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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Objective To observe the effect of thrombophylaxis on pregnancy in women with a history of unexplained recurrent pregnancy loss also carrying the factor V Leiden mutation.

Methods Between 1 January and 31 December 1996, activated protein C (APC) resistance and factor V Leiden mutation were prospectively measured in 56 nonpregnant women, with a history of two or more unexplained recurrent pregnancy losses. During the same study period, seven women carrying the factor V Leiden mutation conceived, and were subsequently followed throughout their pregnancy. Subcutaneous low molecular weight heparin (LMWH, enoxaparin, 40 mg/day) and oral low dose aspirin (100 mg/day) were administered throughout the pregnancies, starting at early first trimester. Ultrasound and Doppler umbilical and fetal middle cerebral arterial flow studies were performed in the second and third trimesters, and the course and outcome of the pregnancies were documented.

Results Activated protein C resistance and factor V Leiden were found in 20 (36%) and 12 (21%) women of the study, respectively. Five of the seven pregnancies occuring progressed uneventfully to term with normal fetal growth, normal Doppler flow studies and uneventful neonatal outcome. Two of the seven women had early missed abortions.

Conclusions Thrombophylaxis, beginning in early pregnancy, in women with unexplained recurrent pregnancy loss associated with factor V Leiden mutation, seems to be safe and allow normal fetal development and good neonatal outcome. To prove the efficacy of thrombophylaxis by LMWH and low dose aspirin in this setting prospective controlled studies seem to be justified.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

In 1993 Dahlback et al.1 introduced a novel explanation for familial thrombophilia, characterised by an inherited resistance to activated protein C (APC). Subsequent studies revealed that hereditary APC-resistance is almost always caused by a single point mutation of guanine to adenine at nucleotide 1691 in the factor V gene, abolishing one of APC-cleavage sites by the substitution of glutamine for arginine at amino acid 506 in factor V2. This mutation has been termed factor V Leiden and it has been found to be a widespread mutation with an estimated prevalence of 2% to 7% in different populations3. Moreover, it has been demonstrated that, in contrast to other known hereditary thrombophilias, APC-resistance is a very common disorder found in 30%–60% of cases with familial thrombophilia3.

With the discovery of this novel genetic factor for thrombosis, a major step forward has been made in understanding the clustering of thrombotic disorders in selected families and young patients. Factor V Leiden has subsequently been found to be a significant risk factor in young women for developing different thrombotic complications, during oral contraception or during pregnancy4,5. Moreover, preliminary studies have suggested that familial thrombophilia, specifically APC-resistance and factor V Leiden mutation, could be associated with recurrent pregnancy loss6,7. These studies postulated that this mutation could lead to haemostatic disturbance in the placental bed, possibly causing microthrombi of the placental vessels, which could, in turn, induce multiple infarctions and thus adversely affect the feto-maternal circulatory system, leading eventually to fetal demise8.

We reported previously that, among a selected group of 39 women with unexplained recurrent fetal loss, 28 (72%) had APC-resistance, and 19 women (49%) had factor V Leiden mutation (3 homozygous and 16 heterozygous). Antithrombin III activity, protein C activity and antigenicity levels and total as well as free protein S antigenicity levels were normal in all patients9.

Other studies examining the association between APC-resistance, factor V Leiden and recurrent pregnancy loss had controversial results10–15. The reason for this controversy is still to be resolved. However, closer scrutiny of these results shows that most of these studies10–13 found some relation between fetal loss and factor V Leiden mutation.

With the awareness of familial thrombophilia, a scientific basis for the ‘thrombosis theory’ of repeated fetal demise is presented for the first time and an objective rationale for thrombophylaxis is provided8. In this preliminary report we have observed the effect of thrombophylaxis on pregnancy outcome in seven women with a history of unexplained recurrent pregnancy loss carrying the factor V Leiden mutation.

METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Fifty-six consecutive women with a history of two or more unexplained successive recurrent pregnancy losses presenting to the Poriya Reproductive Medicine Unit between 1 January 1996 and 31 December 1996 were included in this study. None of them was pregnant at the time of the investigation. Moreover, none was taking oral contraceptives. All women were usually healthy with no previous history of thromboembolic disease, diabetes mellitus or thyroid dysfunction.

Blood samples were obtained following informed consent. All women in the study underwent an investigation for APC-sensitivity ratio (APC-sr), and PCR analysis for factor V Leiden mutation was performed in cases with a APC-sr ≤ 2.4. Blood collection and interpretation of the APC-sr and the method of PCR analysis of factor V has been described in our previous report9. APC-resistance was defined as APC-sr of ≤ 2. Women who had APC-sr ≤ 2, but were found negative for factor V Leiden mutation, were defined to have an acquired APC-resistance.

In addition, our investigation protocol for recurrent pregnancy loss included: levels of fasting glucose, basal FSH, LH and oestradiol on day three of the cycle, TSH and prolactin levels, antinuclear factor, anticardiolipin antibodies, lupus anticoagulant and transvaginal scanning to verify ovarian morphology. Furthermore, women with three of more recurrent pregnancy losses underwent a hysterosalpingography and/or hysteroscopy to confirm uterine cavity normality and both partners were also investigated for chromosomal aberrations. All of these studies were within normal limits for all of the 56 patients.

Twelve of the 56 women with recurrent pregnancy loss were found to have the factor V Leiden mutation, and seven of these, who conceived during the study period, were included in this report. Each of these seven women was put on a thrombophylaxis regimen after informed consent was obtained and was closely followed during the entire pregnancy. The regimen was started early in the first trimester—between 5–8 weeks of gestation—as soon as pregnancy was diagnosed and was continued throughout pregnancy. Six of the seven pregnant women were treated with 40 mg/day SC enoxaparin, a low molecular weight heparin (LMWH) (Clexane, Rhone-Poulenc Rorer, Dagenham, France) and all were treated with oral low dose aspirin (Cartia, Smith, Kline and French, Dandenong, Australia; 100 mg/day). Low molecular weight heparin treatment was continued following abortion or delivery for a further six weeks.

Investigation of the pregnancies included an early transvaginal scan (Acuson 128, Mountain View, California, USA) for gestational age and fetal normality. Thereafter, the 3.5 MHz convex transducer was used for the assessment of fetal growth, fetal biophysical profile and Doppler studies (S/D ratio of the umbilical artery and pulsatility index of the middle cerebral arteries), which were performed at 24–26 weeks and 30–34 weeks of gestation. These studies were performed to reassure about fetal growth and wellbeing.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Mean numbers of first and second trimester abortions were 3.0 (SD 1.5) and 0.9 (SD 1.3), respectively, in the initial group of 56 women. Of these 56 patients with unexplained recurrent pregnancy loss, 20 (36%) had APC-resistance (defined as APC-sr of ≤ 2) and, of those, 12 (21%) were carriers for the factor V Leiden. Nine women were heterozygous and three were homozygous for the mutation. Five of the women with factor V Leiden mutation had no pregnancy other than abortions and seven had had other pregnancies. The incidence of acquired APC-resistance was found to be 14% (8 of 56).

The medical and obstetrical histories of the seven women carrying the factor V Leiden mutation who conceived during the study period are listed in Table 1. Woman number 2 had aspirin treatment advocated only after consultation since she had had four previous uneventful pregnancies and deliveries before her three consecutive first trimester abortions.

Table 1.  Medical and obstetrical histories of pregnant women with factor V Leiden mutation and weight of newborns after thrombophylaxis during pregnancy. Values are given as n, unless otherwise indicated. APC-sr = activated protein C sensitivity ratio; Hetero = heterozygote; Homo = homozygote.
PatientAge (years)Medical historyGravidityFirst trimester abortionSecond trimester abortionLiving childrenAPC-srFactor V Leiden mutationWeight (g)
126Healthy98001.76Hetero2910
234Healthy83041.73Hetero3690
326Healthy62301.84Hetero3220
426Budd-Chiari53011.36Homo
533Healthy42011.25Homo
631Healthy82412.00Hetero2900
719Healthy32001.57Hetero3340

Two women with factor V Leiden (patients 4 and 5) developed missed abortions early in the first trimester—before sonographic appearance of a fetal heart beat—and in each the uterus was evacuated. Chromosomal analysis of the conceptus material was not performed in either case. In these two women thrombophylaxis was started at the end of the sixth week of gestation, and continued for two weeks, before final diagnosis of missed abortion. In each case, a yolk sac appeared in the gestational sac with a fetal pole of 2 and 3 mm, respectively, but a fetal pulse was not documented in either case.

