Comparative incidence of pregnancy outcomes in thrombophilia-positive women from the NOH-APS observational study

Pregnancy outcomes in symptomatic women carrying the F5 rs6025 (factor V Leiden) or F2 rs1799963 (prothrombin gene G20210A) polymorphism are still debated.

The Thrombosis: Risk and Economic Assessment of Thrombophilia Screening (TREATS) study systematically reviewed selected case-control studies to determine the clinical risks associated with thrombophilia in pregnancy.¹  Heterozygous F5 rs6025 and F2 rs1799963 polymorphisms were associated with early pregnancy loss in the first and second trimester (factor V Leiden: 172/243 [70.8%] vs 1632/2689 [60.7%], mean odds ratio [OR], 1.68; prothrombin gene polymorphism: 53/75 [60.3%] vs 657/1356 [48.5%], mean OR, 2.49), with late loss in the third trimester (factor V Leiden: 27/382 [7.1%] vs 124/1121 [11.1%], mean OR, 2.06; prothrombin gene polymorphism: 5/7 [71.4%] vs 35/113 [30.9%], mean OR, 2.66) and with preeclampsia (PE; factor V Leiden: 161/249 [64.7%] vs 1790/3673 [48.7%], mean OR, 2.19; prothrombin gene polymorphism: 42/71 [59.1%] vs 937/2028 [46.2%], mean OR, 2.54). Despite the increase in relative risk (RR), the absolute risk for adverse outcomes in pregnancy remained low. The subsequent meta-analysis of 10 prospective cohort studies only showed that women bearing the F5 rs6025 polymorphism were at a small absolute increased risk for late pregnancy loss (from 511/16 158 [3.2%] in women testing negative for the polymorphism to 34/801 [4.2%] in women testing positive for the polymorphism, mean OR, 1.52). These results could not be further restricted to women with previous pregnancy complications and according to the clinical subtype of previous pregnancy losses.

The Nîmes Obstetricians and Hematologists Antiphospholipid Syndrome (NOH-APS) study observed a cohort of nonthrombotic women with previous pregnancy loss, recurrent unexplained spontaneous abortions, or a single fetal death, focusing on patients with APS.³  The 2 included control groups, the first group consisting of women with a negative thrombophilia screening result and the other group consisting of those with a positive F5 rs6025 or positive F2 rs1799963 polymorphism, gave us the opportunity to evaluate the heterogeneity of new pregnancy outcomes in thrombophilic women with prior abnormal pregnancies, according to the heterogeneity of their clinical history.

Recruitment of patients is presented in Figure 1 and has been described in detail elsewhere.³  Briefly, 6318 women with pregnancy loss during spontaneous pregnancies were referred, during a 10-year period, for investigation to the Outpatient Department of Hematology, University Hospital of Nîmes (a tertiary referral center). Exclusion criteria were any history of thrombotic events (at least 1 clinical episode of venous, arterial, or small-vessel thrombosis, in any tissue or organ except the placenta, thrombosis being confirmed by objective validated criteria, ie, unambiguous findings by appropriate imaging studies or histopathology studies), or any treatment given during previous pregnancies that may have modified the natural evolution of their condition, such as antithrombotics, or immunosuppressive or immunity-modulating drugs. Women with pregnancy losses explained by infectious, metabolic, anatomic, or hormonal factors were also excluded. Women with seropositive results for HIV or hepatitis B or C were also excluded. A total of 4801 women fulfilled one of the 2 following inclusion criteria: (1) 3 unexplained consecutive spontaneous abortions before the 10th week of gestation, not caused by maternal anatomic or hormonal abnormalities or by paternal and maternal chromosomal causes (recurrent embryo loss subgroup); or (2) 1 unexplained death of a morphologically normal fetus (fetal loss) at or after the 10th week of gestation, with normal fetal morphology having been documented by ultrasound scan or direct examination of the fetus (fetal loss subgroup). Patients were categorized as primary aborters (no previous successful pregnancy) or secondary aborters.

The women underwent standardized thrombophilia screening at 3 to 6 months after the last pregnancy loss; screening included a complete whole blood count, and tests for fibrinogen, antithrombin, protein C, protein S, polymorphisms F5 rs6025 (factor V Leiden polymorphism) and F2 rs1799963 (prothrombin gene G20210A polymorphism), the JAK2 V617F mutation, and antiphospholipid antibodies (aPLAbs).

