Genetic risk assessment of thrombophilia in patients with adverse obstetric outcomes
Genetic risk assessment of thrombophilia in patients with adverse obstetric outcomes
M. Fern a?ndez Arias 0 1 2
E. MazaricoID 0 1 2
A. Gonzalez 0 1 2
M. Muniesa 0 1 2
C. MolinetID 0 1 2
L. Almeida 0 1 2
M. D. Go? mez Roig 0 1 2
0 BCNatal-Barcelona Center for Maternal-Fetal and Neonatal Medicine (Hospital Sant Joan de Deu and Hospital Cl ??nic) , Barcelona , Spain , 2 Maternal and Child Health and Development Network II (SAMID II) funded by Instituto de Salud Carlos III (ISCIII)-Sub-Directorate General for Research Assessment and Promotion and the European Regional Development Fund (ERDF) , Madrid , Spain , 3 Institut de Recerca Sant Joan de D e ?u (IR-SJD) , Barcelona , Spain
1 Editor: Dermot Cox, Royal College of Surgeons in Ireland , IRELAND
2 Funding: We thank the Gynecology and Obstetrics Service at the Sant Joan de De ?u University Hospital in Barcelona and the RETICS, funded by the PN I
3 I 2008-2011 (Spain), the ISCIII- Sub-Directorate General for Research Assessment and Promotion, the European Regional Development Fund (ERDF , ref. RD12/0026) , and the Fund for Health Research of the Spanish Social Security Service, for financial support of this project , Exp. PI11/02613
This is a case-control prospective study performed in Hospital Sant Joan de De? u in Barcelona, Spain. Cases had a history of intrauterine growth restriction requiring delivery before 34 weeks gestation, placental abruption before 34 weeks gestation, or severe preeclampsia. Controls had at least two normal, spontaneously conceived pregnancies at term, without complications or no underlying medical disease. At least 3 months after delivery, all case and control women underwent blood collection for standard blood tests for thrombophilias and saliva collection for the genetic test, which enables the diagnosis of 12 hereditary thrombophilias by analyzing genetic variants affecting different points of the blood coagulation cascade.
and a novel genetic test.
To investigate the incidence of inherited thrombophilias in patients with adverse obstetric
outcomes and to compare detection rates of thrombophilias between standard blood tests
The study included 33 cases and 41 controls. There were no statistically significant
differences between cases and controls in the standard blood tests for thrombophilias in plasma
or the TiC test for genetic variables. One clinical-genetic model was generated using
variables with the lowest P values: ABO, body mass index, C_rs5985, C_rs6025, and protein S.
This model exhibited good prediction capacity, with an area under the curve of almost 0.7
(P <0.05), sensitivity of almost 67%, and specificity of 70%.
Although some association may exist between hypercoagulability and pregnancy outcomes,
no significant direct correlation was observed between adverse obstetric outcomes and
Competing interests: The authors have declared
that no competing interests exist.
inherited thrombophilias when analyzed using either standard blood tests or the genetic
test. Future studies with a larger sample size are required to create a clinical-genetic model
that better discriminates women with a history of adverse pregnancy outcomes and an
increased risk of poor outcomes in subsequent pregnancies.
Thrombophilia describes a tendency to develop thromboses because of inherited or acquired
disorders of blood coagulation or fibrinolysis, which lead to a prothrombotic state [
Causes of hereditary thrombophilia include factor V Leiden mutation (activated protein C
[PC] resistance); prothrombin 20210A mutation; PC, protein S (PS), and antithrombin III
(AT III) deficiencies; lupus anticoagulant; anticardiolipin antibodies;
methylenetetrahydrofolate (MTHFR) gene mutation; and hyperhomocysteinemia.
The hemostatic system appears to play an important role in both establishing and
maintaining pregnancy. Development of the placental circulation is ensured by structural modifications
of the spiral arteries and a pregnancy-induced hypercoagulable state resulting from an increase
in procoagulant factors and a decrease in anticoagulant factors and fibrinolysis [
pregnancy itself induces a physiological hypercoagulable state that might be exacerbated by
inherited or acquired thrombophilia [
There has always existed much controversy about the association between inherited
thrombophilias and the possibility of adverse pregnancy outcomes due to uteroplacental thrombosis
]. A number of authors suggest that the presence of any thrombophilic condition may
cause venous or arterial thrombosis and placental circulation abnormalities, leading to a
higher rate of certain obstetrical complications, such as fetal loss, severe preeclampsia, severe
fetal growth restriction, or placental abruption [
2, 5, 8
]. Padmashree et al. reported that
inherited and acquired thrombophilias can be found in 49% to 65% of women with pregnancy
complications, in contrast to 28% to 22% of women with normal pregnancies, suggesting a
three- to eight-fold increased risk of thrombophilia in women with complications.
