Low molecular weight heparin (Fragmin) in pregnant women with a
history of uteroplacental insufficiency and thrombophilia, a randomized
trial
(FRUIT - study)
Investigator: J.J. Kalk, MD
Supervisor: J.I.P. de Vries, MD, PhD
H.P. van Geijn, MD,PhD
Division of Maternal-Fetal Medicine
Department of Obstetrics & Gynecology
P.C. Huijgens, MD,PhD
Department of Hematology
P.D. Bezemer, PhD.
Department of Epidemiology & Biostatistics
University Hospital Vrije Universiteit Amsterdam
Other participants:
A. Franx, MD, PhD
H.W. Bruinse, MD, PhD.
Department of Obstetrics & Gynecology
Academic Hospital Utrecht
J. van Eyck, MD, PhD
H.H. de Haan, MD, PhD
Department of Obstetrics & Gynecology
Isala Hospital Zwolle, locatie Sophia
E. van Beek, MD, PhD.
Department of Obstetrics & Gynecology
Academic Hospital Nijmegen, St Radboud
J.M. Middeldorp, MD
Department of Obstetrics &Gynecology
Leiden University Medical Center
H. Wolf, MD, PhD.
J.A.M. van der Post, MD, PhD
Department of Obstetrics & Gynecology
Academic Medical Center
W. Visser, MD, PhD.
C.J. de Groot, MD, PhD
Department of Obstetrics & Gynecology
Academic Hospital Rotterdam, Dijkzigt
S.G. Oei, MD,PhD
Department of Obstetrics & Gynecology
St Joseph Hospital, Veldhoven
L.L.H. Peeters, MD, PhD.
Department of Obstetrics &Gynecology
Academic Hospital Maastricht
G.A. Dekker, MD, PhD, FRACOG
Bill Hague, MD FRACOG, FRACP
Department of Obstetrics & Gynecology
North Western Adelaide Health Service,
The Lyell McEwin Hospital &
The Queen Elizabeth Hospital
University of Adelaide, South Australia
E.C.M. van Pampus, MD,PhD
Department of Hematology
Academic Hospital Maastricht
J.T.J. Brons, MD, PhD
Department of Obstetrics &Gynecology
Medical Spectrum Twente, Enschede
Protocol 20-01-2000
P. Hummel, MD, PhD
Department of Obstetrics &Gynecology
Medical Center Alkmaar
M.G. van Pampus, MD, PhD
M.P. Heringa, MD, PhD
Department of Obstetrics &Gynecology
Academic Hospital Groningen
Summary
The aim of the study is:
Primary: Does preventive treatment with low-molecular-weight heparin reduce the occurrence
of preeclampsia in women with thrombophilia and a history of preeclampsia before 34 weeks of
pregnancy.
Secondary: A reduction of recurrence of small for gestational age infants in women with a
history of small for gestational age infants before 34 weeks and thrombophilia.
Trial:
Multi-center open two-armed randomized study.
Intervention of the trial:
The verum group will receive low-molecular weight heparin (Fragmin, subcutaneously 1 x
5000 IU AXA per day) + low dose acetylsalicylacid (ASA 80mg). Fragmin is started as early as
possible between six and twelve weeks gestation, after ultrasound confirmation of a viable
intrauterine pregnancy. Fragmin will be continued until six weeks post partum. ASA is given
from 12 weeks gestation till 36 weeks gestation. The control group will receive ASA and
standard care that is normally given to these high-risk pregnancies.
Population
The recurrence rate of preeclampsia is 35% and small for gestational age infants 21% as
recently studied in a Dutch population with a prior pregnancy with severe early onset
preeclampsia or small for gestational age infants (before 34 weeks gestation) and coagulation
abnormalities, being treated with or without low-molecular-weight heparin and aspirin.
