1 Department of Obstetrics, Gynaecology and Reproductive Medicine, 2 Department of Clinical Epidemiology and 3 Haemostasis and Thrombosis Research Center, Leiden University Medical Center, P.O.Box 9600, 2300 RC Leiden, The Netherlands
5 To whom correspondence should be addressed. Email: f.m.helmerhorst{at}lumc.nl
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Factor V Leiden/fecundity/miscarriage/time to pregnancy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To study the effect of factor V Leiden mutation on embryo implantation and human reproduction, we investigated the association of factor V Leiden mutation on fecundity and miscarriages and the trimester in which the miscarriages occurred in 297 women. To be able to construct a large cohort of women with factor V Leiden, we used information from a large study on venous thrombosis. We also investigated the effect of factor V Leiden carriership on term birth rate and birthweight.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For the present analysis, the first 115 female thrombosis patients identified with factor V Leiden mutation were matched by age to 230 female thrombosis patients (controls). Besides the age matching and absence of factor V Leiden, controls were randomly selected from the study participants. All 345 patients, who were unaware of their genotype, were asked to fill in an additional structured questionnaire concerning their reproductive history. Questions consisted of age at first pregnancy attempt, the period of unprotected intercourse until the desired pregnancy occurred, number of pregnancies, and the duration of each pregnancy. When there had been no pregnancies, we enquired whether this was despite efforts to become pregnant (infertility). If there was no response to the initial questionnaire, a written reminder accompanied with an identical questionnaire was sent after 3 weeks. To increase response, patients were contacted by telephone if they did not return the questionnaire after an additional 5 weeks.
Time to pregnancy was defined as self-reported time between unprotected intercourse and the occurrence of pregnancy. Miscarriage ratio was calculated as the total number of miscarriages per number of pregnancies. First trimester miscarriage was defined as embryonic or fetal loss before the completion of 12 weeks gestation; second trimester miscarriage was defined as fetal loss from 13 to 24 weeks gestation; stillbirth was defined as a loss after 24 weeks gestation. A number of factors known to influence miscarriage or fecundity were included in the questionnaire, such as age at first pregnancy attempt, age at first birth, level of education (primary school, secondary school, college or university), smoking habits, and alcohol use. The body mass index [BMI: weight in kg/(height in m)2] was calculated at the time of thrombosis.
DNA was isolated from whole blood or buccal swabs. For the latter, three large cotton swabs in a total of 6 ml sodium dodecyl sulphateproteinase K solution (100 mmol/l NaCl, 10 mmol/l EDTA, 10 mmol/l TrisHCl pH 8.0, 0.5% SDS, 0.1 mg/ml proteinase K) were obtained from each patient. Upon arrival, the proteinase K concentration was raised to 0.2 mg/ml and the sample was incubated for 2 h at 65°C. Subsequently, the suspension was recovered by centrifugation. Potassium acetate was added to a final concentration of 1.6 mol/l. After 15 min incubation on ice, proteins were removed using chloroformisoamylalcohol (24:1) treatment. The DNA in the water phase was subsequently ethanol-precipitated. After centrifugation, the pellet was resuspended in 200 ml 10 mmol/l TrisHCl, 10 mmol/l EDTA pH 8.0 and frozen at 20°C until further analysis. Assessment of the factor V Leiden mutation in DNA retrieved from the buccal swabs was performed identically to the method for DNA from whole blood, and determined by PCR and Mnl I restriction digestion as described elsewhere (Bertina et al., 1994).
Data are presented as simple counts and percentages. Relative risks (RR) were computed as the ratio of these counts, and 95% confidence intervals (CI) were based on a binomial distribution.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of the 102 factor V Leiden carriers who returned the completed questionnaire, 80 (78%) had been pregnant at least once compared to 140 (72%) of the 195 non-carriers (RR 1.1, 95% CI 0.951.2). Patient characteristics of these 220 women are listed in Table I. The reasons for not having had a pregnancy were similar; infertility was reported by 5% (1/22) of factor V Leiden carriers and 11% (6/55) of non-carriers (RR 0.4, 95% CI 0.13.3).
