suggests that treatment with heparin and low-dose aspirin in order to reduce recurrence risk is warranted in this situation, with overall pregnancy success rates of more than 70% (Ernest JM, 2011) (Lockwood C., 2010).

It must also be noted that women with APS are at greater risk of thromboembolism, and anticoagulation should be prescribed for this purpose also.

## **Author details**

Ricardo Barini, Joyce Annichino-Bizzache, Egle Couto, Marcelo Luis Nomura, Adriana Goes Soligo and Isabela Nelly Machado

Faculdade de Ciências Médicas UNICAMP, SP, Brazil

## **References**

quality of the positive associations is questionable, that these associations are modest (3-fold increase) and that large prospective cohort studies did not show a consistent association.

In summary, the most recent evidence points to a weak association between preeclampsia and inherited thrombophilias. Even this evidence derives from small studies, with possible selection and report biases. The recent evidence also discourages treatment with heparin based

Antiphospholipid antibodies are more frequently encountered in patients with preeclampsia, and in this setting they might be a modulator of the severity of the disease rather than a direct

The Sydney Consensus Statement on Investigational Classification Criteria for the Antiphos‐ pholipid Syndrome (Miyakis S, 2006) included eclampsia or severe preeclampsia leading to preterm delivery prior to the 34th week as clinical criteria. To establish the diagnosis of antiphospholipid syndrome (APS) a laboratory criteria must also be present, which might be detection in the plasma of lupus anticoagulant, or anticardiolipin antibodies (IgG or IgM) or anti-beta2-glycoprotein (IgG or IgM), in at least two occasions, 12 weeks apart, and according

Do Prado et al (do Prado AD, 2010) performed a systematic review on the association between preeclampsia and anticardiolipin antibodies. The authors found a significant association with

Abou-Nassar et al. (Abou-Nassar K, 2011) Published a systematic review of antiphospholipid antibodies and preeclampsia and found an inconsistent association, detected only in case-

Although controversial, recent evidence suggests that treatment with heparin and low-dose aspirin in order to reduce recurrence risk is warranted in this situation, with overall pregnancy

It must also be noted that women with APS are at greater risk of thromboembolism, and

an odds ratio of 11.15 for severe preeclampsia and 2.86 for preeclampsia.

control studies but not in cohort studies, and of lower magnitude.

success rates of more than 70% (Ernest JM, 2011) (Lockwood C., 2010).

Ricardo Barini, Joyce Annichino-Bizzache, Egle Couto, Marcelo Luis Nomura,

anticoagulation should be prescribed for this purpose also.

Adriana Goes Soligo and Isabela Nelly Machado

Faculdade de Ciências Médicas UNICAMP, SP, Brazil

on a diagnosis of inherited thrombophilia.

56 Pregnancy Thrombophilia - The Unsuspected Risk

to specific standard laboratory guidelines.

*4.3.2. Acquired thrombophilia*

cause.

**Author details**


[14] Bose P, Black S, Kadyrov M et al. "Heparin and aspirin attenuate placental apoptosis in vitro: implications for early pregnancy failure." Am J Obstet Gynecol, 2005: 23–30.

[26] Cotter AM, et al. "Elevated plasma homocysteine in early pregnancy: A risk factor for the development of severe preeclampsia." Am J Obstet Gynecol, 2001: 781–5.

Main Types of Clinical Appearance of Thrombophilic States During Pregnancy – Target Groups…

http://dx.doi.org/10.5772/56819

59

[27] Couto E, Nomura ML, Barini R, Silva JL. "Pregnancy- associated venous throm‐ boembolism in combined heterozygous factor V Leiden and prothrombin G2022110A

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**Chapter 4**

**Pharmacogenetics and the Treatment of Thrombophilia**

Inherited forms of thrombophilia such as factor V Leiden mutation (FVL), prothrombin gene mutation (PT 20210A), and deficiencies of natural anticoagulants protein C, protein S, and antithrombin are well known. DNA tests for factor V Leiden and PT 20210A mutation have been incorporated in clinical practice for several years [1,2,3]. A number of studies have analyzed how this and other molecular genetic testing alter the clinical management and treatment of patients with thromboembolic disease or pregnancy complications. Data regard‐ ing the influence of the genotype to the disease phenotype as well as pharmacogenetic data

Several topics are of particular interest. Usually genetic tests follow standard investigation of coagulation cascade, but some laboratories perform them in initially. Testing of first-degree relatives of a diagnosed carrier of a thrombophilic trait is still not consecutive. Administration of anticoagulant therapy is followed by genetic tests also; DNA variations are associated with variations in drug efficacy and toxicity, particularly in cases of warfarin and clopidogrel. Investigation of inherited thrombophilia and its treatment in women with reproductive challenges, including *in vitro* fertilization (IVF), is another important question. Finally, recommendation for genetic testing and treatment of thrombophilia in children, as vulnerable

**2. Thromophilia screening and treatment in asymptomatic adult carriers**

Thrombophilia testing is one of the most common genetic tests ordered by clinicians [4]. Current guidelines recommend screening for inherited thrombophilia only in selected group of patients with venous thromboembolism, dependently of the age of onset, the circumstances

and reproduction in any medium, provided the original work is properly cited.

© 2013 Novaković et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

of thrombosis, and the severity of the clinical manifestations [5,6].

Ivana Novaković, Nela Maksimović and Dragana Cvetković

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56566

are still controversial and emerging.

group, should be clarified.

**1. Introduction**


## **Pharmacogenetics and the Treatment of Thrombophilia**

Ivana Novaković, Nela Maksimović and Dragana Cvetković

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56566

## **1. Introduction**

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1998: 175–182.

lipid antibodies." Fertil Steril, 1996: 540–546.

Inherited forms of thrombophilia such as factor V Leiden mutation (FVL), prothrombin gene mutation (PT 20210A), and deficiencies of natural anticoagulants protein C, protein S, and antithrombin are well known. DNA tests for factor V Leiden and PT 20210A mutation have been incorporated in clinical practice for several years [1,2,3]. A number of studies have analyzed how this and other molecular genetic testing alter the clinical management and treatment of patients with thromboembolic disease or pregnancy complications. Data regard‐ ing the influence of the genotype to the disease phenotype as well as pharmacogenetic data are still controversial and emerging.

Several topics are of particular interest. Usually genetic tests follow standard investigation of coagulation cascade, but some laboratories perform them in initially. Testing of first-degree relatives of a diagnosed carrier of a thrombophilic trait is still not consecutive. Administration of anticoagulant therapy is followed by genetic tests also; DNA variations are associated with variations in drug efficacy and toxicity, particularly in cases of warfarin and clopidogrel. Investigation of inherited thrombophilia and its treatment in women with reproductive challenges, including *in vitro* fertilization (IVF), is another important question. Finally, recommendation for genetic testing and treatment of thrombophilia in children, as vulnerable group, should be clarified.