The five other women with factor V Leiden mutation completed their pregnancies successfully and were delivered of healthy newborns. Thrombocytopenia and/or bleeding disorders were not encountered in any of the women. No significant bruising at the site of injection was detected in any of the patients receiving enoxaparin. Vaginal bleeding at delivery was within the normal range and blood transfusion was not required in any of the five women.

Ultrasonographic assessments of fetal growth, as well as Doppler studies of the umbilical and middle cerebral arteries in the second and third trimesters, were within normal limits in each of the ongoing pregnancies. Woman no. 3 developed oligohydramnios at 31 weeks of gestation. Although there was no alteration in blood flow through the umbilical and middle cerebral arteries, we assumed that oligohydramnios might be due to reduced placental perfusion and the dose of enoxaparin was empirically increased to 80 mg/day. The amniotic fluid volume returned to normal and the pregnancy continued uneventful ending in a normal delivery. Amniotic fluid volumes were within normal limits throughout each of the other pregnancies. Newborn weights were all in the normal range (Table 1). The Apgar scores at one and five minutes post delivery were above eight in all neonates. All mothers and neonates were discharged from hospital within four days of delivery.

Woman no. 4, homozygous factor V Leiden, presented three months following the abortion with diffuse abdominal pain and distension due to general ascites which was subsequently ascribed to the occurrence of Budd-Chiari syndrome. This complication occurred despite the continuation of the LMWH treatment after the abortion.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Although the emergence of the antiphospholipid syndrome (APS) in the early 1980's supported a role for the ‘thrombosis theory’ of repeat fetal demise, APS explains no more than 5% to 10% of the cases. Moreover, the exact mechanism of the APS is still not well characterised16. The introduction of APC-resistance as a new aetiology of familial thrombophilia and subsequently of recurrent pregnancy loss cases, has substantiated a rationale for thrombophylaxis in these patients during pregnancy8. Indeed, in our study, five out of seven (71%) pregnant women with factor V Leiden treated with antithrombotic therapy successfully completed uneventful term pregnancies. The normal ultrasound and Doppler studies as well as the lack of bleeding complications throughout gestation were especially reassuring.

Of the seven women in the study, four had at least one successful pregnancy in the past without any treatment (Table 1). On the other hand, only five out of the seven women employing thrombophylaxis, in this study, completed their pregnancy successfully. Therefore, it could be argued that the successes in this study might well have occurred without treatment. Our purpose in this preliminary extended case report presentation was to address the safety of combined thrombophylaxis, starting in early pregnancy, in women with recurrent pregnancy loss associated with factor V Leiden. However, in order to prove a beneficial effect of LMWH and low dose aspirin in this setting, prospective controlled studies seem to be justified.

Both women developing missed abortions in our study, despite LMWH and low dose aspirin therapy, were homozygous for factor V mutation. The other five women heterozygous for the mutation continued their pregnancies uneventfully. It might be argued that a higher dose of LMWH and/or an earlier start of treatment might have improved the outcome. However, since karyotype analysis of the conceptual material was performed in neither of the two abortions no definite conclusion can be drawn.

We preferred to employ the LMWH rather than unfractionated heparin since LMWH has a higher antithrombotic ratio, leading to less bleeding with a better antithrombotic effect. Moreover, LMWH has a longer half-life requiring only one injection per day, a smaller injected volume and causing less heparin-induced thrombocytopenia than unfractionated heparin17.

