Inherited Thrombophilia is Associated with: Recurrent pregnancy losses Preeclampsia Intrauterine growth retardation Stillbirths Preterm labors Maternal deaths

Recurrent Miscarriage Predisposes Women to: Preterm labor Intrauterine growth retardation Preeclampsia Fetal anomalies

New Actions of Antiphospholipid Antibodies Cause increased serum tumor necrosis factor (TNF) alpha Alters the TH1:TH2 balance towards TH1 Decreases interleukin-3 (IL-3) TNF alpha can initiate the release of tissue factor and initiate the clotting cascade

Inherited Thrombophilias Rey et al. in a meta-analysis found: Increased incidence of factor V Leiden mutation Prothrombin gene mutation factor II MTHFR gene mutation in patients with recurrent miscarriage Screening for inherited thrombophilia in women with recurrent spontaneous abortion (RSA), infertility and implantation failure is indicated

Heparins 1 Heparin increases serum TNF binding protein protecting against harmful systemic manifestations Low molecular weight (LMW) heparins inhibit TNF alpha production Heparin and LMW heparin are antiinflammatory

Heparins 2 LMW heparin limits neutrophil extravasion LMW heparin decreases vein wall permeabiolity Prevents accumulation of proliferating vascular smooth muscle that encroaches on the lumen of arteries

Inherited and Acquired Thrombophilia Pregnancy is a hypercoagulable state (Stirling et al., 1984) Antiphospholipid antibodies (aPL), an acquired thrombophilic defect, are an important and treatable cause for pregnancy losses at all gestational ages (Rai et al., 1997) Inherited thrombophilias are a greater cause for pregnancy losses and complications at all gestational ages (Rai et al., 2002)

Factor V Leiden and RSA Outcome of Untreated Pregnancies Early Losses FVL + 37.5% - 69.3% Late Losses FVL 11.1% 48.9%

Factor V Leiden Treatment Increase LMW heparin to twice daily with a positive pregnancy test LMW Heparin 30 or 40 mg once daily started on cycle day 6 Continue until 6 weeks postpartum Aspirin 81 mg daily starting cycle day one of conception cycle

Factor V Leiden Outcome with Treatment on RSA Women Carp et al., 2002 Without treatment 33.2% With treatment 85.7%

Recommended Tests LAC screen APTT and dRVVT Antiphospholipid antibodies Factor V Leiden gene mutation Prothrombin gene factor II gene mutation Methylenetetrahydrofolate reductase (MTHFR) gene mutation Antinuclear Antibody (ANA)

Importance of ANA Testing Infertility, RSA Ogasawara et al., 1999 25% of RSA patients have low titer positive ANA with speckled pattern 21% of Infertility patients have low titer positive ANA with speckled pattern A positive ANA mandates a full immunological workup

Mechanisms of Thrombosis Protein C and S and AT III slow down the forward momentum of clot formation Factor V Leiden is a mutated variant of factor V that is partially resistant to inactivation by activated protein C

Mechanisms of Thrombosis in Placenta Antibodies Stern et al., 1998 Increased localization of prothrombotic proteins Blocking of natural anticoagulants Transmembrane signaling

Treatment for MTHFR Hetero and Homozygous Women Folgard RXTM 2.2 daily (lifetime) Aspirin 81 mg daily (lifetime) LMW heparin 30 – 40 mg daily, cycle day 6, increasing to twice daily until 6 weeks postpartum

Treatment of Prothrombin Gene Factor II Variant Aspirin 81 mg daily LMW heparin 30 – 40 mg daily, cycle day 6, increasing to twice daily until 6 weeks postpartum

MTHFR Outcome with and Without Treatment Carp et al., 2004 With treatment 66.7% Without treatment 3.5%

Different direction in intact vessels... Vessel Wound Factor VII+ Tissue Factor Coagulation Cascade Thrombin Factors V and VIII Fibrinogen ® Fibrin Platelets Activated Factors V and VIII (Va and VIIIa) Further Coagulation Activation Different direction in intact vessels...

Intact Blood Vessel Thrombin + Thrombomodulin (Endothelial Surface) Protein C System Activation Activated C (APC) and Protein S and Factor V Deactivation of Va & VIIIa Normal conditions ® Anticoagulant mechanisms ® Procoagulant mechanisms Wound conditions ® Procoagulant mechanisms ® Anticoagulant mechanisms

European Prospective Cohort on Thrombophilia (Lancet 1996) Pregnancies Fetal Loss Stillbirth/ Miscarriage Stillbirth, Combined Defects Factor V Leiden Thrombophilia (n=843) n=1524 1.35 3.6 14.3 2 Control (n=541) n=1019 1

Missense Mutation G to A in the FV Gene Position 1691, leading to Arg506Gln protein (factor V Leiden) Most frequent genetic defect associated with disease In Europe 3-7% with APC-R, 1% homozygotes “Mitera” study: 4.5% carriers (Greek blood donors)

Hypercoagulability has Positive Genetic (Natural) Selection Effects In the past ® survival advantage (war and hunting wounds, deliveries) Now ® artifacts of modern life style

Mutated Factor Va (FV Leiden) ® Less Sensitive to APC Deactivation APC-Resistance ® frequent to general population, 3-5% Carriers have an increased risk for thromboses Most important factor of venous thromboembolism More than half of the patients with hereditary thrombophilia