## **2. Thromophilia screening and treatment in asymptomatic adult carriers**

Thrombophilia testing is one of the most common genetic tests ordered by clinicians [4]. Current guidelines recommend screening for inherited thrombophilia only in selected group of patients with venous thromboembolism, dependently of the age of onset, the circumstances of thrombosis, and the severity of the clinical manifestations [5,6].

When the results of the index patients are positive asymptomatic relatives often come with requests for thrombophilia testing. To date, there is variety of published guidelines. However, the utility of family testing remains matter of debate and it should be done with caution. It is a general knowledge that genetic testing is justified only if the results are likely to change medical management. American College of Medical Genetics (ACMG) and Evaluation of Genomic Applications in Practice and Prevention (EGAPP) working group published consen‐ sus statements on FVL and FII. According to ACMG it is not recommended to perform random screening of general population or prenatal and routine newborn screening [7]. Based on the current knowledge, identification of thrombophilic disorders in asymptomatic individuals would not lead to long-term treatment with anticoagulants since the risk of bleeding is higher than the risk of venous thromboembolism (VTE) [7]. The overall annual incidence of the first VTE in individuals with antithrombin, protein C or protein S deficiency is ~1.5 %, whereas for the factor V Leiden or prothrombin 20210A mutation heterozygote this risk is ~0.5% [8]. Annual major bleeding risk associated with continuous anticoagulant treatment is around 2% and it overweighs the risk of VTE [9]. The results of Middeldorp et al. on asymptomatic carriers of FVL, are in agreement with the above and since there is no clear evidence of the benefit of thrombophylaxis they do not recommend routine screening of families of symptomatic patients [10]. Also, Coppens et al. do not recommend testing first degree relatives of probands with the prothrombin 20210A mutation based on the results of a large prospective cohort study in which the annual incidence of a first VTE in PT carriers was 0.37% [11]. For asymptomatic family members who are homozygous for FVL mutation the risk increases to closely 2%. According to EGAPP the risk is sufficient to consider anticoagulation therapy but there are still no data about the outcomes [12].

**3. Antithrombotic therapy and the promise of pharmacogenetics**

for individual patients [14-17].

**3.1. Warfarin: A case in point**

of major antithrombotic drugs (e.g. [21]).

to serious adverse effects, such as hemorrhage.

The expansion of pharmacogenetics, the study of genetic variants relevant to variations in drug efficacy and toxicity, and pharmacogenomics, referred to as a whole-genome application of pharmacogenetics, allowed rapid progress towards the goal of personalized therapy, tailored

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The research in this field provides large amounts of individual-specific information concerning risk for adverse reactions or lack of drug efficacy, thus it could have significant influence on clinical practice. The question of how to use the pharmacogenetic information to improve health outcomes gains continuously increasing attention [18-20]. Specifically, it has been shown that pharmacogenetic information has the potential to improve the efficacy and safety

One of the most compelling examples of potential benefits from pharmacogenomic testing is warfarin [22]. Warfarin is a widely prescribed oral anticoagulant; for decades it has been used as standard drug to prevent and treat thrombotic events in patients with deep vein thrombosis, various hypercoagulable states, atrial fibrillation, surgical cardiac valve replacement, etc.

One of the major problems with its use in clinical practice is large interindividual variation – patients differ in sensitivity to warfarin, hence the dose requirements vary widely (up to 20 fold) [19,23]. The consequences of over- or under-anticoagulation can be serious. In patients less sensitive than typical, the standard doses may be too low to achieve anticoagulation and therapeutic failure may occur, while in highly sensitive individuals the same doses may lead

Numerous factors are known to impact dose variation, including age, dietary vitamin K intake, presence of other comorbidities and interactions with other drugs, as well as genetic variants. The identification of these variants, and the potential use of pharmacogenetic testing to predict

Prior work pointed to significant genetic component underlying variations in warfarin sensitivity. Pharmacogenetic studies identified polymorphisms in genes *CYP2C9* and *VKORC1*

*CYP2C9* gene encodes one of the major cytochrome P450 drug-metabolizing enzymes; it is involved in metabolic clearance of S-warfarin, the more potent isomer of warfarin, which is largely responsible for its therapeutic effects. Two common alleles are described, *CYP2C9\*2* and *CYP2C9\*3*, based on non-synonymous SNPs that result in Arg144Cys (\*2) and Ile358Leu (\*3) substitutions; both variants are associated with reduced metabolic clearance of S-warfarin, thus lowering dose requirements [24]. Carriers of these variants show high sensitivity to drug and increased risk for hemorrhagic complications compared to individuals homozygous for allele \*1. It is estimated that SNPs in *CYP2C9* gene account for approximately 12% of the total

the appropriate drug dosing have attracted much research interest [23-28].

as principal genetic determinants of warfarin dose [23,24,29].

variance in required warfarin dose [23] (range 6–18%, [18]).

The practice of family testing has been most useful for women from thrombophilic families who intend to be pregnant. Affected female relatives with antithrombin, protein C and protein S deficiency as well as FVL and 20210A mutation carriers have VTE incidence as high as 4% per pregnancy while women homozygous for FVL have the risk of 16% per pregnancy in the absence of prophylaxis [13]. In these cases anticoagulant therapy, usually low molecular heparin injections, is frequently applied.

Genetic testing is also very useful for women from thrombophilic families who wish to use oral contraceptives. Use of oral contraceptives increases the risk of VTE for women with antithrombin, protein C or protein S protein deficiency or FVL and 20210A mutation. However, it is important to know that women from thrombophilic families are at the increased risk (compared with the general population) even if they do not have these specific deficiencies or mutations, due to the other cosegregating thrombophilic defects [8]. Thus, a negative throm‐ bophilia test may give them false reassurance.

Family testing may also help reduce VTE risk for women who tested positive through avoidance of postmenopausal hormone therapy. Advantages of testing are even higher for women considering postmenopausal hormone therapy than oral contraceptives, due to the much higher absolute risk of VTE in middle-aged than in younger women [13].

## **3. Antithrombotic therapy and the promise of pharmacogenetics**

The expansion of pharmacogenetics, the study of genetic variants relevant to variations in drug efficacy and toxicity, and pharmacogenomics, referred to as a whole-genome application of pharmacogenetics, allowed rapid progress towards the goal of personalized therapy, tailored for individual patients [14-17].

The research in this field provides large amounts of individual-specific information concerning risk for adverse reactions or lack of drug efficacy, thus it could have significant influence on clinical practice. The question of how to use the pharmacogenetic information to improve health outcomes gains continuously increasing attention [18-20]. Specifically, it has been shown that pharmacogenetic information has the potential to improve the efficacy and safety of major antithrombotic drugs (e.g. [21]).