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Miscarriages occurred as often in factor V Leiden carriers (14%) and non-carriers (14%), in percentages that are similar to the general population (1015%) (Zinaman et al., 1996). Published data on factor V Leiden in relation to miscarriages are conflicting. Recent meta-analyses have shown an association between factor V Leiden and recurrent fetal loss, occurring both in early and in late in gestation (respectively OR 2.01, 95% CI 1.133.58 and OR 7.83, 95% CI 2.8321.67) (Rey et al., 2003
; Kovalevsky et al., 2004
). For non-recurrent early loss (<19 weeks gestation) no clear association was found with factor V Leiden (OR 1.40, 0.662.97); and for non-recurrent isolated second/third trimester loss (stillbirth >19 weeks) a positive association was found (OR 3.26, 95% CI 1.825.83) (Rey et al., 2003
; Dudding and Attia, 2004
).
The focus in the literature has mainly been on factor V Leiden in relation to second and third trimester loss, as thrombosis of the placental vessels is assumed to be an important factor in fetal loss. In our study, factor V Leiden carriers experienced more fetal loss in the second trimester, as would be expected by the placental vessel thrombosis theory. A clear increase in third trimester loss would also be expected, with an even higher rate of fetal loss in third than in second trimester for factor V Leiden carriers. However, third trimester loss (stillbirth) was equally distributed over carriers and non-carriers, with a similarly low rate, which contradicts this theory. Moreover, in the present study, mean birthweight was not influenced by factor V Leiden status, which is in line with earlier published data (Lindqvist et al., 1999). If we consider hypercoagulability to lead to obstruction of placental vessels as a major pathological factor among pregnant factor V Leiden carriers, one would not expect an effect on first trimester miscarriages, since the placental circulation in the first trimester has not yet been fully established (Hustin and Schaaps, 1987
; Burton et al., 1999
). Therefore, impaired placental perfusion might not be critical for the embryonic development and implantation during (very) early gestation. We cannot differentiate between the types of early pregnancy loss in this study (e.g. presence or absence of fetal heart prior to the loss), as all the data were provided to us by the patients through interview. Furthermore, most would not have had an early first trimester scan, as they are not routinely done in The Netherlands.
Our results indicate a clear reduction in first trimester loss in factor V Leiden carriers, which is subsequently compensated by an increased loss in the second trimester. Similar findings were reported in a recent study where a decreased risk of recurrent pregnancy losses at <10 weeks gestation was found in factor V Leiden carriers (OR 0.23, 95% CI 0.070.77) with a subsequent increase in losses after 10 weeks (Roqué et al., 2004). This suggests a protective effect on the embryo during the first trimester in factor V Leiden carriers, including less viable embryos that will eventually abort in the second trimester. This may explain the similar overall frequency of miscarriages in factor V Leiden carriers and non-carriers. A lower frequency of miscarriages in the first trimester may thus reflect a successful implantation. Approximately 5070% of all miscarriages are attributable to detectable chromosomal abnormalities, furthermore 1520% is thought to be due to morphological defect(s) in the embryo. A recent study confirmed this with a transcervical embryoscopy at the time of the curette and cytogenetic analysis of the products of conception (Philipp et al., 2003
). We have no cytogenetic information in our study, as karyotyping of the products of conception is not a routine consideration in The Netherlands. The possible protective effect of factor V Leiden early in pregnancy will require further study with, among other things, cytogenetic testing of the miscarriage products and possibly transcervical embryoscopy prior to evacuation.