Accordingly, these authors recommended antenatal screening for congenital and acquired
thrombophilias among women with prior adverse pregnancy outcomes, with the intention of offering
treatment with anticoagulants or antiplatelet agents. Most studies supporting this approach
hypothesize that microthrombi, thrombosis, and infarction of the placenta contribute to
pregnancy complications or loss [
]. In addition, there is evidence that women with any type
of thromboembolic defect have a higher prevalence of pregnancy complications [
Nevertheless, many current guidelines do not recommend screening unless a personal or
strong family history of venous thromboembolism is present. Some reasons can be found
against screening women with a history of adverse pregnancy outcome. First of all, some
studies fail to demonstrate a strong association between hypercoagulability and pregnancy
outcomes. And moreover, most pregnant women with inherited thrombophilia have normal
pregnancy outcomes [
Anticoagulants and aspirin are the best studied and most commonly used therapeutic
agents to prevent pregnancy complications in thrombophilic women [
], and they have
been demonstrated to reduce the incidence of adverse obstetrical events in women with
previous adverse pregnancy outcomes and inherited or acquired thrombophilias [
molecular weight heparin is often the main treatment for women with thrombophilia and
pregnancy complications to prevent adverse pregnancy outcomes [
2 / 9
In addition to conventional thrombophilias (Factor V Leiden mutation; prothrombin
20210A mutation; PC, PS, and AT III deficiencies; lupus anticoagulant; and anticardiolipin
antibodies), novel thrombophilias have been recently investigated and also appear to
potentially affect pregnancy outcomes [
]. It is a new technique that analyzes genetic variants
affecting different points of the blood coagulation cascade, enabling the diagnosis of 12
hereditary thrombophilias. These variants involve Factor V Leiden (Arg506Gln FV conventional,
Arg306Thr FV Cambridge, Arg306Gly FV Hong Kong), Factor II (G20210A), Factor XII
(46C>T), Group ABO (rs8176719, rs7853989, rs8176743, rs8176750), Serpina A10
(Arg67Stop), Serpina C1 (Cambridge Ala384Ser II), and Factor XIII (Val34Leu).
The aim of this study was to investigate the incidence of inherited thrombophilias in
patients with adverse obstetric outcomes (severe fetal growth restriction, placental abruption,
or severe preeclampsia) and to compare detection rates of thrombophilia between standard
blood tests and the genetic test, with the goal of improving prediction of patients who could
benefit from anticoagulant therapy in subsequent pregnancies.
Materials and methods
This is a case-control prospective study performed in Hospital Sant Joan de Deu? in Barcelona,
Spain. The study protocol was approved by the Institutional Review Board of Sant Joan de De?u
University Hospital. Written informed consent was obtained from each participant.
Cases and controls were recruited over a 12-month period from women above age 18 (22?45
years old) attending the outpatient clinic in the obstetrics department of the Hospital Sant
Joan de De?u and who also gave birth in the same hospital. Gestational ages for all cases and
controls were determined by measuring the crown-rump length during the first trimester [
Women were eligible for inclusion as cases in this study if they met the following criteria:
1. Intrauterine growth restriction: birth weight <10th percentile with an abnormal uterine
arterial Doppler study (pulsatility index >95th percentile [
]) or cerebroplacental ratio
(pulsatility index <5th percentile [
]), which led to the need for delivery before 34 weeks
2. Placental abruption before 34 weeks gestation.
3. Severe preeclampsia [
]: systolic blood pressure (BP) 140 mm Hg and/or diastolic BP
90 mm Hg measured twice in 6 h after 10 min of rest (determined in the sitting position
with the arm at heart level), >300 mg protein in a 24-hour urine collection, and one or
more of the following:
? Systolic BP 160 mm Hg and/or diastolic BP 110 mm Hg measured on two occasions
separated by 6 h, with the patient at rest. Systolic BP >180 and/or diastolic BP >120 mm
Hg on two separate occasions in 30 min.