Prospective randomised studies addressing APS patients have shown that treatment with heparin plus low dose aspirin results in a significantly better outcome than low dose aspirin alone18,19. Whether combined therapy will be similarly superior to single drug therapy in APC-resistant patients should be investigated in prospective randomised clinical trials.

The incidence of APC-resistance in our unselected population of patients with recurrent pregnancy loss was 36% (20 out of 56), while the incidence of factor V Leiden mutation was 21% (12 out of 56). This rate of thrombophilic mutation is much higher than the 6% prevalence reported in the general Israeli population20 and is in line with our previous reports of women with recurrent pregnancy loss7,9. Twelve of the 20 women with APC-resistance were found to have the single point mutation of factor V Leiden. The remaining eight women had APC-resistance, but no mutation. The significance of such an occurrence in recurrent pregnancy loss—currently defined as acquired APC-resistance—is still to be determined.

These results strengthen our previous preliminary data7,9 and are in accordance with other recent publications6,10,12,13 indicating an association between factor V Leiden mutation and pregnancy loss. Lately the EPCOT study, a multicentre collaborative evaluation of fetal loss in patients with familial thrombophilia, revealed that while first and second trimester abortions were not more common in an unselected population of thrombophilic patients with factor V Leiden, the odds ratio for stillbirth in the third trimester was increased twofold11. The discrepancy between the EPCOT study and our results may be related to the methodologies of the two studies. While the EPCOT study involved a retrospective review of unselected patients with factor V Leiden, our study prospectively investigated a specific group suffering from recurrent pregnancy loss. It is possible that only a minority of patients with factor V Leiden may experience repeated fetal loss. This is similar to the reported findings in APS—a well known disorder associated with recurrent pregnancy loss16— even though the majority of patients with APS may not have gestational abnormalities21.

As seven of the twelve women with factor V Leiden in our study had uneventful pregnancies in the past, it must be assumed that by itself the mutation is not incompatible with normal pregnancy outcome. A possible explanation could be that factor V Leiden acts as a mild risk factor for thrombosis and that additional hereditary or acquired thrombophilic factors are required for expression of the thrombosis17. It has also been shown that factor V Leiden as well as other mutations related to protein C, protein S and antithrombin III deficiencies are associated with an increasing cumulative risk for thrombosis as a function of age3. Therefore, some women with factor V Leiden mutation may have uneventful pregnancies at an early age and pregnancies complicated by fetal loss when they are older.

Recently, two separate studies, one from Spain14 and the other from Utah, USA15, have claimed no association between recurrent abortions and factor V Leiden mutation. Since we are addressing a genetic disease—a single point mutation with different prevalence in distinct populations—it could be suggested that this difference is the main cause for the discrepancy found between these two reports14,15 and others6,7,9–13. Indeed, the incidence of factor V Leiden mutation was 2%–2.5% in the control groups of these two studies, whereas it is 6% in the general Israeli population20. Moreover, since karyotype analysis of the aborted material was not performed in any of the studies published so far, it is still difficult to draw any firm conclusion concerning pregnancy loss and factor V Leiden.

It is important to note that the vast majority of women with factor V Leiden and recurrent pregnancy loss are healthy and have no past medical or family histories of thrombo-embolic phenomena. Only one of the 12 (8%) women with factor V Leiden developed a thrombotic disorder (woman no. 4) 3 months following the end of her pregnancy. In our previous report five of the nineteen women (26%) found positive for Leiden mutation had prior episodes of deep vein thrombosis9, but these comprised a selected group of women suspected of having haemostatic disturbances. We therefore conclude that tests for APC-resistance and factor V Leiden should be considered in cases of recurrent pregnancy loss, despite absence of previous thrombotic medical history.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References

Thrombophylaxis by LMWH and low dose aspirin, beginning in early pregnancy, in women suffering from recurrent pregnancy loss associated with factor V Leiden mutation seems to be safe and allow normal fetal development and good neonatal outcome. To the best of our knowledge this is the first report that describes the pregnancy outcome in such a group of patients undergoing thrombophylaxis. Larger, prospective randomised studies are required to substantiate the efficacy of such treatment in this setting.