To detect a 50% reduction from 35% recurrence rate of preeclampsia 262 patients will be
randomized, aiming for 131 + 131 patients in the 2 arms of the study (2-tailed, an alpha of 0.05
and a power of 80%). Recurrence rate of uteroplacental insufficiency in women with
antiphospholipid antibodies is 60%. To detect a 50% reduction from 60% recurrence rate of
uteroplacental insufficiency 84 patients will be randomized, aiming for 42 + 42 patients in the 2
arms of the study (2-tailed, an alpha of 0.05 and a power of 80%).
1
Analysis
Differences between the groups treated with low-molecular weight heparin and aspirin versus
aspirin only will be assessed, accounting for prognostic factors, with the Mantel-Haenszel
method or with logistic regression. Quantitative analysis will be assessed with linear regression.
2
Introduction
The aim of this trial is to evaluate the effects of low-molecular-weight heparin in pregnant
women with thrombophilia and a history of uteroplacental insufficiency before 34 weeks.
Occurrence
Nowadays, uteroplacental insufficiency resulting in preeclampsia, hemolysis, elevated liver
enzymes and low platelets (HELLP syndrome), eclampsia, intra-uterine growth restriction and
preterm birth is one of the major problems in perinatal medicine. Hypertensive disorders of
pregnancy occur in 15-20% of pregnancies and preeclampsia in approximately 3-5%. The latter
is a major cause of maternal mortality in the United States and other developed countries.1 In
the Netherlands, it is estimated that about 2000 (1%) of all pregnant women per year (200.000
births) develop severe complications due to uteroplacental insufficiency.
Clinical features
Maternal and fetal morbidity and mortality are a major problem especially in early
preeclampsia before 34 weeks. Uteroplacental insufficiency is associated with endothelial cell
dysfunction resulting in widespread vasoconstriction, plasmavolume reduction, hypertension
and localized intravascular coagulation. Eventually the end result is hypoperfusion of maternal
organs and further impairment of placental bloodflow. 2
Maternal risks vary from headache, impaired vision, edema, nausea, upper abdominal pain to
eclampsia, intracranial hemorrhage, liver rupture, disseminated intravascular coagulation, adult
respiratory distress syndrome and multiple organ failure. 3 These complications still account for
the major proportion of the maternal mortality rate in Western countries.4
Perinatal morbidity and mortality are related to both the intra-uterine growth restriction as well
as to (often iathrogenic) prematurity. Intra-uterine growth restriction is a major health issue
since impaired fetal growth is not only associated with short term morbidity, but also with an
increased incidence of hypertension, coronary artery disease and diabetes in adult life, the socalled
insulin
resistance
syndrome
(syndrome-X)
initially described
by Barker.5;6
Uteroplacental insufficiency with or without abruptio placentae may also cause intrauterine
fetal death.
3
Etiology
The exact cause of uteroplacental insufficiency is unknown, a multifactorial origin is suggested.
Immune maladaption (fetal-maternal) as well as genetic factors are probably involved in its
etiology7, and there is more or less consensus that endothelial cell dysfunction/activation
represents the final common pathway in the maternal syndrome preeclampsia. It is known that
in women with diseases inducing vessel wall damage (diabetes mellitus, chronic hypertension,
systemic lupus erythematosis) the incidence of uteroplacental insufficiency is increased. More
recently several investigators confirmed the presence of thrombophilic disorders in a substantial
percentage of women with a history of severe early onset preeclampsia, severe intra-uterine
growth restriction and/or abruptio placentae in the absence of a history of arterial or venous
thrombosis. 8-10
The thrombophilic factors examined are coagulation abnormalities, hyperhomocysteinemia and
antiphospholipid antibodies. For this study we focus on coagulation abnormalities. To elucidate
inclusion and exclusion criteria the current knowledge on hyperhomocysteinemia and
antiphospholipid antibodies in relation to pregnancy is described as well.