? Persistent symptoms of eclampsia, including one or more of the following: hyperreflexia
with clonus, severe headache, visual disturbances, stupor, epigastric or right upper
quadrant pain, nausea, or vomiting.
? Oliguria: 500 mL in 24 h or <100 mL in 3 h, or evidence of renal insufficiency (serum
creatinine >1.2 mg/dL or blood urea nitrogen >40 mg/dL).
? Acute pulmonary edema
3 / 9
? Hepatic dysfunction
? Thrombocytopenia (<100,000/mm )
? Laboratory signs of hemolysis
Women were eligible for inclusion as controls if they met the following criteria: at least two
normal, spontaneously-conceived pregnancies; delivery at term without any complications;
and no underlying medical disease. Patients with a history of one or more abortions were
Women were excluded from this study if they exhibited any of the following: illicit drug
use, endocrine pathologies that might interfere with fetal growth, previous diagnosis of
thrombophilia, treatment with aspirin and/or heparin during their pregnancy, and failure to follow
clinical protocols. Other exclusion criteria were multiple-fetus pregnancies, fetal infections,
and fetal malformations or genetic anomalies.
Standard blood tests for thrombophilias were performed in all cases and controls at least 3
months after pregnancy. These included tests for Factor V Leiden mutation; prothrombin
20210A mutation; PC and PS deficiencies; lupus anticoagulant; and anticardiolipin antibodies.
At the same time blood was obtained for the standard blood tests, a sample of saliva was
obtained from all cases and controls for the genetic test. The Thrombo inCode test was
used, as it enables the diagnosis of 12 hereditary thrombophilias (F5 rs6025/rs118203906/
rs118203905, F2 rs1799963, F12 rs1801020, F13 rs5985, SERPINC1rs121909548, and
SERPINA10 rs2232698 plus the A1 blood group (rs8176719, rs7853989, rs8176743, rs8176750) by
analyzing genetic variants affecting different points of the blood coagulation cascade.
A bivariate table was constructed to show the distribution of population characteristics
between cases and controls. Normally distributed variables are described as mean and
standard deviation and were analyzed using the Student?s t-test. Continuous non-normally
distributed variables are presented as median and interquartile range and were analyzed using the
Kruskal-Wallis test. Categorical variables are shown as absolute frequency and percentage
and were analyzed using the chi-squared test (or Fisher?s exact test when the expected cell
frequency was <5). Multivariate logistic regression analysis was performed to explore the
relationships between potential risk factors and adverse obstetric events.
The area under the receiver operating characteristic curve (AUC) was used to evaluate the
predictive performance of multivariate logistic models. Sensitivity, specificity, and positive and
negative likelihood ratios were based on the point of the curve that maximized the Youden
index. All calculations were performed using R statistical software (version 3.1.3) (R
Development Core Team, 2015). P values <0.05 were considered statistically significant.
A total of 33 patients were identified as cases and 41 as controls. Table 1 shows the baseline
characteristics of each group. There were no significant differences between groups. Cases had
a higher body mass index (BMI), but the difference between groups did not reach statistical
significance (p = 0.064).
4 / 9
The incidence of thrombophilias in women with adverse obstetric outcomes (cases group)
was 15.2% versus 18.2% in controls. There were no significant differences between both
No significant differences in the results of any standard blood test for thrombophilia were
observed between cases and controls (Table 2). Lupus anticoagulant and anticardiolipin
antibodies were not compared, as these tests were positive in only 2 and 0 patients, respectively.
Moreover, no statistically significant differences in genetic variables were found between the
cases and controls (Table 3).
We analyzed multivariate prediction models that considered only thrombophilic variables
of usual clinical practice or only genetic variables. None of these models were individually
predictive of pregnancy complications. We then generated one clinical-genetic model combining
variables with the lowest P values (Tables 4 and 5). This model included ABO, BMI, C_rs5985,
C_rs6025, and PS. It exhibited good prediction capacity, with an AUC of almost 0.7 (P <0.05),
sensitivity of almost 67%, and specificity of 70%.