The 152 women with an incomplete thrombophilia screening were not included. Women with antithrombin, protein C, or protein S deficiency, and women with an abnormal fibrinogen or the JAK2 V617F mutation were not included.

Women with completely negative thrombophilia screening results (N = 3741) and who agreed to participate in the observational study (N = 3604) were provisionally assigned to the “negative” group and were numbered according to the order in which they entered the study. Women with an isolated F5 rs6025 polymorphism or isolated F2 rs1799963 polymorphism (N = 301) and who agreed to participate in the observational study (N = 279) constituted the “thrombophilia” group.

Women who initially tested positive for aPLAbs, with or without the F5 rs6025 or F2 rs1799963 polymorphism, who agreed to participate in the observational study and who persistently tested positive for aPLAbs at the 6-month test (N = 517) were definitively assigned to the “APS” group. As each APS or thrombophilia woman entered the study, the next woman on the list of the negative group was definitively assigned to the “no-thrombophilia” group and was included in the study.

We do not discuss APS women in our present work. In total, 796 women in the no-thrombophilia group and 279 women in the thrombophilia group were studied (Table 1).

The study was approved by the University Hospital of Nîmes Institutional Review Board and ethics committee and by the local Comité de Protection des Personnes soumises à la Recherche Biomédicale. This clinical investigation was performed in accordance with the Helsinki declaration of 1975 as revised in 1996. All of the women gave their informed consent to participate in this study.

All patients were informed at the start of the study about the advisability of early blood testing in case of suspicion of a new pregnancy; they were then observed until 18 months after the final recruitments and any new pregnancy noted. Women with any positive pregnancy test result were instructed to contact their general practitioner for evaluation and, after discussion with a study physician, the possible initiation of prophylaxis.

Patients were clinically reevaluated each year in our outpatient department. Patient loss to follow-up was minimized by directly contacting the general practitioners and the patients themselves. A complete clinical check-up was performed. Any suspicion of developing systemic disease led to further adapted investigations for diagnosis. Symptoms were evaluated, and treatments received during the year were recorded. There was no systematic aPLAbs assessment if a new pregnancy occurred.

Women in the thrombophilia and no-thrombophilia groups were not taking any chronic prophylaxis for thrombosis.

Low-molecular-weight heparin (LMWH; enoxaparin, 40 mg/day ie, 4000 U/day) was given to all patients from the thrombophilia group with any fetal loss antecedent,⁴  from the positive pregnancy test to delivery, whereas thrombophilic women with previous recurrent abortions received no antithrombotic treatments. Compliance with the LMWH treatment was monitored only by self-declaration by the patient-companion couples and systematic examination of subcutaneous injection sites at each medical examination. Monitoring and investigation for heparin-induced thrombocytopenia was performed according to published recommendations.⁵  Enoxaparin injections were stopped at the onset of uterine contractions indicating imminent delivery. Patients of the no-thrombophilia group had no drug-mediated prophylaxis during pregnancy.

Events occurring during the first pregnancy during the observational period were systematically reviewed.

Thrombotic events were objectively confirmed as described elsewhere.³ 

Fetal death was defined as the intrauterine demise of a conceptus known to be alive at or beyond 10 completed weeks of gestation (WG) documented by (1) detection of fetal heart tones, (2) ultrasonic detection of a conceptus with biometric measurements indicating a gestational age at or beyond 10 WG, or (3) delivery of a dead conceptus whose size indicated a gestational age at or beyond 10 WG. All pregnancy losses before 10 WG were considered spontaneous abortions of either an anembryonic or embryonic nature.

