1 Department of Obstetrics and Gynecology and 2 Haematology unit, Bnai-Zion Medical Center, Rappaport Faculty of Medicine, Technion, 47 Golomb St, PO Box 4049, Haifa 31048, Israel
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: activated protein C resistance/factor V mutation/repeated miscarriages/thrombophilia
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Early development of a conception is a multifactorial process and involves among other things, formation of adequate vascular support. Ineffective blood flow caused by vascular impairment or thrombotic events in the trophoblast may have a deleterious effect on the developing pregnancy. Evidence for such a vascular insult resulting sometimes in pregnancy loss is found in patients with the antiphospholipid syndrome (Haywood and Brown, 1991). Recently, attention has been drawn to the possible association of early and late pregnancy loss and hypercoagulable states such as seen in different types of thrombophilia (Rai et al., 1995
; Preston et al., 1996
; Balasch et al., 1997
; Brenner et al., 1997
). Thus, a 20% activated protein C resistance (APCR) rate was found in habitual aborters if at least one of the miscarriages was in the second trimester and 5.7% if all the miscarriages were in the first trimester, which was comparable with the control group (4.3%) (Rai et al., 1995
). A carrier state of Leiden mutation (factor V) was reported in 19 of 39 patients (48%) with repeated pregnancy loss ranging from the first to the third trimester (Brenner et al., 1997
). Moreover, in nine of their patients acquired APC-resistance was demonstrated with no evidence for factor V mutation. The European multicentre study, EPCOT (Preston et al., 1996
), revealed an odds ratio of 2.0 in women with factor V mutation who delivered a stillbirth but an odds ratio of 0.9 in women with the same mutation who miscarried.
The prevalence of the Leiden mutation in the European and American Caucasian population ranges from 35% (Preston et al., 1996) yet some ethnic groups have a higher carrier rate for the Leiden mutation, such as some population sectors in Israel in whom the Leiden mutation heterozygous carrier state is about 10% (U. Seligsohn, personalcommunication).
When should investigation for APC-resistance begin in patients with pregnancy failure? We planned a prospective case-controlled study to assess the prevalence of APC-resistance in patients referred to our infertility clinic with one or more first or second trimester pregnancy losses.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A control group consisted of 125 women who had one or more live births but no pregnancy failures and were matched with the study group patients for age and ethnicity.
The age range of the 125 patients in the study group was 2142 years with a mean of 31.4 ± 5.6 years, and of those in the control group 1941 years with a mean of 30.7 ± 4.2 years.
Blood samples were collected by venipuncture into two plastic tubes, one containing 1/10 volume 3.8% sodium citrate for coagulation assays and the other containing 1/10 volume of 0.5 mol/l sodium EDTA for DNA extraction. None of the women in the study or control group was pregnant at the time of blood sampling and an interval of at least 2 months had passed between the last pregnancy and blood sampling. Women in the control group had coagulation assays only for activated protein C resistance and polymerase chain reaction (PCR) for detection of factor V mutation.
Coagulation assay
Protein C antigen was determined by enzyme immunoassay, using a specific Asserachrom® kit. Protein C activity was determined by a chromogenic assay, using the protein C Stachrom® kit. Total protein S antigen was determined by electro-immunoassay using Asseroplate® protein S kit. Antithrombin III activity was measured by chromogenic assay using the Antithrombin III Asserochrom® kit (all kits diagnostic, Stago, Asnières, France).
Lupus anticoagulant was detected by two different tests. Diluted Russell Viper Venom test (DRVVT) was performed using the Gradipore La Screen, Northryde, Australia (DRVVT)® kit. Values >1.2 were considered positive and in those cases, presence of lupus anticoagulant was verified by using the Gradipore La-confirm (DRVVT)® kit, which is a phospholipid-rich reagent for the specific correction of lupus anticoagulant.
A solid phase immunoassay technique was used to quantify anticardiolipin concentrations. IgG anticardiolipin concentrations were measured and a concentration of >16 U was considered positive.
