LABORATORY EVALUATION OF
THROMBOPHILIA IN RECURRENT PREGANCY LOSS
Kathleen E. Puca,
M.D., ITxM Fellow
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 1st trimester pregnancy loss as well.1,2
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 (2nd and 3rd trimester) pregnancy loss.4
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 2nd and 3rd 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 2nd and 3rd 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.
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 1st
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
HYPERHOMOCYSTEINEMIA AND MTHFR
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).
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 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.
APS and certain inherited
thrombophilia are potential causes for fetal loss. Laboratory evaluation is
indicated in women with RPL, particularly in cases of 2nd and 3rd
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.
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5. Rai R, et al. Hum Reprod.
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7. Gris JC, et al. Thromb
Institute For Transfusion Medicine