Kathleen E. Puca, M.D., ITxM Fellow

INTRODUCTION

In normal pregnancy, levels of most procoagulant factors (factors VII, VIII, X, XII, and fibrinogen) rise, while anticoagulant proteins either remain unchanged (protein C, antithrombin) or decrease (protein S).  The fibrinolytic system is also altered, with increased levels of plasminogin activator inhibitor (PAI). These hemostatic changes can result in a hypercoagulable state. It has been postulated that maternal thrombophilic defects may exaggerate this hemostatic response and lead to an adverse pregnancy outcome.    

Recurrent pregnancy loss (RPL) occurs in up to 5% of couples with two or more losses. RPL has various causes, but in many women the cause cannot be found.    While acquired antiphospholipid syndrome (APS) is a well-recognized cause; recent evidence indicates that hereditary thrombophilia, which may predispose to thrombosis within the placental vasculature and inadequate placental circulation, may also result in RPL.

ANTIPHOSPHOLIPID SYNDROME (APS)

APS is an autoimmune disorder characterized by the presence of persistent significant levels of antiphospholipid antibodies (APA) and one or more clinical features, including RPL, unexplained fetal death or thrombosis. APA are a heterogenous group of antibodies directed against negatively charged phospholipids or phospholipid-protein complexes, categorized by their  laboratory detection as lupus anticoagulant (LA) and/or anticardiolipin antibodies (ACA).  Likely targets of APA are b2-glycoprotein I (b2-GPI) and prothrombin. Recent studies have suggested that APA may displace or reduce annexin-V (a thromboprotective agent) on the surface of placental villi and thereby promote thrombosis and lead to placental insufficiency.¹ 

Although, the frequency of APA in the general population is estimated at 1-2%, up to 20% of women with unexplained RPL have either LA or ACA.  In untreated women with persistent APA and a history of RPL, the prospective fetal loss rate has been as high as 90%.  Though it is well established that elevated levels of APA are associated with unexplained midtrimester fetal loss, some studies have reported increased prevalence of APA in 1^st trimester pregnancy loss as well.^1,2

HEREDITARY THROMBOPHILIA

Over the past few years, several studies have reported a potential association of inherited thrombophilia with RPL.   The European Prospective Cohort on Thrombophilia (EPCOT) study revealed that significantly more women with thrombophilia than controls had experienced fetal loss. The association was highest for stillbirth (>28 weeks gestation) and in patients with combined thrombophilic defects.³ Similarly, a recent study showed that at least one thrombophilic defect was found in 66% of the 145 women with unexplained RPL compared with only 28% of the 145 matched parous controls.  Further, combined thrombophilic defects were more common in women with RPL, and women with thrombophilia had a higher percentage of late (2^nd and 3^rd trimester) pregnancy loss.⁴ 

APC RESISTANCE AND FACTOR V LEIDEN

Protein C, when activated, inhibits the actions of factors V and VIII.  Resistance to the anticoagulant properties of activated protein C is known as APC resistance (APCr) and may be either congenital or acquired.  Congenital APCr is almost exclusively due to a single point mutation  in the factor V gene, known as factor V Leiden (FVL). FVL mutation (FVLm) is the most common inherited thrombophilia, with 3-8% of Caucasians being heterozygous and accounting for 20-25% of venous thrombosis. Acquired APCr is associated with APS and high concentrations of factor VIII. APCr may also be acquired during the 2^nd and 3^rd trimester of normal pregnancy, as a result of increased factor VIII levels and decreased levels of protein S. 

APCr with or without FVLm is significantly more common in women with either early or late RPL.^4,5  Although the association of FVLm and early pregnancy loss is controversial, several studies have confirmed that FVL is associated with a 2 to 3-fold increase in risk of RPL in the 2^nd and 3^rd trimester.^2,4,6 In women with RPL, there appears to be a high incidence of APCr, which cannot be explained solely by FVLm, and may be related to other factor V polymorphisms, exaggerated physiologic changes in pregnancy, APS, or other unknown prothrombotic factors.

PROTHROMBIN GENE MUTATION

The prothrombin G20210A mutation (PGM) is a point mutation at nucleotide position 20210 in the prothrombin gene.  This mutation is associated with elevated plasma prothrombin levels (>130%), and increased risk of venous and arterial thrombosis.  It is present in 2 to 3% of Caucasians, and has been identified in 6% of subjects presenting with a first venous thromboembolism.  After FVL, it is the second most common identified independent risk factor for venous thrombosis.  