Resistance to activated protein C caused by factor V Leiden mutation was determined using Coatest APC-resistance (chromogenix® kit, Molendal, Sweden). About half of the tests were performed using the above kit and approximately half of the tests were performed using the more specific kit in which plasma samples are prediluted in factor V diluted plasma in order to avoid the influence of other plasma proteins on the assay (this kit was not available during the first year of the study). In both assays, response to APC was expressed by the sensitivity ratio: (SR) = clotting time + APC + CaCl/clotting time + CaCl. When the activated protein C sensitivity ratio (APCSR) was less than 2.2 a PCR analysis for factor V mutation was done. According to our experience (unpublished data) the Leiden mutation was never found if the APCSR was higher than 2.2.
Acquired APC-resistance (factor V Leiden negative) was defined when the APCSR was less than 1.8. The inter-assay coefficient of variation for normal controls in our laboratory is 6.6%.
Molecular diagnosis of the Leiden mutation in factor V was made by PCR on DNA extracted from whole blood. The reaction was used to amplify exon 10 of the factor V gene, followed by allele-specific restriction with Mnl I for mutation detection as described elsewhere (Zoller et al., 1994).
Chromosomal analysis
Chromosomal analysis of fetal material was performed after two or more miscarriages whenever possible. Preclinical pregnancies were not analysed for chromosomal abnormalities because of the paucity of material available for examination in these cases. Cytogenetic analysis was performed as described previously (Eiben et al., 1990).
The 2 and t-tests were used for statistical analysis of discrete and continuous variables respectively.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Table I relates pregnancy outcome and results of APC-resistance studies. Preclinical pregnancy failure was significantly more frequent in Leiden mutation carriers, 17/48, than in patients with no APC-resistance, 25/214 (P < 0.001).
|
Table II shows the occurrence of APC-resistance after one, two and three or more pregnancy losses per patient. Of 47 patients with one pregnancy loss, APC-resistance (due to the Leiden mutation or acquired) was revealed in seven (14.9%), two with the mutation and five acquired. Forty three patients had two pregnancy losses and 11 of them (25.6%) had APC-resistance, 10 with the mutation and one acquired. Of 35 patients with three or more abortions, seven (20%) had APC-resistance, six with the mutation and one acquired. Therefore, in our study group APC-resistance occurred in ~1/7 patients with only one pregnancy loss, in 1/4 after two pregnancy losses and in 1/5 after three or more pregnancy losses.
|
Fetal material analysis was performed in 79 miscarriages in the group of patients with no APCR and 35 had chromosomal abnormalities (44.3%). In the group of patients with APCR due to the factor Leiden mutation, five cases of chromosomal abnormalities were found in 22 miscarriages analysed (22.7%). Of three trophoblastic materials analysed in patients with acquired APCR, one had a chromosomal abnormality.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our study group was comprised of patients with 18 pregnancy losses (mean 2.9 ± 1.8) in whom a routine work-up had not revealed a cause for the pregnancy failure. We therefore postulated that a thrombotic event due to thrombophilia could be a possible reason for these patients' pregnancy losses, as recently suggested (Rai et al., 1995; Preston et al., 1996
; Balasch et al., 1997
; Brenner et al., 1997
) and that was the basis for our study.
Pregnancy losses were divided into preclinical, first trimester clinical and second trimester. We found a significantly increased rate of preclinical pregnancy failure in Leiden mutation carriers than in no APCR patients (P < 0.001). To the best of our knowledge this is the first report to show this difference. Whether hypercoagulability (due to the Leiden mutation) may lead to such an early pregnancy loss is merely speculative. However, development of the fetal arterial supply to the placenta begins as early as 1820 days after conception and soon thereafter the fetoplacental villous circulation becomes established (King, 1987; Demir et al., 1989
). During this early period of conception, detection of a pregnancy is made by serum ßHCG measurement because it is too early for imaging by transvaginal ultrasonography. Either way, this was the period when our patients' preclinical pregnancy losses took place.