Women were classified as having hypertension if they were using hypertensive medication or had either a systolic blood pressure of ≥140 mm Hg or a diastolic blood pressure of ≥90 mm Hg, both on 2 readings taken with the patient in a supine position 5 minutes apart, and on 2 separate occasions. PE was defined as diastolic blood pressure increasing to >90 mm Hg, or systolic blood pressure increasing to >140 mm Hg on 2 occasions at least 4 hours (h) apart after 20 WG, accompanied by a significant proteinuria (>0.3 g in a 24-h urine sample). Women with preexisting hypertension were scored as having PE if there was a new onset of proteinuria. PE was classified as severe if 1 or more of the following was observed: severe hypertension (diastolic >110 mm Hg or systolic >160 mm Hg), eclampsia (seizures), pulmonary edema, proteinuria >5g per 24 h, renal insufficiency as revealed by abnormal creatininemia levels, abnormally high liver enzyme levels (aspartate aminotransferase or alanine aminotransferase levels >70 IU/L) with abdominal pain, or low platelet counts (<100 G/L). HELLP syndrome was defined according to the criteria established at the University of Tennessee: hemolysis on peripheral blood smear (schistocytes) with serum lactate dehydrogenase levels >600 IU/L, serum aspartate aminotransferase levels >70 IU/L, and platelet count <100 000/μL. PE with onset before 34 WG was defined as early-onset PE.

Biometry-based gestational age was assessed according to charts derived from published data,^6,7  and newborn weight percentiles were taken from standard French birth weight charts. Babies with birth weights lower than the corresponding gestational age- and sex-adjusted, customized 10th percentile were scored as small-for-gestational age (SGA). Severe SGA was defined as weight below the fifth percentile, very severe SGA as below the third percentile.

Placental abruption (PA) was defined according to classic clinical prenatal signs and symptoms: vaginal bleeding accompanied by nonreassuring fetal status or uterine hypertonicity, or visualization of abruption by ultrasound scanning, and evidence of retroplacental clots during examination of the delivered placenta. Cases were confirmed by histopathological diagnosis.

Neonatal mortality (NM) was defined as death before age 28 days; death before age 8 days was recorded as early neonatal mortality and death after age 8 days as late neonatal mortality.

Major bleeding events were classified according to the definitions of the Subcommittee on Control of Anticoagulation of the International Society on Thrombosis and Haemostasis⁸ : fatal bleeding; and/or symptomatic bleeding in a critical area or organ, such as intracranial, intraspinal, intraocular, retroperitoneal, intra-articular, or pericardial bleeding; intramuscular bleeding with compartment syndrome; and/or bleeding that led to the hemoglobin concentration decreasing to 20 g/L or lower, or that led to transfusion of 2 or more units of red blood cells.

All events were adjudicated by a committee of independent experts blinded to the thrombophilia screening results.

Quantitative data were described by median, interquartile range, and range values. Qualitative data were described by values and percentages. Comparisons between baseline characteristics and risk factors were performed using the Student ttest, the Mann-Whitney test, the Kruskal-Wallis test, the χ² test, and the Fisher exact test when appropriate.

Definition of groups and analysis of the impact of biological covariates on pregnancy outcome incidences were based on values obtained at the initial inclusion.

All analyses were based on pregnancy outcomes that occurred during the first pregnancy attempt, treated as described above, after initial investigations and recruitment.

We compared the pregnancy outcomes between groups estimating a RR using the Mantel-Haenszel method, with a 95% confidence interval (CI), providing a P value from the associated Mantel-Haenszel χ² test.

Putative predictors of various outcomes among the clinical predictors (age, body mass index, thrombotic familial antecedents, ethnicity, smoking history, varicose veins, preexisting hypertension, embryonic/fetal pregnancy loss, primary/secondary pregnancy loss, and initial inflammatory disease), the metabolic markers at inclusion (hypercholesterolemia defined as a fasting cholesterol concentration >5.2 mmol/L and hypertriglyceridemia as a fasting triglyceride concentration >1.7 mmol/L) and the biological predictors (positive F5 rs6025 or F2 rs1799963 polymorphism) were evaluated in women from the Constitutional Thrombophilia group, first by univariate analysis then by multivariate analysis. For multivariate models, a stepwise variable selection was performed, starting with all of the variables from the univariate models having P < .20 as potential predictors, with adjustment being finally performed for all variables with P < .20 in the univariate models. The final model included only main effects with P < .10.

All tests were 2 sided and were assessed at the 5% significance level.

Statistical analyses were performed using SAS-Windows software (version 9.1).