Coagulation abnormalities
Deficiencies and/or some mutations in the pro- and anticoagulant route are known to be
associated with a marked increase in thrombotic disorders. From the procoagulant route the
Factor (f) XII deficiency, f II mutation and from the anticoagulant pathway protein S
deficiency, protein C deficiency, activated protein C (aPC) resistance (with or without the
factor V Leiden mutation) are most frequently involved. Antithrombin III deficiency is rare,
which probably reflects the fact that antithrombin is the main physiological coagulation
inhibitor. Percentages of coagulation abnormalities in general population are described in tabel
1. Percentages of coagulation abnormalities in women with normal progeniture are described in
tabel 2. Table 3 and 4 summarize the coagulation abnormalities in women with a history of
preeclampsia and small for gestational age, respectively. The data are derived from the
following studies.
Kupferminc et al10 studied 110 women who had one of the obstetrical complications such as
severe preeclampsia, abruptio placentae, fetal growth retardation, and stillbirth associated with
intervillous or spiral artery thrombosis and inadequate placental perfusion. These women were
compared with 110 women with normal pregnancies. Women with serious obstetrical
complications have an increased incidence of mutations predisposing them to thrombosis and
other inherited and acquired forms of thrombophilia (tabel 2-4).
4
Dekker et al
8
showed that in a group of 101 patients with severe early-onset preeclampsia
38.6% had chronic hypertension. Of the 85 patients tested for protein S deficiency 24.7% were
positive, of the 50 patients tested for activated protein C resistance, 16% were positive. 95
patients were tested for anticardiolipin antibodies; 29.4% had detectable immunoglobulin G
and/or M anticardiolipin antibodies. Riyazi11 enlarged this group and found the same
percentages (tabel 3 and 4). Van Pampus et al12 also found a high incidence in coagulation
abnormalities and hyperhomocysteinemia in women with severe preeclampsia and HELLPsyndrome.
De Vries et al
9
demonstrated in a group of 62 women with a history of placental abruption,
intrauterine fetal death or small for gestational age infants without hypertensive disorders that
there was a high incidence of coagulation abnormalities, anti-phospholipid antibodies and
hyperhomocysteinemia (tabel 4).
>From the above mentioned coagulation abnormalities all will be included in this study except
for antithrombin deficiency. In contrast to the other anomalies the latter is known to induce
recurrent thrombosis under low-risk thrombotic circumstances. All women with antithrombin
deficiency and pregnancy will be treated with anticoagulants.
Hyperhomocysteinemia has also been identified as an additional risk factor to develop
preeclampsia, small for gestational age infants/or abruptio placentae. In most patients the
metabolic defect can be treated with vitamin suplementation (folic acid with or without
pyridoxine) 13. The reason to describe this metabolic disease lays in the fact that women with
hyperhomocysteinemia in combination with one of the earlier described coagulation
abnormalities are included in the study, whereas women with only hyperhomocysteinemia are
excluded.
A randomized study of Rai et al
14
demonstrated the positive effects of unfractionated heparin
(Calciparin) and low-dose aspirin on pregnancy outcome in women with recurrent miscarriages
associated with antiphospholipid antibodies, however with unknown coagulation abnormalities
or hyperhomocysteinemia.
5
Tabel 1. Thrombophilia in men and women in a general population
Incidence
Antithrombin deficiency
0.1% 15
Protein C deficiency
0.1-0.3% 16
Activated
C 3.0-7.0% 17
protein
resistance
Protein S deficiency
0.2-2.0% 18
Factor II mutation
0.0-4.0% 19
Hyperhomocysteinemia
2.0-5.0% 20-22
Anticardiolipin antibodies
1.0-3.0% 23;24
Tabel 2. Two studies of thrombophilia in women with uncomplicated pregnancies.