We found in our study a decreased rate of clinical first trimester miscarriages in patients with hereditary APCR compared with patients with no APCR. Similarly in a group of 19 patients with APCR (Brenner et al., 1997) no increase in the rate of first trimester abortions was found. Rai et al. (1995) took a different approach in their analysis but also did not find a difference in the prevalence of APCR between first trimester aborters and a control group.
The increased rate of second trimester abortion in Leiden mutation carriers revealed in our study group is in accordance with the study of Brenner et al. (1997) who reported an increased risk for second trimester abortions in their patients with APC-resistance. Rai et al. (1995), looking specifically at second trimester pregnancy losses, found a 20% rate of Leiden mutation carriers in that group of patients, significantly more than in the control group (4.3%). Grandone et al. (1997) found seven cases with the Leiden mutation in a sample of 43 Caucasian women (16.8%) with a history of two or more unexplained fetal losses compared with five carriers among 118 controls with uneventful pregnancies (4.2%; P = 0.011). This association was stronger for late events than for first trimester abortions. Ridker et al. (1998) have recently evaluated 113 women with recurrent pregnancy losses for the factor V mutation and found a 2.2-fold increase in the prevalence of the mutation among the aborters compared with controls (P = 0.026).
In contrast, other reports do not support an association of APCR with miscarriage.
The EPCOT study (Preston et al., 1996), defining a miscarriage as a pregnancy loss before 28 weeks of gestation, could not detect an increased risk for such a fetal loss among carriers of the factor V mutation (odds ratio 0.9). Balasch et al. (1997) compared a group of 55 patients with unexplained first trimester repeated abortions with 50 healthy control women who had at least one child but no previous abortion. In each group they detected one heterozygous Leiden mutation carrier. They concluded that first trimester repeated miscarriages are not associated with APCR.
Leiden mutation carriers and patients with no APC-resistance in our study group showed comparable live birth rates, 20/71 (28.2%) and 104/319 (32.6%) respectively. This is in contrast to the report by Brenner et al. (1997) who found 19% live births in carriers of the Leiden mutation compared with 33% in patients with normal APC-resistance (P < 0.03). This may be due to the fact that their study group comprised 39 patients with at least three miscarriages whereas our study included patients with a various number of miscarriages.
Why do some pregnancies in patients with APCR end as an early or late pregnancy loss whereas other pregnancies result in live births? A recent study (Dizon-Townson et al., 1997) is interesting in this regard and may shed some light on the answer to that question. They found a twofold increase in the carrier frequency for Leiden mutation in abortuses compared with unselected pregnant women and, even more remarkable, a 10-fold increase in the carrier state for the factor V mutation when the pathologically examined placentae of women at birth contained more than 10% infarctions compared with those with less than 10% infarctions. Therefore the existence of a carrier state in the conceptus may play a more important role regarding the fate of the pregnancy than the maternal carrier state since the inheritance is considered autosomal dominant (Zoller, 1994) and thus some fetuses may be affected while the others may not.
The 14.4% of Leiden mutation carriers in our study group was significantly higher than in the control group (5.6%). Since matching was done for ethnicity and age it is quite obvious that the group of patients with repeated miscarriages had a higher prevalence of hereditary thrombophilia than women with no history of miscarriage. Thus, we found an APCR rate of approximately 1/7 after one pregnancy loss occurring any time between the preclinical stage and the second trimester. After two or more pregnancy losses the APCR rate was close to 1/5.
APCR may be associated not only with an increased rate of pregnancy loss but also with a five to 10-fold increased risk of maternal thrombotic events in the heterozygous and 50 to 100-fold in homozygous patients (Dalback, 1996). We maintain therefore that searching for this thrombophilic factor after repeated spontaneous abortions for which no reason had been revealed after a thorough work-up is appropriate because it may be the first clue to the existence of thrombophilia. We do not suggest, however, screening of women who plan pregnancy for the existence of APC-resistance, in agreement with others (Rouse et al., 1997
).