## **3.1. Warfarin: A case in point**

When the results of the index patients are positive asymptomatic relatives often come with requests for thrombophilia testing. To date, there is variety of published guidelines. However, the utility of family testing remains matter of debate and it should be done with caution. It is a general knowledge that genetic testing is justified only if the results are likely to change medical management. American College of Medical Genetics (ACMG) and Evaluation of Genomic Applications in Practice and Prevention (EGAPP) working group published consen‐ sus statements on FVL and FII. According to ACMG it is not recommended to perform random screening of general population or prenatal and routine newborn screening [7]. Based on the current knowledge, identification of thrombophilic disorders in asymptomatic individuals would not lead to long-term treatment with anticoagulants since the risk of bleeding is higher than the risk of venous thromboembolism (VTE) [7]. The overall annual incidence of the first VTE in individuals with antithrombin, protein C or protein S deficiency is ~1.5 %, whereas for the factor V Leiden or prothrombin 20210A mutation heterozygote this risk is ~0.5% [8]. Annual major bleeding risk associated with continuous anticoagulant treatment is around 2% and it overweighs the risk of VTE [9]. The results of Middeldorp et al. on asymptomatic carriers of FVL, are in agreement with the above and since there is no clear evidence of the benefit of thrombophylaxis they do not recommend routine screening of families of symptomatic patients [10]. Also, Coppens et al. do not recommend testing first degree relatives of probands with the prothrombin 20210A mutation based on the results of a large prospective cohort study in which the annual incidence of a first VTE in PT carriers was 0.37% [11]. For asymptomatic family members who are homozygous for FVL mutation the risk increases to closely 2%. According to EGAPP the risk is sufficient to consider anticoagulation therapy but there are

The practice of family testing has been most useful for women from thrombophilic families who intend to be pregnant. Affected female relatives with antithrombin, protein C and protein S deficiency as well as FVL and 20210A mutation carriers have VTE incidence as high as 4% per pregnancy while women homozygous for FVL have the risk of 16% per pregnancy in the absence of prophylaxis [13]. In these cases anticoagulant therapy, usually low molecular

Genetic testing is also very useful for women from thrombophilic families who wish to use oral contraceptives. Use of oral contraceptives increases the risk of VTE for women with antithrombin, protein C or protein S protein deficiency or FVL and 20210A mutation. However, it is important to know that women from thrombophilic families are at the increased risk (compared with the general population) even if they do not have these specific deficiencies or mutations, due to the other cosegregating thrombophilic defects [8]. Thus, a negative throm‐

Family testing may also help reduce VTE risk for women who tested positive through avoidance of postmenopausal hormone therapy. Advantages of testing are even higher for women considering postmenopausal hormone therapy than oral contraceptives, due to the

much higher absolute risk of VTE in middle-aged than in younger women [13].

still no data about the outcomes [12].

68 Pregnancy Thrombophilia - The Unsuspected Risk

heparin injections, is frequently applied.

bophilia test may give them false reassurance.

One of the most compelling examples of potential benefits from pharmacogenomic testing is warfarin [22]. Warfarin is a widely prescribed oral anticoagulant; for decades it has been used as standard drug to prevent and treat thrombotic events in patients with deep vein thrombosis, various hypercoagulable states, atrial fibrillation, surgical cardiac valve replacement, etc.

One of the major problems with its use in clinical practice is large interindividual variation – patients differ in sensitivity to warfarin, hence the dose requirements vary widely (up to 20 fold) [19,23]. The consequences of over- or under-anticoagulation can be serious. In patients less sensitive than typical, the standard doses may be too low to achieve anticoagulation and therapeutic failure may occur, while in highly sensitive individuals the same doses may lead to serious adverse effects, such as hemorrhage.

Numerous factors are known to impact dose variation, including age, dietary vitamin K intake, presence of other comorbidities and interactions with other drugs, as well as genetic variants. The identification of these variants, and the potential use of pharmacogenetic testing to predict the appropriate drug dosing have attracted much research interest [23-28].

Prior work pointed to significant genetic component underlying variations in warfarin sensitivity. Pharmacogenetic studies identified polymorphisms in genes *CYP2C9* and *VKORC1* as principal genetic determinants of warfarin dose [23,24,29].

*CYP2C9* gene encodes one of the major cytochrome P450 drug-metabolizing enzymes; it is involved in metabolic clearance of S-warfarin, the more potent isomer of warfarin, which is largely responsible for its therapeutic effects. Two common alleles are described, *CYP2C9\*2* and *CYP2C9\*3*, based on non-synonymous SNPs that result in Arg144Cys (\*2) and Ile358Leu (\*3) substitutions; both variants are associated with reduced metabolic clearance of S-warfarin, thus lowering dose requirements [24]. Carriers of these variants show high sensitivity to drug and increased risk for hemorrhagic complications compared to individuals homozygous for allele \*1. It is estimated that SNPs in *CYP2C9* gene account for approximately 12% of the total variance in required warfarin dose [23] (range 6–18%, [18]).

Larger proportion of the dose variance, up to 30%, is explained by SNPs in the gene *VKORC1* [25,29]. *VKORC1* encodes vitamin K epoxide reductase complex, the target enzyme inhibited by warfarin; this enzyme is necessary for the recycling of vitamin K and consequently for activation of several clotting factors. Currently, several *VKORC1* SNPs are described (the major one being *VKORC1* -1639G>A, a common polymorphism of the promoter sequence) that define two common haplotypes, A and B. Haplotype A is associated with higher warfarin sensitivity, and hence lower mean drug doses required, contrary to B haplotype [29].

**4. Clinical application of pharmacogenetic testing — Promises and**

What are the promises and problems of the genotype-guided antithrombotic therapy? Pharmacogenetic testing has the potential to improve the efficacy and safety of warfarin and

Pharmacogenetics and the Treatment of Thrombophilia

http://dx.doi.org/10.5772/56566

71

Recognizing the significance of the genetic information, US FDA added it to warfarin label in 2007 and suggested that clinicians considered genetic testing before initiating therapy. Genetic tests for *CYP2C9* and *VKORC1* 'sensitivity' variants are available for clinical use, and so are dosing algorithms that combine genetic and clinical data [35,36]. Including *CYP4F2* rs2108622

However, the question of routine adoption of pharmacogenetic testing for warfarin sensitivity into clinical practice has led to vigorous debates. Numerous problems and challenges arise, from cost-effectiveness analyses, possibility of development of alternative drugs [27], com‐ plexity, quality and time demands, the need for additional education and training, to ethical

The major issue for clinical application of pharmacogenetic testing is that this approach must provide significant benefit to patients compared to nongenetic approach only. Cost-effective‐ ness emerges as another important question in modern health care; currently, discussions are focused on the cost of genetic testing *vs.* potential savings by reducing severe health compli‐ cations [18,19,31,37]. Also, the aim is to identify specific groups of patients who will benefit most from the pharmacogenetic testing [20], and to obtain diversity of warfarin dosing