8.9% 4.2% 30% 20% 0.9% 10% 3% 4.1% FV Leiden Subjects No Mutation Severe Pre-eclampsia Normotensive Women on Oral Contraception and Thromboses Oral Contraception and Cerebral-Vein Thromboses Stroke in Young Women FV Leiden Subjects 8.9% 4.2% 30% 20% 0.9% No Mutation 10% 3% 4.1% Domna S. Dizon-Townson et al, Salt Lake City, Utah, Am J Obstet Gynecol, 1996 Margareta Hellgren et al, Mohndal and Malmo, Sweden, Am J Obstet Gynecol, 1994 Longstreth WT Jr et al, Washington, Seattle, Stroke, 1998 Martinelli I et al, Milan, N Engl J Med, 1998

First Trimester Abortions Br J Haematol, 1997 Brenner B et al., Bruce Rappaport Faculty of Medicine, Haifa, Israel Women with Fetal Losses (n=39) Pregnancies First Trimester Abortions Late Abortions Intrauterine Death Live Births FV Leiden (n=19) 128 50% 17% 47% 18% With APC-R (n=9) 56

Prothrombin Gene G20210A Variant Inherited in an autosomal dominant manner Gene on chromosome 11p11-q12 (21kb, 14 exons) encodes a plasma glycoprotein, activated to thrombin by FXa and Fva Point mutation G20210A in the 3' untranslated region is associated with elevated prothrombin levels

Title Needed??? Accounts for ~18% of inherited thrombophilia Accounts for ~6% of unselected patients with deep-vein thrombosis Frequently co-inherited with other genetic risk factors like factor V Leiden

Title Needed??? Carriers have a 2 to 5 fold increased risk of venous thrombosis, including cerebral-vein thrombosis Carriers using oral contraceptives have a highly elevated risk of cerebral-vein thrombosis

5.1 20% 1.9% 1.6 3% 1.6% Prothrombin Gene Variant Subjects No Mutation Women with Myocardial Infarction Women on Oral Contraception and Cerebral-Vein Thromboses Stroke in Young Women Prothrombin Gene Variant Subjects 5.1 20% 1.9% No Mutation 1.6 3% 1.6% Longstreth WT Jr et al., Washington, Seattle, Stroke, 1998 Rosendaal FR et al., Leiden, Netherlands, Blood, 1997 Martinelli I et al., Milan, N Engl J Med, 1998

Title Needed??? Carrier frequency of 1 to 4% in different populations Carrier frequency in southern Europe is nearly twice As high as in northern Europe "Mitera" study: 3.8 % among healthy Greek blood donors Geographical distribution probably due to founder effect

Hyperhomocysteinemia Cystathionine ß-synthase (CBS) deficiency causes the metabolic disorder "homocystinuria" (including premature arteriosclerosis and thrombosis) MTHFR deficiency is an inborn error of folate metabolism (with increased risk of arteriosclerosis and thrombosis)

Hyperhomocysteinemia (Cont.) Mild hyperhomocysteinemia has been recognized as a risk factor for arteriosclerosis and thrombosis Mild hyperhomocysteinemia due to a thermolabile variant of the MTHFR enzyme has been associated with premature vascular disease Homocysteine is presumed to damage the endothelial cell, but the exact mechanism of its toxicity is unknown

Title Needed??? Homozygous individuals show reduced MTHFR activity, increased thermolability and hyperhomocysteinemia Homozygotes may have an increased risk of cardiovascular disease and neural tube defects

Title Needed??? Relatively high frequency throughout the world: allele frequency from 6% in Africa to 40% in some European populations Selective advantage to explain high frequency

25% 8% 38% Women with Mild Hyperhomocysteinemia Placental Abruption (n=31) Intrauterine Fetal Death (n=18) Small for Gestational Age Infants (n=13) Women with Mild Hyperhomocysteinemia 25% 8% 38% J. I. P. de Vries et al., Amsterdam British Journal of Obstetrics and Gynaecology, 1997

“Mitera” Study, ASH 1998 Haematology Dept, Haemostasis Lab Group Aborters > 2, (n=37) Non-Aborters, (n=102) Age Median (range) 34 (19-51) 34 (20-54) Platelets All Normal PT INR APTT Fibrinogen > 400 mg/dl 30%, n=11 14%, n=14 Fibrinogen < 400 mg/dl 70%, n=26 86%, n=88 LA Positive (by confirmative test) 16%, n=16 9%, n=9 AT III < 75% 3%, n=1 3%, n=3 PC < 75% PS < 75% Unreliable Test Increased APC-R (suggesting FV Leiden) 22%, n=8 11%, n=11

Consultation Extended family screening Anticoagulant prophylaxis in selected cases Inherited Thrombophilia (Prothrombotic Gene Mutation) Acquired Thrombophilia Hypercoagulability Thrombosis

LMW Heparin Given subcutaneously to maintain trough anti-Xa activity of 0.15-0.2U/ml and 2 h post injection levels of 0.4-0.6 U/ml Checking of levels are no longer recommended May check monthly during pregnancy

LMW Heparin Complications Epidural anesthesia is managed by omitting a dose or inserting the needle 6 hours after the previous dose There were no thromboembolic events or excessive hemorrhages There is one report of osteoporotic vertebral collapse post partum Hunt BJ et al., 1997

LMW Heparin Complications No local or systemic side effects reported No excessive intrapartum or postpartum bleeding in vaginally or c-sectioned delivered patients Epidural was done without complications No intraventricular hemorrhages in any infant No effect on platelet count Dulitzke M. et al., 1996