Consecutive miscarriages in a given couple show a non-random distribution with respect to chromosomal complements, thus the high percentage (44.3%) of chromosomal abnormalities found in our patients who had no APCR is expected. These correlate well with the 50% chromosomal aberrations associated with fetal loss between 8 and 15 weeks of gestation. The much lower percentage (22.7%) of chromosomal abnormalities revealed in Leiden mutation carriers may suggest that another factor was also involved in causing the miscarriages. However, statistical analysis seems irrelevant here since for obvious reasons only a part of the miscarriages in the two groups could have been analysed.
We have a preliminary experience (unpublished ) with 14 patients who had 16 pregnancy losses before APCR was diagnosed (none had anticardiolipin antibodies) and were administered a low molecular weight heparin, clexane 40 mg/day (Rhône-Poulenc Rorer, Netanya, Israel) throughout their next conception. Only two of the 14 women aborted and the other 12 delivered healthy newborns. No complications known to be related to the anticoagulant occurred in those 14 patients. Although we think that these results are encouraging it is premature to conclude that enoxaparine is of any benefit in preventing recurrent miscarriages. Certainly, prospective randomized trials are warranted to answer the question of the efficacy of antithrombotic treatment in preventing pregnancy loss in these patients.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brenner, B., Mandel, H., Lanir, N. et al. (1997) Activated protein C resistance can be associated with recurrent fetal loss. Br. J. Haematol., 97, 551554.[ISI][Medline]
Cunningham, F.G., MacDonald, P.C., Gant, N.F. et al. (eds) (1997) Williams obstetrics. Appelton and Lange, Stamford.
Dalback, B. (1996) Are we ready for factor V Leiden screening? Lancet, 347, 13461347.[ISI][Medline]
Demir, R., Kaufman, P., Castelluci, M. et al. (1989) Fetal vasculogenesis and angiogenesis in human placental villi. Acta Anat. (Basel), 136, 190203.[Medline]
Dizon-Townson, D.S., Meline, L., Nelson, L.M. et al. (1997) Fetal carriers of the factor V Leiden mutation are prone to miscarriage and placental infarction. Am. J. Obstet. Gynecol., 177, 402405.[ISI][Medline]
Eiben, B., Bartels, I., Bahr-Porsch, S. et al. (1990) Cytogenetic analysis of 750 spontaneous abortions with the direct-preparation method of chorionic villi and its implication for studying genetic causes of pregnancy wastage. Am. J. Hum. Genet., 47, 656663.[ISI][Medline]
Grandone, E., Margaglione, M., Colaizzo, D. et al. (1997) Factor V Leiden is associated with repeated and recurrent unexplained fetal loss. Thromb. Haemost., 77, 822824.[ISI][Medline]
Haywood, L. and Brown, M.D. (1991) Antiphospholipid antibodies and recurrent pregnancy loss. Clin. Obstet. Gynecol., 34, 117.[ISI]
King, B.F. (1987) Ultrastructure differentiation of stromal and vascular components in early macaque placental villi. Am. J. Anat., 178, 3044.[ISI][Medline]
Preston, F.E., Rosendaal, F.R., Walker, I.D. et al. (1996) Increased fetal loss in women with heritable thrombophilia. Lancet, 348, 913916.[ISI][Medline]
Rai, R.S., Regan, L., Hadley, E. et al. (1995) Second trimester pregnancy loss is associated with activated protein C resistance. Br. J. Haematol., 92, 489490.[ISI]
Ridcker, P.M., Miletich, J.P., Buring, J.E. et al. (1998) Factor V Leiden mutation as a risk factor for recurrent pregnancy loss. Ann. Intern. Med., 128, 1001003.
Rouse, D.J., Goldenberg, R.L. and Wenstrom, K.D. (1997) Antenatal screening for Factor V Leiden mutation: a critical appraisal. Obstet. Gynecol., 90, 848851.
Stray-Pederson, B. and Stray-Pederson, S. (1984) Etiologic factors and subsequent reproductive performance in 195 couples with a prior history of habitual abortion. Am. J. Obstet. Gynecol., 148, 140146.[ISI][Medline]
Zoller, B., Sevensson, P.J., He, X. and Dahlback, B. (1994) Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J. Clin. Invest., 94, 25212524.[ISI][Medline]
Submitted on October 9, 1998; accepted on March 8, 1999.