A multicenter study, published in 2009 by the International Warfarin Pharmacogenetics Consortium, demonstrated that algorithms for warfarin dosing that incorporate pharmaco‐ genomic information were better than those using clinical data alone [35]. The greatest benefits were observed in patients with extreme (very low or very high) dose requirements. A recent Medco-Mayo Warfarin Effectiveness study demonstrated that application of warfarin geno‐ typing significantly reduced the incidence of hospitalizations due to bleeding and throm‐ boembolism [37]. Eckman and colleagues analyzed cost-effectiveness of using pharmacogenetic approach for patients with atrial fibrillation and concluded that genotype-

However, general consensus regarding these questions is lacking. The results of the ongoing studies and trials, conducted on large scales and diverse populations, are expected to clarify

With the current pace of pharmacogenetic discoveries, integrating the growing amount of individual-specific data into clinical practice to improve health outcome will remain the

**problems**

other antithrombotic drugs [21].

and regulatory issues [19,21,36].

these issues [21].

challenging task.

in testing procedures and algorithms is also suggested [27].

algorithms that should reflect genetic diversity of populations [28].

guided warfarin therapy might be cost effective in a high-risk group [31].

With respect to frequencies of these variants, genetic differences between populations are also a matter of great interest. The common *CYP2C9* alleles \*2 and \*3, associated with high warfarin sensitivity, are present in approximately 30% of people of European descent (range 13-35%), but are less frequent in those of Asian (1-12%) and African descent (0-12%) [21,25,30]. *VKORC1* B haplotype, associated with low warfarin sensitivity, is more common in European and African populations, while 'high sensitivity' A haplotype predominates in Asian populations. The frequency of A is reported as 75–92% in Asians, compared to approximately 40% in Europeans or 9–12% in people of African descent [21].

To predict response to treatment, considering polymorphisms in both genes simultaneous‐ ly is of great importance. Carriers of variants associated with 'high sensitivity' at both loci are at much higher risk of over-anticoagulation [31]. On the other hand, individuals who are *CYP2C9*\*1\*1-*VKORC1*BB show less warfarin sensitivity and require higher drug dose for therapeutic anticoagulation [25]. The associated variants in both genes are thought to account for approximately 45% of response variance in European and 30% in African populations [21].

The frequency of *VKORC1* and *CYP2C9* alleles was also investigated in Serbian population, among patients under oral anticoagulant therapy [32,33]. In a group of patients with extremely unstable anticoagulant response, 89.7% were carriers of 'sensitivity' alleles, and 25% carried these variants at both *CYP2C9* and *VKORC1* loci [33].

A recent genome wide association study (GWAS) by Takeuchi et al. confirmed polymorphisms in genes *VKORC1* and *CYP2C9* as principal genetic determinants of warfarin dose and also found weaker, but still significant effect of polymorphism in another CYP gene, *CYP4F2* [23]. The effect of *CYP4F2* rs2108622 was confirmed by other authors (e.g. [26,34]).

The results concerning possible contribution of other candidate genes are still inconsistent. The investigation of other SNPs and CNVs (copy number variations) did not reveal new significant warfarin associations [23], however, limited positive data was obtained for polymorphisms in additional candidate genes such as *POR* (encoding cytochrome P450 oxidoreductase) or *CALU* (encoding calumenin) (review in [27]).

The additional polymorphisms in these or other genes relevant to blood coagulation may be worth further investigation, especially in non-European populations that were less studied pharmacogenetically [27,28].

## **4. Clinical application of pharmacogenetic testing — Promises and problems**

Larger proportion of the dose variance, up to 30%, is explained by SNPs in the gene *VKORC1* [25,29]. *VKORC1* encodes vitamin K epoxide reductase complex, the target enzyme inhibited by warfarin; this enzyme is necessary for the recycling of vitamin K and consequently for activation of several clotting factors. Currently, several *VKORC1* SNPs are described (the major one being *VKORC1* -1639G>A, a common polymorphism of the promoter sequence) that define two common haplotypes, A and B. Haplotype A is associated with higher warfarin sensitivity,

With respect to frequencies of these variants, genetic differences between populations are also a matter of great interest. The common *CYP2C9* alleles \*2 and \*3, associated with high warfarin sensitivity, are present in approximately 30% of people of European descent (range 13-35%), but are less frequent in those of Asian (1-12%) and African descent (0-12%) [21,25,30]. *VKORC1* B haplotype, associated with low warfarin sensitivity, is more common in European and African populations, while 'high sensitivity' A haplotype predominates in Asian populations. The frequency of A is reported as 75–92% in Asians, compared to approximately 40% in

To predict response to treatment, considering polymorphisms in both genes simultaneous‐ ly is of great importance. Carriers of variants associated with 'high sensitivity' at both loci are at much higher risk of over-anticoagulation [31]. On the other hand, individuals who are *CYP2C9*\*1\*1-*VKORC1*BB show less warfarin sensitivity and require higher drug dose for therapeutic anticoagulation [25]. The associated variants in both genes are thought to account for approximately 45% of response variance in European and 30% in African

The frequency of *VKORC1* and *CYP2C9* alleles was also investigated in Serbian population, among patients under oral anticoagulant therapy [32,33]. In a group of patients with extremely unstable anticoagulant response, 89.7% were carriers of 'sensitivity' alleles, and 25% carried

A recent genome wide association study (GWAS) by Takeuchi et al. confirmed polymorphisms in genes *VKORC1* and *CYP2C9* as principal genetic determinants of warfarin dose and also found weaker, but still significant effect of polymorphism in another CYP gene, *CYP4F2* [23].

The results concerning possible contribution of other candidate genes are still inconsistent. The investigation of other SNPs and CNVs (copy number variations) did not reveal new significant warfarin associations [23], however, limited positive data was obtained for polymorphisms in additional candidate genes such as *POR* (encoding cytochrome P450 oxidoreductase) or *CALU*

The additional polymorphisms in these or other genes relevant to blood coagulation may be worth further investigation, especially in non-European populations that were less studied

The effect of *CYP4F2* rs2108622 was confirmed by other authors (e.g. [26,34]).

and hence lower mean drug doses required, contrary to B haplotype [29].

Europeans or 9–12% in people of African descent [21].

70 Pregnancy Thrombophilia - The Unsuspected Risk

these variants at both *CYP2C9* and *VKORC1* loci [33].

(encoding calumenin) (review in [27]).

pharmacogenetically [27,28].

populations [21].

What are the promises and problems of the genotype-guided antithrombotic therapy? Pharmacogenetic testing has the potential to improve the efficacy and safety of warfarin and other antithrombotic drugs [21].