This study included 1075 nonthrombotic women with unexplained pregnancy loss (Table 1), the dominant inclusion criterion being recurrent unexplained embryonic pregnancy loss in the no-thrombophilia group but fetal loss in the thrombophilia group. The heterozygous F5 rs6025 polymorphism was the main thrombophilia (n = 176, 63%), and the heterozygous F2 rs1799963 polymorphism was evident in 103 women (37%). There were no homozygote women and 11 double-heterozygote women (3.9%). Fetal death or venous thrombotic histories were also more frequent in the first-degree relatives of women included in the Constitutional Thrombophilia group. The interval between the inclusion and the observed new pregnancy attempt was not different between the groups. A globally normal fecundity rate was evidenced, nearly all included women (1069/1075) initiating a new pregnancy attempt during the observational period.

Almost all women had a new positive pregnancy test result after inclusion, with no difference between thrombophilic and nonthrombophilic women (Table 2). None of the women in this subgroup received any antithrombotics during pregnancy.

Thrombophilic women demonstrated similar spontaneous abortion recurrence rates as nonthrombophilic women (13/93 [14%] vs 92/480 [19.2%], respectively), but as pregnancies evolved, higher rates of fetal death were observed (7/80 [8.8%] vs 9/388 [2.3%], respectively; P = .0063). A nonsignificant trend for higher PE rates (5/75 [6.6%] vs 11/384 [2.9%], respectively) and early-onset PE rates (3/75 [4%] vs 4/384 [1%], respectively), higher preterm live birth rates (12/73 [16.4%] vs 35/379 [9.2%], respectively), and higher early neonatal mortality rates (2/73 [2.7%] vs 2/379 [0.5%], respectively) was also evidenced in thrombophilic women.

A new pregnancy could be diagnosed after inclusion in almost all women (Table 3).

Treated thrombophilic women in this subgroup experienced fewer fetal deaths than untreated nonthrombophilic women (19/185 [10.3%] vs 72/311 [23.2%], respectively; P = .0007). Consequently, they more frequently gave birth to living neonates (130/185 [70.3%] vs 156/311 [50.2%], respectively; P < .0001), with fewer cases of preterm living births before 37 weeks (16/130 [12.3%] vs 39/156 [25%], respectively; P = .0106).

Whereas the comparison of the rates of all PE cases between thrombophilic and nonthrombophilic women did not reach statistical significance, treated thrombophilic women experienced less cases of severe PE (3/139 [2.2%] vs 15/180 [8.3%], respectively; P = .0242) and less cases of early-onset PE before 34 weeks (1/139 [0.7%] vs 11/180 [6.1%], respectively; P = .0394) than untreated nonthrombophilic women. Finally, we observed a nonsignificant trend toward lower rates of SGA babies (19/139 [13.7%] vs 37/180 [20.6%], respectively; P = .115) and placenta-mediated complications as a whole (26/139 [18.7%] vs 50/189 [27.8%], respectively; P = .064) in these LMWH-treated thrombophilic women compared with the non-LMWH–treated, nonthrombophilic women.

Prophylactic LMWH treatments were given to 185 thrombophilic pregnant women. No treatment had to be stopped for safety reasons; we observed no major bleeding events.⁸  There were no cases of heparin-induced thrombocytopenia, symptomatic osteoporosis, or thrombosis. Patients who received epidural or spinal analgesia or anesthesia experienced no complications, with no case of epidural hematoma or hemorrhagic or neurologic complications.

Three patients (1.6%) experienced allergic skin reactions after enoxaparin injections, and these symptoms resolved after switching for another prophylactic LMWH. Easy bruising, at least at the injection site, was frequent (n = 97 [52.4%]), and sometimes also in cutaneous friction or contact zones (n = 39 [21.1%]).

Primary postpartum hemorrhage occurred in 15 (8.1%) of the 185 treated patients and in 51 (6.4%) of the 796 nonthrombophilic women (P = .41); severe primary postpartum hemorrhage with an estimated blood loss of >1000 mL occurred in 3 treated patients (1.6%) and in 10 nonthrombophilic women (1.3%, P = .97).

Heterogeneous therapeutic treatment of the thrombophilic patients during their new pregnancy led us to evaluate the putative predictors of various pregnancy outcomes: first, in cases of previous recurrent abortions; second, in cases of previous fetal loss. None of the predictors reached statistical significance for any outcome after univariate analyses was performed in each subgroup defined by its inclusion criterion. P values systematically exceeded .20 in the recurrent abortion group. P values between .10 and .20 were rarely observed in the fetal death group: they remained higher than .10 when a multivariate analysis was possible (data not shown).