It is unclear whether this genetic mutation is a significant independent risk factor for fetal loss. Several studies have evaluated the potential role of PGM in RPL, and while some have demonstrated PGM to be more common in women with late fetal loss; other studies have shown no difference in the prevalence of PGM between women with RPL (primarily 1^st trimester) and matched controls.^1,2,4,6  Until additional studies verify the association of PGM and RPL,  PGM is potentially a risk factor in late fetal loss and may contribute to pregnancy loss when present with other thrombophilic defects.

HYPERHOMOCYSTEINEMIA AND MTHFR

Methylene tetrahydrofolate reductase (MTHFR) is an enzyme involved in regulating the circulating levels of homocysteine.  Folic acid, vitamin B6 and B12 are also important cofactors in this metabolic pathway.  A mutation in the MTHFR gene causes the enzyme to be thermolabile with mild loss of activity.  Homozygosity for this mutation is present in approximately 11% of the Caucasian population and can lead to hyperhomocysteinemia when there is concomitant nutritional folate deficiency.  Deficiencies of vitamin B6, B12, and folic acid can also result in elevated plasma homocysteine levels (>15 mmol/L).   

In women, hyperhomocysteinemia is associated with increased risk of having a child with a neural tube defect or RPL.  However, folate supplementation during pregnancy lowers homocysteine concentration and tends to reduce the risk of these adverse pregnancy outcomes. The association of MTHFR gene mutation with recurrent pregnancy loss is not well established, and may be related to the fact that even in the presence of homozygosity for this mutation, dietary folic acid supplement will lower the homocysteine level. Earlier studies showed that women with recurrent pregnancy losses before 17 weeks gestation (with associated high homocysteine levels and lower folate concentrations) were 2-3 times more likely to be homozygous for MTHFR than matched controls.  However, more recent studies have demonstrated that homozygosity for MTHFR mutation contributes to pregnancy loss only in the presence of other thrombophilic risk factors.^1,4,7 

PROTEIN C, PROTEIN S, AND ANTITHROMBIN

Protein C (PC) and its cofactor, Protein S (PS), are naturally occurring anticoagulants. Together, they inactivate Factor V and Factor VIII, thereby preventing further clot formation.  The prevalence of heterozygous PC deficiency in the general population is approximately 0.3%, whereas the exact prevalence of heterozygous PS is unknown, but is likely to be similar to PC.  Deficiency of PC and PS are present in approximately 3% of patients with thrombosis.  Antithrombin (AT) is the most important physiological inhibitor, inactivating thrombin and factors Xa, IXa, and XIa.  AT deficiency is present in 0.02-0.17% of patients with venous thrombosis and is the most thrombogenic of the inherited thrombophilias, with a 70-90% lifetime risk of thrombosis.   

There is wide consensus regarding the relationship between AT, PC, or PS abnormalities and the risk for fetal loss.  Data from the EPCOT study showed  increased fetal loss, particularly late fetal loss (>28 weeks gestation), among women with AT, PC and PS deficiency. The risk of fetal loss was higher for women with AT deficiency, or with combined defects.³ Overall, AT deficiency appears to be a more significant risk factor for RPL (probably due to it being more thrombogenic) than either PC or PS deficiency.  Due to their low prevalence, though, they are rarely seen.

CONCLUSION

APS and certain inherited thrombophilia are potential causes for fetal loss.  Laboratory evaluation is indicated in women with RPL, particularly in cases of 2^nd and 3^rd trimester loss.  Initial laboratory evaluation may include: LAC, ACA IgG and IgM, APCr, FVL, PGM, and, although the prevalence is low, PC, PS, and AT.  Depending on these results, additional studies for b2-GPI and annexin-V antibodies or MTHFR may be warranted.

REFERENCES

1.  Mousa HA, Alfirevic Z. Croat Med J.  2001;42(2):135-145 (Review)

2.  Arkel YS, Ku DH. Clin Appl Thromb Hemost. 2001;7(4):259-268. (Review)

3.  Preston FE, et al. Lancet. 1996;348:913-916.

4.  Sarig G, et al. Fertil Steril. 2002;77(2):342-347.
5.  Rai R, et al. Hum Reprod. 2001;16(5);961-965.
6.  Eldor A. Thromb Haemost. 2001;86:104-111. (Review)
7.  Gris JC, et al. Thromb Haemost. 1999;81(6):891-899.