Recognizing the significance of the genetic information, US FDA added it to warfarin label in 2007 and suggested that clinicians considered genetic testing before initiating therapy. Genetic tests for *CYP2C9* and *VKORC1* 'sensitivity' variants are available for clinical use, and so are dosing algorithms that combine genetic and clinical data [35,36]. Including *CYP4F2* rs2108622 in testing procedures and algorithms is also suggested [27].

However, the question of routine adoption of pharmacogenetic testing for warfarin sensitivity into clinical practice has led to vigorous debates. Numerous problems and challenges arise, from cost-effectiveness analyses, possibility of development of alternative drugs [27], com‐ plexity, quality and time demands, the need for additional education and training, to ethical and regulatory issues [19,21,36].

The major issue for clinical application of pharmacogenetic testing is that this approach must provide significant benefit to patients compared to nongenetic approach only. Cost-effective‐ ness emerges as another important question in modern health care; currently, discussions are focused on the cost of genetic testing *vs.* potential savings by reducing severe health compli‐ cations [18,19,31,37]. Also, the aim is to identify specific groups of patients who will benefit most from the pharmacogenetic testing [20], and to obtain diversity of warfarin dosing algorithms that should reflect genetic diversity of populations [28].

A multicenter study, published in 2009 by the International Warfarin Pharmacogenetics Consortium, demonstrated that algorithms for warfarin dosing that incorporate pharmaco‐ genomic information were better than those using clinical data alone [35]. The greatest benefits were observed in patients with extreme (very low or very high) dose requirements. A recent Medco-Mayo Warfarin Effectiveness study demonstrated that application of warfarin geno‐ typing significantly reduced the incidence of hospitalizations due to bleeding and throm‐ boembolism [37]. Eckman and colleagues analyzed cost-effectiveness of using pharmacogenetic approach for patients with atrial fibrillation and concluded that genotypeguided warfarin therapy might be cost effective in a high-risk group [31].

However, general consensus regarding these questions is lacking. The results of the ongoing studies and trials, conducted on large scales and diverse populations, are expected to clarify these issues [21].

With the current pace of pharmacogenetic discoveries, integrating the growing amount of individual-specific data into clinical practice to improve health outcome will remain the challenging task.

## **5. Genetics and treatment of reproductive adversity in thrombophilia**

Clinical manifestations and morbidity associated with thrombophilia in pregnancy include pregnancy loss, as well as other adverse outcomes eg. preeclampsia, placental abruption, and intrauterine growth restriction. Pregnancy-related thromboembolism is also part of thrombo‐ philia spectrum making the influence of thrombophilia in pregnancy is an important and interesting research topic.

were analyzed: factor V Leiden, factor V H1299R (R2), factor V Y1702C, prothrombin gene G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, human platelet alloantigen a/b (L33P), methylenetetrahydrofolate reductase C677T and A1298C [45]. There were no differences in the frequency of specific mutations in women with recurrent miscarriage compared to healthy control. However, the prevalence of homozygous mutations and total gene mutations was significantly higher in patients compared to controls. Homozygous mutations were found in 59% of women with a history of recurrent pregnancy loss vs. 10% of control women. More than three gene mutations were observed in 68% of women with recurrent miscarriage compared to 21% of controls. It would be of especial interest to explore how number of detected mutations influences effects of prophylactic therapy and further

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The possible connection between inherited thrombophilia and outcomes of *in vitro* fertili‐ zation (IVF) is another challenging topic. A number of investigations suggest no associa‐ tion of thrombophilic mutations and IVF pregnancy failure [46,47]. Rudick et al. found a very low prevalence of FVL mutation in women in their IVF program (1.6%), and sug‐ gested a positive association between this genetic marker and pregnancy [47]. The au‐ thors suggested that routine testing in a general IVF population for FVL mutation as a cause of IVF failure and infertility is not indicated. Ricci et al. compared the prevalence of FVL and PTG mutation in women undergoing IVF to women with spontaneous pregnan‐ cy, as well as IVF outcomes and the risk of complications in FVL and PTG carriers to non-carriers [48]. In this prospective cohort study they found the same prevalence of thrombophilic mutations in women requiring IVF and in women with spontaneous preg‐ nancy. The results of this study also suggested the presence of FVL and PTG in asympto‐ matic women and in the absence of other risk factors did not influence IVF outcome, represent a risk for ovarian hyperstimulation syndrome, or favor thrombosis after IVF. According to these authors, screening for FVL and PTG does not appear to be justified to

identify the patients at the risk for IVF failure or associated complications.

**6. Thrombophilia screening in asymptomatic children**

However, some studies have shown positive effects of LMWH treatment for women with thrombophilia and recurrent IVF- embrio transfer failures [49,50]. In one prospective random‐ ized placebo-controlled trial Qublan et al. observed that implantation rate, pregnancy and live birth rates are significantly increased with LMWH compared to placebo [49]. At this moment, diagnostic tools to identify patients at risk of implantation failure are still limited and thera‐ peutic options to improve implantation rates are far from being established. In addition to genetic markers of thrombophilia and thromboprophylaxis, different immunological mecha‐ nisms and consecutive immunomodulatory treatments are the subjects of intensive investiga‐

Parents with known specific thrombophilic defect frequently ask whether or not their child(ren) should also be screened for thrombophilia. Many of them are concerned about their

reproductive outcome.

tions [51].

The effect of preventive anticoagulant therapy during the pregnancy in women with inherited thrombophila is still controversial. Early investigations were characterized by small partici‐ pant numbers, poor study design and heterogeneity. The debate on the efficacy of aspirin and heparin has advanced with recently published randomised-controlled trials. One large Italian study encompassed 1011 pregnancies of 416 women who were carriers of factor V Leiden (FVL) mutation and/or prothrombin gene variant G20210A (PTG) [38]. The outcome was evaluated according to the type of treatment (low molecular weight heparin and/or aspirin) and the period of pregnancy when the treatment started. The results showed that low molecular weight heparin (LMWH) had a protective effect on miscarriages (odds ratio, OR 0.52) and venous thromboembolism (OR 0.05) while aspirin administration showed no advantage on the prevention of obstetric complications and venous thromboembolism (OR 2.2 and 0.48, respectively). These results suggest that LMWH prophylaxis reduces the risk of obstetric complications in carriers of FVL and/or PTG, particularly in those with previous obstetric events. Mitic et al. also reported significant improvement of pregnancy outcome after imple‐ mentation of thromboprophylaxis in Serbian patients with inherited thrombophilia and previous pregnancy losses [39].

One Bulgarian group reported their first experience with management of inherited thrombo‐ philia during pregnancy [40]. After the testing for factor V Leiden, prothrombin G20210A, plasminogen activator inhibitor-1 (PAI-1) 4G/4G and PAI-1 4G/5G they established a diagnosis of inherited thrombophilia in 72% (24 out of 38) patients with history of an abnormal pregnancy (miscarriage, still birth, placental abruption, preeclampsia and intrauterine fetal growth restriction). All diagnosed patients were treated with aspirin (75mg) prior to conception and low molecular heparin after detection of fetal heart sounds. Anticoagulant treatment of these patients was deemed successful with 87.5% (21 out of 24) giving birth to a term newborn.