Our observational study of nonthrombotic F5 rs6025 or F2 rs1799963 heterozygous patients with pregnancy loss shows heterogeneous results.

Thrombophilic patients with prior recurrent abortions, who did not receive any antithrombotic treatment during their new pregnancy attempt, had an increased risk for fetal death comparatively to untreated thrombophilia-negative women sharing the same obstetric history.

Thrombophilic patients with prior fetal death, who received LMWH treatment (prophylactic dosage) during their new pregnancy attempt, were at lower risk for the development of fetal death recurrence, premature live birth, PE, and early-onset PE. They more frequently gave birth to a living neonate than untreated thrombophilia-negative women sharing the same obstetric history.

These observational results must be interpreted with caution. The absence of academic control groups (untreated thrombophilic women with prior fetal loss and treated nonthrombophilic women with prior fetal loss) cannot lead to definitive conclusions. LMWH treatments may have had no effect or even some deleterious effects if thrombophilias are protective factors against bad pregnancy outcomes. However, this is not the described tendency,^1,2  and our thrombophilia-positive untreated patients with prior recurrent abortions experienced more fetal death cases than their thrombophilia-negative untreated control patients.

We studied previously symptomatic patients. We may have selected a group of women in whom thrombophilia is only one of the individual characteristics cooperating with a series of biological cofactors and mechanisms, yet unknown, altogether contributing to the risk for abnormal pregnancy outcomes. LMWH use in a secondary prophylaxis setting may be the main determinant of our results, which cannot apply to primary prophylaxis. The suspected interactions may per se explain most of the clinical effects that we observed. Experimental in vitro data show that LMWH may promote extravillous trophoblast development, being able to stimulate their invasive properties.^9,10  Series of placental bed biopsies from late, sporadic miscarriage with a normal karyotype showed reduced trophoblast invasion features,¹¹  which were negative in cases of early embryonic demise.¹²  Heparin-binding epidermal growth factor–like growth factor triggers chemotaxis of endometrial stromal cells,¹³  which may be modified by heparin. The classic complement pathway inhibition in pregnant women treated with heparin may also be relevant,¹⁴  altered complement regulation being the rule in PE.¹⁵  However, invasion of the decidua by extravillous trophoblasts is accompanied by thrombin generation from decidual cell–expressed tissue factor.¹⁶  In thrombophilic women, excess thrombin formation may abnormally enhance expression of soluble fms-like tyrosine kinase-1,¹⁶  a potent inhibitor of angiogenesis involved into PE, thus prone to be regulated by LMWH.

Our results in nonthrombophilic women show 79.6% (95% CI, 76%-83%) of new pregnancy attempts ending in live births in women with prior recurrent abortions, but only 50% (95% CI, 45%-56%) of these attempts leading to a living neonate in women with prior fetal death. In this latter group, 26.7% (95% CI, 22%-32%) and 23.2% (95% CI, 18%-28%) of new pregnancy attempts ended in abortion or fetal death, respectively. Our observations are on line with available results.^17-19  In a retrospective Dutch study on pregnancy after a first loss in women with the F5 rs6025 or F2 rs1799963 polymorphism, the live birth rates were 80% (95% CI, 49%-94%) in noncarriers after a late loss after 12 WG and 76% (95% CI, 57%-89%) after an early loss, thus overlapping our CIs despite a nonsimilar time frame–related dichotomization of events.²⁰  Thus, prior unexplained fetal loss defines a population of women with a poor natural prognosis in the subsequent pregnancy. Dichotomization of pregnancy losses according to the 10 WG threshold, as in APS clinical criteria,²¹  should also be regarded as more systematic outside of any aPLAb context.