Kupferminc (n=110)10
V Pampus (n=65) 12
AT def
1/110 = 0.9%
-
aPCR
-
0/65 = 0.0%
fVL mut
7/110 = 6.4%
1/65= 1.5%
PS-def
1/110 = 0.9%
6/65 = 9.2%
fII-mut
3/110 = 2.7%
-
HHC
9/110 = 8.2%
3/65 = 4.6%
ACA  10 GPL/MPL
5/67 = 7.5%
ACA  20 GPL/MPL
1/67 = 1.5%
ACA
0/110 = 0%
-
AT def = antithrombin deficiency; aPCR = activated protein C resistance; fVL = factor V
Leiden mutation; PS-def = protein S deficiency; fII-mut = factor II mutation; HHC =
hyperhomocysteinemia; ACA= anticardiolipin antibodies
6
Tabel 3. Four studies in women with a history of preeclampsia
Kupferminc
(n=34)
10
Van Pampus
(n=251)
12
Riyazi
(n=242)
Leeda
11
(n=204)13
ATIII-def
-
-
4/216 = 1.9%
-
PC-def.
-
-
1/215 = 0.5%
-
APCR
-
<28 weeks 16%
18/179 = 10.1%
-
-
-
52/217 =23.5%
-
>28 weeks 10%
FVL-mut.
9/34 = 26.5%
<28 weeks 8%
>28 weeks 6%
PS-def.
-
<28 weeks 19%
>28 weeks 10%
FII-mut
2/34 = 5.9%
-
-
-
HHC
7/34 = 20.6%
<28 weeks 19%
-
32/181 = 17.7%
>28 weeks 11%
ACA 10 GPL/MPL
<28weeks 27.4%
>28weeks 19.3%
ACA 20 GPL/MPL
<28weeks 1.6%
>28weeks 3.5%
ACA
-
-
52/216 = 24.1%
AT def = antithrombin deficiency; PC-def = protein C deficiency; aPCR = activated protein C
resistance; fVL = factor V Leiden mutation; PS-def = protein S deficiency; fII-mut = factor II
mutation; HHC = hyperhomocysteinemia; ACA= anticardiolipin antibodies
7
Tabel 4. Four studies in women with a history of small for gestational age infants
Kupferminc10
De Vries 9
Leeda 13
Riyazi 11
(n=44)
(n=13)
(n=26)
(n=34)
ATIII-def
-
0/13 = 0%
-
0/29 = 0.0%
PC-def
-
1/13 = 7.7%
-
2/29 = 6.9%
APCR
-
2/8 = 25.0%
-
2/24 = 8.3%
fVL-mut
5/44 = 11.4%
1/8 = 12.5%
-
-
PS-def
-
3/13 = 23.1%
-
7/29 = 24.1%
fII-mut
5/44 = 11.4%
-
-
-
HHC
12/44 = 27.3%
5/13 = 38.5%
5/26 = 19.2%
-
ACA
-
1/13 = 7.7%
-
7/31 = 22.6%
AT def = antithrombin deficiency; PC-def = protein C deficiency; aPCR = activated protein C
resistance; fVL = factor V Leiden mutation; PS-def = protein S deficiency; fII-mut = factor II
mutation; HHC = hyperhomocysteinemia; ACA= anticardiolipin antibodies
8
Recurrence rate
The recurrence rate is derived from a Dutch retrospective study. Obstetricians from 9 hospitals
in The Netherlands who decided to participate in the prospective trial evaluated the outcome of
the pregnancy after the index pregnancy with preeclampsia or small for gestational age infants
before 34 weeks (Kalk et al). 25 In total 1146 women with such an obstetric history were tested
and 410 (35%) were found to have coagulation abnormalities and/or anticardiolipin antibodies
and/or hyperhomocysteinemia. Only 51 women with coagulation abnormalities, but no
anticardiolipin antibodies had a consecutive pregnancy and in 35% preeclampsia and in 21%
small for gestational age infants recurred. The treatment in the following pregnancy was with
either low-molecular-weight heparin (Fraxiparin 2x 2850 units per day) plus aspirin (80 mg)
(n=22) or aspirin only (n=19) or nothing (n=10). It is not known if the medical treatment in the
different clinics was started at the same gestational age. Within the limits of this small
retrospective studied population no significant difference was found in the recurrence rate of
preeclampsia or small for gestational age infants between the group who received lowmolecular weight heparin and the group that did not.