However, several investigators have reported confounding experiences [41-43]. In a recently published review, de Jong et al suggest that the association between inherited thrombophilia and recurrent miscarriage is not very strong, and the evidence does not indicate that the use of anticoagulants improves the chance of live birth in these women [41]. The authors conclude that by the current state of evidence, testing for inherited thrombophilia should not lead to altered clinical management and so, should not be performed routinely in women with recurrent miscarriage. In light of the available data, a well-designed, multi-center collaboration is required to ascertain the effect of inherited thrombophilia on early pregnancy loss and to establish evidence-based treatment recommendations [44].

It may be possible that in women with recurrent pregnancy loss multiple thrombophilic gene mutations rather than specific single gene changes play a role. In one study, 10 gene mutations were analyzed: factor V Leiden, factor V H1299R (R2), factor V Y1702C, prothrombin gene G20210A, factor XIII V34L, beta-fibrinogen -455G>A, PAI-1 4G/5G, human platelet alloantigen a/b (L33P), methylenetetrahydrofolate reductase C677T and A1298C [45]. There were no differences in the frequency of specific mutations in women with recurrent miscarriage compared to healthy control. However, the prevalence of homozygous mutations and total gene mutations was significantly higher in patients compared to controls. Homozygous mutations were found in 59% of women with a history of recurrent pregnancy loss vs. 10% of control women. More than three gene mutations were observed in 68% of women with recurrent miscarriage compared to 21% of controls. It would be of especial interest to explore how number of detected mutations influences effects of prophylactic therapy and further reproductive outcome.

**5. Genetics and treatment of reproductive adversity in thrombophilia**

interesting research topic.

72 Pregnancy Thrombophilia - The Unsuspected Risk

previous pregnancy losses [39].

establish evidence-based treatment recommendations [44].

Clinical manifestations and morbidity associated with thrombophilia in pregnancy include pregnancy loss, as well as other adverse outcomes eg. preeclampsia, placental abruption, and intrauterine growth restriction. Pregnancy-related thromboembolism is also part of thrombo‐ philia spectrum making the influence of thrombophilia in pregnancy is an important and

The effect of preventive anticoagulant therapy during the pregnancy in women with inherited thrombophila is still controversial. Early investigations were characterized by small partici‐ pant numbers, poor study design and heterogeneity. The debate on the efficacy of aspirin and heparin has advanced with recently published randomised-controlled trials. One large Italian study encompassed 1011 pregnancies of 416 women who were carriers of factor V Leiden (FVL) mutation and/or prothrombin gene variant G20210A (PTG) [38]. The outcome was evaluated according to the type of treatment (low molecular weight heparin and/or aspirin) and the period of pregnancy when the treatment started. The results showed that low molecular weight heparin (LMWH) had a protective effect on miscarriages (odds ratio, OR 0.52) and venous thromboembolism (OR 0.05) while aspirin administration showed no advantage on the prevention of obstetric complications and venous thromboembolism (OR 2.2 and 0.48, respectively). These results suggest that LMWH prophylaxis reduces the risk of obstetric complications in carriers of FVL and/or PTG, particularly in those with previous obstetric events. Mitic et al. also reported significant improvement of pregnancy outcome after imple‐ mentation of thromboprophylaxis in Serbian patients with inherited thrombophilia and

One Bulgarian group reported their first experience with management of inherited thrombo‐ philia during pregnancy [40]. After the testing for factor V Leiden, prothrombin G20210A, plasminogen activator inhibitor-1 (PAI-1) 4G/4G and PAI-1 4G/5G they established a diagnosis of inherited thrombophilia in 72% (24 out of 38) patients with history of an abnormal pregnancy (miscarriage, still birth, placental abruption, preeclampsia and intrauterine fetal growth restriction). All diagnosed patients were treated with aspirin (75mg) prior to conception and low molecular heparin after detection of fetal heart sounds. Anticoagulant treatment of these patients was deemed successful with 87.5% (21 out of 24) giving birth to a term newborn. However, several investigators have reported confounding experiences [41-43]. In a recently published review, de Jong et al suggest that the association between inherited thrombophilia and recurrent miscarriage is not very strong, and the evidence does not indicate that the use of anticoagulants improves the chance of live birth in these women [41]. The authors conclude that by the current state of evidence, testing for inherited thrombophilia should not lead to altered clinical management and so, should not be performed routinely in women with recurrent miscarriage. In light of the available data, a well-designed, multi-center collaboration is required to ascertain the effect of inherited thrombophilia on early pregnancy loss and to

It may be possible that in women with recurrent pregnancy loss multiple thrombophilic gene mutations rather than specific single gene changes play a role. In one study, 10 gene mutations The possible connection between inherited thrombophilia and outcomes of *in vitro* fertili‐ zation (IVF) is another challenging topic. A number of investigations suggest no associa‐ tion of thrombophilic mutations and IVF pregnancy failure [46,47]. Rudick et al. found a very low prevalence of FVL mutation in women in their IVF program (1.6%), and sug‐ gested a positive association between this genetic marker and pregnancy [47]. The au‐ thors suggested that routine testing in a general IVF population for FVL mutation as a cause of IVF failure and infertility is not indicated. Ricci et al. compared the prevalence of FVL and PTG mutation in women undergoing IVF to women with spontaneous pregnan‐ cy, as well as IVF outcomes and the risk of complications in FVL and PTG carriers to non-carriers [48]. In this prospective cohort study they found the same prevalence of thrombophilic mutations in women requiring IVF and in women with spontaneous preg‐ nancy. The results of this study also suggested the presence of FVL and PTG in asympto‐ matic women and in the absence of other risk factors did not influence IVF outcome, represent a risk for ovarian hyperstimulation syndrome, or favor thrombosis after IVF. According to these authors, screening for FVL and PTG does not appear to be justified to identify the patients at the risk for IVF failure or associated complications.

However, some studies have shown positive effects of LMWH treatment for women with thrombophilia and recurrent IVF- embrio transfer failures [49,50]. In one prospective random‐ ized placebo-controlled trial Qublan et al. observed that implantation rate, pregnancy and live birth rates are significantly increased with LMWH compared to placebo [49]. At this moment, diagnostic tools to identify patients at risk of implantation failure are still limited and thera‐ peutic options to improve implantation rates are far from being established. In addition to genetic markers of thrombophilia and thromboprophylaxis, different immunological mecha‐ nisms and consecutive immunomodulatory treatments are the subjects of intensive investiga‐ tions [51].