Our results in untreated women with the F5 rs6025 or F2 rs1799963 polymorphism and prior recurrent early losses, which show 78.5% (95% CI, 76%-83%) of new pregnancy attempts ending in live births, are also in agreement with published data in women with a first pregnancy loss before 12 WG describing a 77% success rate (95% CI, 62%-87%).¹⁹ 

Our data are globally in line with a recent multicentric observational Italian study suggesting that LMWH prophylaxis may reduce the risk for spontaneous miscarriages in F5 rs6025 or F2 rs1799963-positive women, particularly in those with previous obstetric events.²²  They also, at least partly, resemble the results of a multicentric randomized controlled trial performed in women with inheritable thrombophilia and prior PE, mainly F5 rs6025 or F2 rs1799963 heterozygosity, which showed a strong effect of LMWH on the occurrence of early-onset PEs.²³ 

Our study had some limitations. First, we included heterogeneous patients; this study was not a randomized controlled trial but only an observational study. Genome-wide scan in affected sibling pairs with miscarriages has suggested genetic linkage,²⁴  and such a genetic background may act as a strong confounder. Second, our study was not an academic multicenter study, even if several primary and secondary centers from our administrative area participated in the initial screening of patients. Some patients may have been treated independently from our network. Third, most of the lost embryos were not submitted for karyotype analysis, which may have explained some losses. Our study could not evaluate preclinical, very early losses. We probably did not always study the first pregnancy occurring during follow-up. Fourth, in treated patients, therapeutic compliance was assessed only through self-reporting.

Our study also had several strengths. We could rely on the Nîmes Obstetricians and Haemotologists administrative region-hospital medical network,⁴  which could recruit a substantial number of patients.³  A new pregnancy was the rule after inclusion, without any losses to follow-up, because of limited time between inclusion and new attempts. Pregnancy outcomes could be objectively diagnosed using international criteria or definitions. Therefore, we believe that the number of events that was not registered into the database was minimal.

Because of the lack of definitive randomized controlled trials giving strong conclusions, we understand the latest American College of Chest Physicians grade 2C conservative recommendation suggesting that women with inherited thrombophilia and a history of pregnancy complications not use antithrombotic prophylaxis.²⁵  However, observational evidence obtained in our current study and in other studies^22,23  may give some valuable clues and keys for a precise successful design of future randomized controlled trials, if any. Thrombophilic women with a history of fetal death, not abortions (and probably also late pregnancy complications) should probably be the most promising clinical target. Waiting for any substantial progress of precision medicine in the obstetric field may allow classification of pregnancy complications on causal molecular determinants, thus defining accurate groups of patients more prone to targeted therapeutic randomized controlled trials.

An initial miscarriage has been associated with a higher risk for obstetric complications in the next continuing pregnancy.²⁶  The F5 rs6025 or F2 rs1799963 polymorphism affects pregnancy outcomes.¹  Thrombophilic women with prior recurrent abortions experienced more fetal death than nonthrombophilic women. Thrombophilic women with prior fetal loss, receiving LMWH treatment during a new pregnancy attempt, experienced less pregnancy complications than untreated nonthrombophilic women.

We thank all of the study participants, the patients who finally agreed to join us in this long-distance running adventure. We thank H. Bres for technical assistance. We are also grateful to the Nîmes Obstetricians and Haematologists network of gynecologists, obstetricians, and general practitioners who actively contributed to the study. We thank the adjudication committee (S. Ripart-Neveu and E. Arnaud). We also thank M.L. Tailland, D. Dupaigne, C. Ferrer, V. le Touzey, M. Hoffet, A. Cornille, B. Fatton, E. Mousty, F. Masia, L. Boileau, F. Grojean, R. de Tayrac, and A. Sotto for their efficient help in management of the patients. We thank the research staff of the “Direction de la Recherche Clinique et de l’Innovation” of the University Hospital of Nîmes: S. Clément, C. Meyzonnier, N. Best, S. Granier, B. Lafont, H. Obert, H. Léal, O. Albert, C. Suehs, P. Rataboul, and M.P. Francheschi. We thank the staff of the “Departement de Biostatistiquee, Epidémiologie, Santé Publique et Information Médicale BESPIM” of the University Hospital of Nîmes: P. Landais, J.P. Daurès, and S. Leroy.

Contribution: J.-C.G., J.-P.B., and P.M. designed the research and wrote the paper; P.F.-P. performed the statistical analysis and wrote the paper; S.B., J.-C.G., G.L.-L., J.-P.B., and P.M. performed the research and contributed to the analysis of the data; and S.B., E.C.-N., E.M., and G.L.-L. contributed analytical tools and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Jean-Christophe Gris, Laboratory of Hematology, University Hospital Caremeau, Place du Pr. Robert Debré, 30029 Nîmes cedex 9, France; e-mail: jean.christophe.gris@chu-nimes.fr.