Over the past years, there has been much debate regarding the association between inherited
thrombophilias and the risk of placenta-related adverse obstetric outcomes. Many case-control
studies and systematic reviews [
] have suggested an association. Moreover, an increased
risk of recurrent severe pregnancy complications has been reported in women with inherited
]. However, our results did not show a high incidence of inherited
thrombophilias in patients with adverse obstetric outcomes (severe fetal growth restriction,
placental abruption, or severe preeclampsia). Furthermore, we found that analysis of inherited
thrombophilias using the genetic test did not increase detection rates compared with the
standard blood tests in patients with adverse obstetric outcomes.
ALL PATIENTS(N = 77)
CONTROLS (N = 44)
Similar to our results, other studies failed to confirm an association between inherited
thrombophilias and adverse obstetric outcomes [
], and only weak associations have been
found between hypercoagulability and pregnancy outcomes. The lack of association is likely
attributable to the multifactorial nature of adverse obstetric outcomes, involving the
interaction of epidemiological, clinical, and genetic risk factors. Outcomes do not appear to be solely
caused by genetic factors; these factors are common in the general population and do not lead
to adverse outcomes in the absence of other risk factors.
6 / 9
Because of discrepancies in the literature regarding the importance of thrombophilias in
placenta-related adverse obstetric outcomes, an even larger controversy has recently arisen
regarding the use of screening for inherited thrombophilias in women with a history of adverse
pregnancy outcomes or loss [
]. The ultimate goal is to be able to identify women with
adverse obstetric outcomes who may be candidates for prophylactic heparin or antiplatelet
agents in subsequent pregnancies. As our results suggest, the best predictive model includes
both clinical and genetic variables. When studying the association between genetic variants
and a phenotype (or event), it is often convenient to analyze the association by grouping
genetic variants, since in many diseases, the genetic risk consists of multiple variants in
different genes; analysis of variants individually usually lacks adequate statistical power because
their individual contribution to risk is very low, leading to inconsistent results. This was
demonstrated in the current study, as we found no significant differences between cases and
controls when variants were evaluated individually. But when this genetic information was
grouped together and complemented with clinical information, the test had good predictive
capacity for adverse obstetric outcomes.
We note limitations to our study. The main limitation was the small sample size, which
may have rendered our study underpowered to evaluate differences in detecting inherited
thrombophilias between groups. Another limitation is that sticky platelet syndrome was not
included in the evaluation of thrombophilias, on one hand because it is an expensive and
operator-dependent test and on the other hand because the vast majority of published studies on
hereditary thrombophilias do not consider it.
In conclusion, our study shows that, although some association may exist between
hypercoagulability and pregnancy outcomes, no significant direct correlation was observed between
adverse obstetric outcomes and inherited thrombophilias when analyzed using either standard
blood tests or the genetic test. Future studies with a larger sample size are required to create a
clinical-genetic model that better discriminates women with a history of adverse pregnancy
outcomes and an increased risk of poor outcomes in following pregnancies.
Conceptualization: E. Mazarico, M. D. Go?mez Roig.
Data curation: A. Gonzalez, M. Muniesa, C. Molinet, L. Almeida.
Investigation: M. Ferna?ndez Arias, E. Mazarico, C. Molinet, L. Almeida, M. D. Go?mez Roig.
Methodology: M. Ferna?ndez Arias, E. Mazarico, M. D. Go?mez Roig. Project administration: M. Ferna?ndez Arias, E. Mazarico, M. D. Go?mez Roig.
7 / 9
Supervision: E. Mazarico, M. D. Go?mez Roig.
Validation: E. Mazarico, M. D. Go?mez Roig.
Writing ? original draft: M. Ferna?ndez Arias, E. Mazarico.
Writing ? review & editing: M. Ferna?ndez Arias.
8 / 9
1. Kosar A. , Kasapoglu B. , Kalyouncu S. , Turan H. , Balcik S. , Emime I. , et al. Treatment of adverse perinatal outcome in inherited thrombophilias: a clinical study . Blood Coagulation and Fibrinolysis 2011 , 22 : 14 - 18 . https://doi.org/10.1097/MBC.0b013e32834013f2 PMID: 21042205
2. Bates S. Preventing thrombophilias-related complications of pregnancy: an update . Exert Rev. Hematol . 2013 , 6 ( 3 ), 287 - 300 .