## **6. Thrombophilia screening in asymptomatic children**

Parents with known specific thrombophilic defect frequently ask whether or not their child(ren) should also be screened for thrombophilia. Many of them are concerned about their children's health, mostly the risk of having VTE or reproductive issues, especially if the mother was diagnosed during pregnancy or after several pregnancy losses. Genetic testing is partic‐ ularly controversial in children since their decision-making capability is non-existent or is limited [52].

limited cases, the presence of inherited thrombophilia might lead to targeted thrombophylaxis in high risk situations, e.g., after a femur fracture in an obese teenager, though there are few

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75

It is of major importance to provide genetic counseling to patients as well as to their asymp‐ tomatic family members who are interested in thrombophilia testing, including pharmacoge‐ netic tests. Based on detailed information about a family history, personal history and the reasons for testing genetic counselor should provide education and support for the family members. During the pre-test genetic counseling patient or family member should understand that the testing is optional and that it will be performed only after signed informed consent. It must be clarified that this is a testing for susceptibility gene and not for the disease state and that an individual's thrombotic risk is determined by a complex interplay of genetic, acquired and circumstantial risk factors [1]. It must be clear to the family member that if thrombophilia mutation is inherited the risk of VTE is higher than it is in the general population but although the inheritance pattern is dominant the penetrance of the mutation is not 100%. In order to achieve a better understanding of potential risk when counseling a family member regarding the risk of thrombosis it is most useful to provide the absolute risk (e.g., incidence) of throm‐ bosis among persons with particular thrombophilia [61]. Pre-test genetic counseling should include discussion not only about the risks but also about the benefits and limitations of testing for the patient and for the entire family. Asymptomatic family member should understand that testing for thrombophilia may have lower benefit to risk ratio as compared to symptomatic relative [62]. Post-test counseling is equally as important for family members who tested positive and negative. In case when the result is negative family members should understand that currently available tests might not identify all inherited risk factors for thrombosis [52]. In the other case discussion should include signs and symptoms of thrombosis, risk factors to avoid and the risks and benefits of prophylactic therapy [63]. Clinical geneticist should also be aware of psychological response of the tested individual. Results of the study of Louzada et al. do not support the concern that asymptomatic relatives are at risk of psychological distress as a consequence of thrombophilia screening [64]. However it is general conclusion that characteristics of the genetic predisposition, including the likelihood of developing the disease, perceived severity and availability of treatments for the condition are likely contrib‐ utors to the psychological response [64]. It means that adequate genetic counseling is of key importance for education of family members, in order to increase their awareness of risk factors

As a conclusion, genetic tests are part of modern management and treatment of thrombophilia, but several medical and ethical dilemmas are still open. Healthcare professionals should apply evidence-based guidelines regarding indications for genetic and pharmacogenetic testing, as well as principles of genetic counseling in thrombophilia. In the upcoming era of personalized genomic medicine, genetic tests day after day become more available, but their real power and

data to document the efficacy of this approach [60].

and effective interventions to prevent VTE.

relevance is fully expressed in the context of clinical data.

**7. Genetic counseling**

The recommendation of The American Academy of Pediatrics (AAP) and the ACMG is that predictive genetic testing for late-onset disorders should not be performed unless there is a specific intervention during childhood that will reduce morbidity or mortality [53,54]. Also, the AAP does not support the broad use of carrier testing or screening in children or adolescents. As for any genetic testing, a medical benefit should be the pri‐ mary justification for testing in children and adolescents. It is very important for parents to understand the limitations of testing before they sign informed consent for their chil‐ dren. The results of thrombophilia testing rarely influence medical management decisions and at the moment there is no evidence that thrombophilia testing could benefit a young healthy child. The incidence of venous thrombosis in healthy children is extremely low (0.07/100000), and the long-term use of anticoagulants in an asymptomatic healthy child would be unjustified [55].

Tormene et al. performed a prospective cohort study of children aged 1-14 years from families with a single identified inherited thrombophilia. The children were tested for FVL, prothrom‐ bin G20210A mutations and antithrombin, protein C and protein S deficiency and followed for the evidence of thrombosis 1-8 years (mean 5 years). No children with or without throm‐ bophilia developed VTE during the study period [56]. Thrombophilia testing could show more benefit for children with the acute or chronic medical conditions. The overwhelming majority of pediatric TEs are associated with central venous lines (CVLs) [52].

Other acquired risk factors depend on the age of the child. Within the entire childhood population neonates are at the greatest risk of thromboembolism (5.1/100 000 live births per year in white children) [57]. Neonatal risk factors include birth asphyxia, respiratory distress syndrome, maternal diabetes, infections, necrotizing enterocolitis, dehydration, congenital nephrotic syndrome and polycythemia [57]. Children of any age may have an‐ tiphospholipid or anticardiolipin antibodies which are associated with thrombophilia [52]. Meta-analysis of Young et al. on impact of inherited thrombophilia on venous throm‐ boembolism in children showed significant association with recurrent VTE for all inherit‐ ed thrombophilia traits except the factor V variant and elevated lipoprotein (a) [58]. A second peak of incidence of thrombosis is during adolescence [59]. Adolescents may have the same risk factors as the adults including smoking, pregnancy, obesity, and oral con‐ traceptives which increase the risk of thrombosis [52]. Adolescents identified with an in‐ herited thrombophilia may benefit from avoiding high-risk situations (prolonged immobility, dehydration), pursuing healthy lifestyles (regular exercise and weight con‐ trol), and recognizing early signs and symptoms of VTE [60].

There are some situations in which the presence of an inherited defect may influence medical decision making. The first is in an adolescent female who is interested in using oral contra‐ ceptive pills (OCPs). Knowledge of a congenital thrombophilia provide the opportunity to consider lower-risk alternatives for contraception, such as progesterone-only preparations. In limited cases, the presence of inherited thrombophilia might lead to targeted thrombophylaxis in high risk situations, e.g., after a femur fracture in an obese teenager, though there are few data to document the efficacy of this approach [60].