Our study did not show a clearly increased fecundity in factor V Leiden carriers. Many factors affect fecundity, including physiological, behavioural and environmental factors. Known factors, such as age at pregnancy attempt, regularity of the menstrual cycle, smoking habits and educational level were similar in factor V Leiden carriers and non-carriers. As fecundity is a manifestation of both conception and implantation, it is important to further distinguish these. For conception, factors such as sperm quality, coital frequency and timing are of great importance. However, this study was not designed to examine these factors and it remains unclear whether factor V Leiden has any effect on these factors. Aspects influencing implantation have not yet been fully elucidated; however, improved implantation could be due to an increase in the hypercoagulable state, related to the factor V Leiden mutation. This was suggested by a study that omitted conception by reporting only on ICSI pregnancies, reflecting implantation success (Göpel et al., 2001). A higher incidence of implantation success was found if either the mother or the fetus was a factor V Leiden carrier. It is possible that the beneficial effect of factor V Leiden on implantation alone has a less clear affect on fecundity compared to various factors concerning conception, and therefore, in our study, fails to show an overall difference in fecundity.
We investigated the reproductive histories of women who had suffered venous thrombosis. The choice of this design was opportunistic, in that it offered the opportunity to study a large cohort of factor V Leiden carriers. We have considered whether this choice, rather than the ideal study of factor V Leiden carriers without thrombosis, could have distorted our results. First, the period about which questions were asked preceded the thrombotic event, in most cases by many years. Hence, the thrombotic event itself did not influence our results. As the patients developed thrombosis, they will have had more risk factors for thrombosis than other women. It is known that this is not only true for women without factor V Leiden but also for women with factor V Leiden (the majority of people with factor V Leiden never develop thrombosis, and there must be causes why some do). For this reason, we chose thrombosis cases without factor V Leiden as controls rather than healthy women without factor V Leiden, and therefore differences between the group can be attributed to factor V Leiden. One could argue that the groups differed more: women with thrombosis without factor V Leiden probably had more additional risk factors than those with factor V Leiden, for instance another, possibly still unknown, gene defect. This could, if that other prothrombotic factor also affected implantation and fetal loss, explain the absence of a difference between the groups in the frequency of miscarriage. However, we did find a difference.
As the reproductive data in the present study were collected by interview, recall bias is possible, but seems unlikely. Validation studies of fecundity and miscarriages have shown a good match between long-term recall of personal reproductive history through interview and medical data (Joffe et al., 1993). Moreover, the women were unaware of their factor V Leiden status at the time of the questionnaire.
In conclusion, factor V Leiden mutation may support embryo implantation, as factor V Leiden carriers reported significantly fewer miscarriages in the first trimester. This was not reflected in an increased fecundity. The overall miscarriage proportion was not influenced by factor V Leiden status. These results suggest that factor V Leiden offers a protective effect on early pregnancy and that the miscarriage of embryos with poor viability in factor V Leiden carriers is postponed until the second trimester.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Burton GJ, Jauniaux E and Watson AL (1999) Maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the Boyd collection revisited. Am J Obstet Gynecol 181, 718724.[ISI][Medline]
De Groot CJ, Bloemenkamp KW, Duvekot EJ, Helmerhorst FM, Bertina RM, Van Der Meer F, De Ronde H, Oei SG, Kanhai HH and Rosendaal FR (1999) Preeclampsia and genetic risk factors for thrombosis: a casecontrol study. Am J Obstet Gynecol 181, 975980.[ISI][Medline]
Dilley A, Benito C, Hooper WC, Austin H, Miller C, El-Jamil M, Cottrell S, Benson J, Evatt BL, Patterson-Bamett A et al. (2002) Mutations in the factor V, prothrombin and MTHFR genes are not risk factors for recurrent fetal loss. J Matern Fetal Neonat Med 11, 176182.