3. Grandone MD. , Tomaiuolo M. , Colaizzo D. , Ames P. , Margaglione M. Role of thrombophilia in adverse obstetric outcomes and their prevention using antithrombotic therapy . Seminars in Thrombosis and hemostasis 2009 , 35 ( 7 ).
4. Redline R . Thrombophilia and placental pathology . Clinical Obstetrics and Gynecology 2006 , 46 ( 4 ), 885 - 894 .
5. Brenner B. , Aharon A . Thrombophilia and adverse pregnancy outcome . Clin Pernatol 2007 , 34 , 527 - 541 .
6. The American College of Obstetricians and Gynecologists , Inherited Thrombophilias in Pregnancy, Practice Bulletin, September 2013 , Vol 22 .
7. Said J. , Higgins J. , Moses E. , Walker S. , Monagle P. , Brennecke S. Inherited thrombophilias and adverse pregnancy outcomes: a case-control study in an Australian population . Acta Obstetricia et Gynecologica Scandinavica , 2012 , 91 , 250 - 255 . https://doi.org/10.1111/j.1600- 0412 . 2011 . 01293 . x PMID : 21985600
8. De Vries J. , Pampus MG. , Hague WM. , Bezemer PD. , Joosten JH . Low-molecular-weight heparin added to aspirin in the prevention of recurrent early-onset pre-clampsia in women with inheritable thrombophilia: the FRUIT-RCT , Journal of Thrombosis and Haemostasis 2011 , 10 : 64 - 72 .
Woodham P. , Bogerss K. , Gardner M. , Doyle N. Routine antenatal thrombophilia screening in high-risk preganancies: a decision analysis . Am J Perinatol 2011 ; 278 : 495 - 500 .
10. Bates SM , Greer IA , Pabinger I , et al. Venous thromboembolism, thrombophilia, antithrombotic therapy, and pregnancy: American College of Chest Physicians -based clinical practice guidelines (8th edition) . Chest 2008 ; 133 ( Suppl 6 ): 844S - 86S .
11. Dizon-Townson DS , Meline L , Nelson LM , et al. Fetal carriers of the factor V Leiden mutation are prone to miscarriage and placental infarction . Am J Obstet Gynecol 1997 ; 177 ( 2 ): 402 - 5 . PMID: 9290458
12. Rey E , Kahn SR , David M , et al. Thrombophilic disorders and fetal loss: a metaanalysis . Lancet 2003 ; 361 ( 9361 ): 901 - 8 . https://doi.org/10.1016/S0140- 6736 ( 03 ) 12771 - 7
13. Rodger MA , Paidas M , Mclintock C , et al. Inherited thrombophilia and pregnancy complications revisited . Obstet Gynecol 2008 ; 112 ( 2 Pt 1 ): 320 - 4 . https://doi.org/10.1097/AOG.0b013e31817e8acc PMID: 18669729
14. Branch DW . The truth about inherited thrombophilias and pregnancy . Obstet Gynecol 2010 ; 115 ( 1 ): 2 - 4 . https://doi.org/10.1097/AOG.0b013e3181c879ca PMID: 20027026
15. Aracic N. , Roje D. , Hofman I. , Capkun V. , Stefanovic V . Low molecular weight heparin treatment and impact of inherited thrombophilia type in pregnancies with previous adverse outcome . J Matern Fetal Neonatal Med , 2015 ; 28 ( 3 ): 306 - 310 . https://doi.org/10.3109/14767058. 2014 .916268 PMID: 24749801
16. Mello G , Paretti E , Fatini C , et al. Low molecular-weight heparin lowers the recurrence rate of preeclampsia and restores the physiological vascular changes in angiotensin-converting enzyme DD women . Hypertension 2005 ; 45 : 86 - 91 . https://doi.org/10.1161/01.HYP. 0000149950 .05182.a3
17. Facchinetti F , Marozio L , Frusca T , et al. Maternal thrombophilia and the risk of recurrence of preeclampsia . Am J Obstet Gynecol 2009 ; 200 : 46 . e1 - e5 .