## **7. Genetic counseling**

children's health, mostly the risk of having VTE or reproductive issues, especially if the mother was diagnosed during pregnancy or after several pregnancy losses. Genetic testing is partic‐ ularly controversial in children since their decision-making capability is non-existent or is

The recommendation of The American Academy of Pediatrics (AAP) and the ACMG is that predictive genetic testing for late-onset disorders should not be performed unless there is a specific intervention during childhood that will reduce morbidity or mortality [53,54]. Also, the AAP does not support the broad use of carrier testing or screening in children or adolescents. As for any genetic testing, a medical benefit should be the pri‐ mary justification for testing in children and adolescents. It is very important for parents to understand the limitations of testing before they sign informed consent for their chil‐ dren. The results of thrombophilia testing rarely influence medical management decisions and at the moment there is no evidence that thrombophilia testing could benefit a young healthy child. The incidence of venous thrombosis in healthy children is extremely low (0.07/100000), and the long-term use of anticoagulants in an asymptomatic healthy child

Tormene et al. performed a prospective cohort study of children aged 1-14 years from families with a single identified inherited thrombophilia. The children were tested for FVL, prothrom‐ bin G20210A mutations and antithrombin, protein C and protein S deficiency and followed for the evidence of thrombosis 1-8 years (mean 5 years). No children with or without throm‐ bophilia developed VTE during the study period [56]. Thrombophilia testing could show more benefit for children with the acute or chronic medical conditions. The overwhelming majority

Other acquired risk factors depend on the age of the child. Within the entire childhood population neonates are at the greatest risk of thromboembolism (5.1/100 000 live births per year in white children) [57]. Neonatal risk factors include birth asphyxia, respiratory distress syndrome, maternal diabetes, infections, necrotizing enterocolitis, dehydration, congenital nephrotic syndrome and polycythemia [57]. Children of any age may have an‐ tiphospholipid or anticardiolipin antibodies which are associated with thrombophilia [52]. Meta-analysis of Young et al. on impact of inherited thrombophilia on venous throm‐ boembolism in children showed significant association with recurrent VTE for all inherit‐ ed thrombophilia traits except the factor V variant and elevated lipoprotein (a) [58]. A second peak of incidence of thrombosis is during adolescence [59]. Adolescents may have the same risk factors as the adults including smoking, pregnancy, obesity, and oral con‐ traceptives which increase the risk of thrombosis [52]. Adolescents identified with an in‐ herited thrombophilia may benefit from avoiding high-risk situations (prolonged immobility, dehydration), pursuing healthy lifestyles (regular exercise and weight con‐

There are some situations in which the presence of an inherited defect may influence medical decision making. The first is in an adolescent female who is interested in using oral contra‐ ceptive pills (OCPs). Knowledge of a congenital thrombophilia provide the opportunity to consider lower-risk alternatives for contraception, such as progesterone-only preparations. In

of pediatric TEs are associated with central venous lines (CVLs) [52].

trol), and recognizing early signs and symptoms of VTE [60].

limited [52].

74 Pregnancy Thrombophilia - The Unsuspected Risk

would be unjustified [55].

It is of major importance to provide genetic counseling to patients as well as to their asymp‐ tomatic family members who are interested in thrombophilia testing, including pharmacoge‐ netic tests. Based on detailed information about a family history, personal history and the reasons for testing genetic counselor should provide education and support for the family members. During the pre-test genetic counseling patient or family member should understand that the testing is optional and that it will be performed only after signed informed consent. It must be clarified that this is a testing for susceptibility gene and not for the disease state and that an individual's thrombotic risk is determined by a complex interplay of genetic, acquired and circumstantial risk factors [1]. It must be clear to the family member that if thrombophilia mutation is inherited the risk of VTE is higher than it is in the general population but although the inheritance pattern is dominant the penetrance of the mutation is not 100%. In order to achieve a better understanding of potential risk when counseling a family member regarding the risk of thrombosis it is most useful to provide the absolute risk (e.g., incidence) of throm‐ bosis among persons with particular thrombophilia [61]. Pre-test genetic counseling should include discussion not only about the risks but also about the benefits and limitations of testing for the patient and for the entire family. Asymptomatic family member should understand that testing for thrombophilia may have lower benefit to risk ratio as compared to symptomatic relative [62]. Post-test counseling is equally as important for family members who tested positive and negative. In case when the result is negative family members should understand that currently available tests might not identify all inherited risk factors for thrombosis [52]. In the other case discussion should include signs and symptoms of thrombosis, risk factors to avoid and the risks and benefits of prophylactic therapy [63]. Clinical geneticist should also be aware of psychological response of the tested individual. Results of the study of Louzada et al. do not support the concern that asymptomatic relatives are at risk of psychological distress as a consequence of thrombophilia screening [64]. However it is general conclusion that characteristics of the genetic predisposition, including the likelihood of developing the disease, perceived severity and availability of treatments for the condition are likely contrib‐ utors to the psychological response [64]. It means that adequate genetic counseling is of key importance for education of family members, in order to increase their awareness of risk factors and effective interventions to prevent VTE.

As a conclusion, genetic tests are part of modern management and treatment of thrombophilia, but several medical and ethical dilemmas are still open. Healthcare professionals should apply evidence-based guidelines regarding indications for genetic and pharmacogenetic testing, as well as principles of genetic counseling in thrombophilia. In the upcoming era of personalized genomic medicine, genetic tests day after day become more available, but their real power and relevance is fully expressed in the context of clinical data.

## **Acknowledgements**

This work was supported by Ministry of Education and Science, Republic of Serbia (Grant No. 175091).

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Pharmacogenetics and the Treatment of Thrombophilia

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77

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## **Author details**

Ivana Novaković<sup>1</sup> , Nela Maksimović<sup>1</sup> and Dragana Cvetković<sup>2</sup>

1 Faculty of Medicine, University of Belgrade, Belgrade, Serbia

2 Faculty of Biology, University of Belgrade, Belgrade, Serbia

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**Acknowledgements**

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**Chapter 5**

**Antiphospholipid Antibodies Syndrome and**

**Aspirin and Heparin**

Nicoletta Di Simone

**1. Introduction**

http://dx.doi.org/10.5772/56862

criterion is required [2] (Table 1).

Chiara Tersigni, Silvia D'Ippolito and

Additional information is available at the end of the chapter

glycoprotein I [β2GPI] or lupus anticoagulants) [1-2].

**Reproductive Failures: New Therapeutic Trends Beyond**

The most investigated thrombophilia related to obstetrical complications is the antiphospho‐ lipid antibodies syndrome (APS), also known as Hughes' syndrome. APS is characterized by recurrent thrombosis (arterial or venous, or both) and/or morbidity during pregnancy (losses during early and late pregnancy and pre-eclampsia) associated with moderate to high plasma levels of antiphospholipid (aPL) antibodies (anticardiolipin antibodies, antibodies to β2

According to the last International consensus statement for APS diagnostic criteria, in order to make diagnosis of the syndrome, the combination of at least one clinical and one laboratory

Since aPL antibodies have thrombogenic properties, intraplacental thrombosis with maternal– fetal blood exchange impairment was traditionally suggested to be the main pathogenic mechanism responsible of fetal loss in patients with APS, providing the rationale for the use

Although the management of aPL antibodies-positive pregnant patients is controversial due to the limited well-designed controlled trials, the current recommendation is to use low-dose aspirin and prophylactic or therapeutic doses of heparin for patients fulfilling the updated Sapporo APS classification criteria [2] and no treatment for asymptomatic (no history of pregnancy complications and/or thrombosis) persistently aPL antibodies-positive patients [6].

and reproduction in any medium, provided the original work is properly cited.

© 2013 Tersigni et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

of aspirin or heparin to prevent adverse pregnancy outcomes in APS [3-5].