Acknowledgements

We would like to thank Dr M. Matilsky for his intelligent remarks during the preparation of this manuscript.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. References
  • 1
    Dahlback B, Carlsson M, Svensoon PJ. Familial thrombophilia due to a previously unrecognised mechanism characterised by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci 1993; 90: 10041008.
  • 2
    Bertina RM, Koeleman BPC, Koster T et al. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 6467.
  • 3
    Bauer KA. Management of patients with hereditary defects predisposing to thrombosis including pregnant women. Thromb Haemostas 1995; 74: 94100.
  • 4
    Vandenbroucke JP, Koster T, Briet E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation 1994; 344; 14531457.
  • 5
    Dahlback B. Physiologic anticoagulation. Resistance to activated protein C and venous thromboembolism. J Clin Invest 1994; 94: 25212524.
  • 6
    Rai R, Regan L, Hadley E, Dave M, Cohen H. Second trimester pregnancy loss is associated with activated protein C resistance. Thromb Haemostas 1995; 73: 1300.
  • 7
    Younis JS, Ben-Ami M, Brenner B, Lanir N, Rothbart H, Ohel G. The association of repeat fetal demise with activated protein C resistance and factor V Leiden. Gynecol Endocrinol 1996; 10: 164.
  • 8
    Younis JS, Ohel G, Brenner B, Ben-Ami M. Familial thrombophilia—the scientific rationale for thrombophylaxis in recurrent pregnancy loss Hum Reprod 1997; 12: 13891390.
  • 9
    Brenner B, Mandel H, Lanir N et al. Activated protein C resistance can be associated with recurrent fetal loss. Br J Haematol 1997; 97: 551554.
  • 10
    Rai R, Regan L, Hadley E, Dave M, Cohen H. Second-trimester pregnancy loss is associated with activated protein C resistance. Br J Haematol 1996; 92: 489490.
  • 11
    Preston FE, Rosendaal FR, Walker ID et al. Increased fetal loss in women with heritable thrombophilia. Lancet 1996; 348: 913916.
  • 12
    Grandone E, Margaglione M, Colaizzo D et al. Factor V Leiden is associated with repeated and recurrent unexplained fetal losses. Thromb Haemostas 1997; 77: 822824.
  • 13
    Ridker PM, Miletich JP, Buring JE et al. Factor V Leiden mutation as a risk factor for recurrent pregnancy loss. Ann Intern Med 1998; 128: 10001003.
  • 14
    Balash J, Reverter JC, Fabregues F et al. First-trimester repeated abortion is not associated with activated protein C resistance. Hum Reprod 1997; 12: 10941097.
  • 15
    Dizon-Townson DS, Kinney S, Branch DW, Ward K. The factor V Leiden mutation is not a common cause of recurrent miscarriage. J Reprod Immunol 1997; 34: 217223.
  • 16
    Silver RM, Branch DW. Recurrent miscarriage: autoimmune considerations. Clin Obstet Gynecol 1994; 37: 745760.
  • 17
    Brenner B, Blumenfeld Z. Thrombophilia and fetal loss. Blood Reviews 1997; 11: 7279.
  • 18
    Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with heparin and low-dose aspirin is superior to low-dose aspirin alone. Am J Obstet Gynecol 1996; 174: 15841589.
  • 19
    Rai R, Cohen H, Dave M, Regan L. Randomised controlled trial of aspirin and aspirin plus heparin in pregnant women with recurrent miscarriage associated with phospholipid antibodies (or antiphospholipid antibodies). BMJ 1997; 314: 253257.
  • 20
    Seligsohn U, Zivelin A. Thrombophilia as multigenic disorder. Thromb Haemostas 1997; 78: 297301.
  • 21
    Italian Registry of Antiphospholipid Antibodies (IR-APA). Thrombosis and thrombocytopenia in antiphospholipid syndrome (idiopathic and secondary to SLE): first report from the Italian Registry. Haematologica 1993; 78: 313318.