18. Grandone E , De Stefano V , Rossi E , et al. Antithrombotic prophylaxis during pregnancy in women with deficiency of natural anticoagulants . Blood Coagul Fibrinolysis 2008 ; 19 : 226 - 30 . https://doi.org/10. 1097/MBC.0b013e3282f54545 PMID: 18388503
19. Kupferminc MJ , Rimon E , Many A , et al. Low molecular weight heparin treatment during subsequent pregnancies of women with inherited thrombophilia and previous severe pregnancy complications . J Matern Fetal Neonatal Med 2011 ; 24 : 1042 - 5 . https://doi.org/10.3109/14767058. 2010 .545911 PMID: 21231837
20. Robinson HP , Fleming JE . A critical evaluation of sonar ?crown-rump length? measurements . Br J Obstet Gynaecol . 1975 ; 82 : 702 - 710 .
21. Arduini D , Rizzo G . Normal values of pulsatility index from fetal vessels: a cross-sectional study on 1556 healthy fetuses . J Perinat Med . 1990 ; 18 : 165 - 172 . PMID: 2200862
22. Baschat AA , Gembruch U. The cerebroplacental Doppler ratio revisited . Ultrasound Obstet Gynecol . 2003 ; 21 : 124 - 127 . https://doi.org/10.1002/uog.20 PMID: 12601831
23. Brown MA , Lindheimer MD , de Swiet M , Van Assche A , Moutquin JM . The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP) . Hypertens Pregnancy . 2001 ; 20: IX-XIV . https://doi.org/10.1081/ PRG-100104165 PMID: 12044323
24. Alfirevic Z , Roberts D , Martlew V . How strong is the association between maternal thrombophilia and adverse pregnancy outcome? A systematic review . Eur J Obstet Gynecol Reprod Biol 2002 ; 101 ( 1 ): 6 - 14 . PMID: 11803092
25. Lykke JA , Bare LA , Olsen J , et al. Thrombophilias and adverse pregnancy outcomes: results from the Danish National Birth Cohort . J Thromb Haemost 2012 ; 10 : 1320 -5 https://doi.org/10.1111/j.1538- 7836 . 2012 . 04773 . x PMID : 22578003
26. Robertson L , Wu O , Langhorne P , et al. Thrombophilia in pregnancy: a systematic review . Br J Haematol 2006 ; 132 : 171 - 96 https://doi.org/10.1111/j.1365- 2141 . 2005 . 05847 . x PMID : 16398652
27. Kist WJ , Janssen NG , Kalk JJ , Hague WM , Dekker GA , de Vries JI. Thrombophilias and adverse pregnancy outcome-a confounded problem! Thromb . Haemost. 99 ( 1 ), 77 - 85 ( 2008 ). https://doi.org/10. 1160/TH07-05-0373
28. Facchinetti F , Marozio L , Frusca T , et al. Maternal thrombophilia and the risk of recurrence of preeclampsia . Am J Obstet Gynecol 2009 ; 200 : 46 .e1- 5 .
29. Kalk JJ , Huisjes AJ , de Groot CJ , et al. Recurrence rate of preeclampsia in women with thrombophilia: influenced by low molecular weight heparin treatment? Neth J Med 2004 ; 62 : 83 - 7
30. Dizon-Townson D , Miller C , Sibai B , et al. for the National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. The relationship of the factor V Leiden mutation and pregnancy outcomes for mother and fetus . Obstet Gynecol 2005 ; 106 : 517 - 24 https://doi.org/10.1097/01. AOG. 0000173986 .32528.ca
31. Kahn SR , Platt R , McNamara H , et al. Inherited thrombophilia and preeclampsia within a multicenter cohort: the Montreal Preeclampsia Study . Am J Obstet Gynecol 2009 ; 200 : 151 . e1- 9
32. Silver RM , Zhao Y , Spong CY , et al. for the Eunice Kennedy Shriver National Institute of Child Health and Human Development Units (MFMU) Network. Prothrombin gene G20210A mutation and obstetric complications . Obstet Gynecol 2010 ; 115 : 14 - 20 https://doi.org/10.1097/AOG.0b013e3181c88918 PMID: 20027028
33. Davenport WB , Kutteh W. Inherited thrombophilias and adverse pregnancy outcomes . Obstet Gynecol Cin N Am 41 ( 2014 ) 133 - 144 .