Dudding TE and Attia J (2004) The association between adverse pregnancy outcomes and maternal factor V Leiden genotype: a meta-analysis. Thromb Haemost 91, 700711.[ISI][Medline]
Göpel W, Ludwig M, Junge AK, Kohlmann T, Diedrich K and Moller J (2001) Selection pressure for the factor-V-Leiden mutation and embryo implantation. Lancet 358, 12381239.[CrossRef][ISI][Medline]
Hundsdoerfer P, Vetter B, Stover B, Bassir C, Scholz T, Grimmer I, Monch E, Ziemer S, Rossi R and Kulozik AE (2003) Homozygous and double heterozygous Factor V Leiden and Factor II G20210A genotypes predispose infants to thromboembolism but are not associated with an increase of foetal loss. Thromb Haemost 90, 628635.[ISI][Medline]
Hustin J and Schaaps JP (1987) Echographic [corrected] and anatomic studies of the maternotrophoblastic border during the first trimester of pregnancy. Am J Obstet Gynecol 157, 162168.[ISI][Medline]
Joffe M, Villard L, Li Z, Plowman R and Vessey M (1993) Long-term recall of time-to-pregnancy. Fertil Steril 60, 99104.[ISI][Medline]
Kovalevsky G, Gracia CR, Berlin JA, Sammel MD and Barnhart KT (2004) Evaluation of the association between hereditary thrombophilias and recurrent pregnancy loss: a meta-analysis. Arch Intern Med 164, 558563.
Lindqvist PG, Svensson PJ, Marsaal K, Grennert L, Luterkort M and Dählback B (1999) Activated protein C resistance (FV:Q506) and pregnancy. Thromb Haemost 81, 532537.[ISI][Medline]
Lindqvist PG, Zöller B and Dählback B (2001) Improved hemoglobin status and reduced menstrual blood loss among female carriers of factor V Leidenan evolutionary advantage? Thromb Haemost 86, 11221123.[ISI][Medline]
Majerus PW (1994) Human genetics. Bad blood by mutation. Nature 369, 1415.[CrossRef][ISI][Medline]
Morrison ER, Miedzybrodzka ZH, Campbell DM, Haites NE, Wilson BJ, Watson MS, Greaves M and Vickers MA (2002) Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review. Thromb Haemost 87, 779785.[ISI][Medline]
Pauer HU, Voigt-Tschirschwitz T, Hinney B, Burfeind P, Wolf C, Emons G and Neesen J (2003) Analyses of three common thrombophilic gene mutations in German women with recurrent abortions. Acta Obst Gynecol Scand 82, 942947.[CrossRef][ISI][Medline]
Philipp T, Philipp K, Reiner A, Beer F and Kalousek DK (2003) Embryoscopic and cytogenetic analysis of 233 missed abortions: factors involved in the pathogenesis of developmental defects of early failed pregnancies. Hum Reprod 18, 17241732.
Rai R, Shlebak A, Cohen H, Backos M, Holmes Z, Marriott K and Regan L (2001) Factor V Leiden and acquired activated protein C resistance among 1000 women with recurrent miscarriage. Hum Reprod 16, 961965.
Rees DC (1996) The population genetics of factor V Leiden (Arg506Gln). Br J Haematol 95, 579586.[ISI][Medline]
Rey E, Kahn SR, David M and Shrier I (2003) Thrombophilic disorders and fetal loss: a meta-analysis. Lancet 361(9361), 901908.[CrossRef][ISI][Medline]
Roqué H, Paidas MJ, Funai EF, Kuczynski E and Lockwood CJ (2004) Maternal thrombophilias are not associated with early pregnancy loss. Thromb Haemost 91, 290295.[ISI][Medline]
Rosendaal FR, Koster T, Vandenbroucke JP and Reitsma PH (1995) High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 85, 15041508.
Zinaman MJ, Clegg ED, Brown CC, O'Connor J and Selevan SG (1996) Estimates of human fertility and pregnancy loss. Fertil Steril 65, 503509.[ISI][Medline]
Submitted on May 25, 2004; resubmitted on September 7, 2004; accepted on November 9, 2004.