This multi-center study is supported by the smaller study of Riyazi et al. 11 They tested a total of
276 patients with a history of preeclampsia and/or fetal growth restriction for the presence of
coagulation abnormalities and anticardiolipin antibodies. Ninety patients with preeclampsia and
15 patients with isolated fetal growth restriction had haemostatic abnormalities. In 26 patients a
subsequent pregnancy occurred and they were treated with low-molecular-weight heparin and
low-dose aspirin. Preeclampsia recurred in 38%, and intra-uterine-growth restriction in 15 % of
pregnancies.
From the analyzed women 95 had anticardiolipin antibodies and 20 women had consecutive
pregnancies. Fifteen women had positive anticardiolipin antibodies only, one woman had
activated protein C resistance in combination with anticardiolipin antibodies and 4 women had
protein S deficiency and anticardiolipin antibodies. In the subsequent pregnancy 15 women
received LMWH and aspirin and 5 women received aspirin or no medication. In women with
positive anticardiolipin antibodies and coagulation abnormalities there was a significant
difference in birthweight in the subsequent pregnancy in favour of women treated with LMWH
and aspirin (p= 0.042). There was no significant difference in gestational age (p=0.466). The
recurrence rate of uteroplacental insufficiency was 6% (1 of 15) in women receiving LMWH
and aspirin and 60% (3 of 5) in women receiving aspirin or no medication. This is a statistically
significant difference (95% CI -0.0857 to -0.981). From the 15 women who had positive
9
anticardiolipin antibodies only, 11 women received LMWH and aspirin in the subsequent
pregnancy and 4 women received aspirin or no medication. There was no significant difference
in gestational age (p=0.551) or in birthweight (p=0.095) in the subsequent pregnancy. For
details see table VI. Uteroplacental insufficiency recurred in 1 (9%) woman receiving LMWH
and aspirin and in 2 (50%) women receiving aspirin or no medication. This difference is not
statistically significant (95% CI –.110 to .928).
Two studies reported the recurrence rate of severe preeclampsia or HELLP syndrome in a
population with unknown thrombophilic status. Sibai et al26 described a group of 125 women
with a history of severe preeclampsia in the second trimester (range 18-27 weeks gestation), of
which 108 women had 169 subsequent pregnancies (range, 1-4 per woman): 59 (35%) had a
normotensive pregnancy and 110 (65%) developed preeclampsia. Sullivan et al27 studied 481
patients with a history of HELLP-syndrome. Subsequent gestations (n=195) occurred in 122 of
481 patients. Available data in 161 pregnancies showed a recurrence rate of 43%.
Treatment
For years, numerous clinical reports and randomized trials described the use of various methods
to prevent or reduce the incidence and severity of preeclampsia.
In the 80’s it was thought that an imbalance between prostacyclin and thromboxane was the
major pathogenetic pathway in the causation of preeclampsia and/or fetal growth restriction.
These findings and the evidence for early involvement of the platelets have led to the use of
low-dose aspirin for prevention of preeclampsia and/or fetal growth restriction. 28 In the CLASP
study29 9364 women were randomly assigned 60 mg aspirin daily or matching placebo. 74%
were entered for prophylaxis of preeclampsia, 12 % for prophylaxis of intrauterine growth
retardation, 12% for treatment of preeclampsia and 3% for treatment of intrauterine growth
retardation.
The use of aspirin was associated with a reduction of only 12% in the incidence of
preeclampsia, which was not significant. According to the CLASP investigators low-dose
aspirin is only indicated in pregnant women with a high risk for recurrent early-onset
preeclampsia. Nor was there any significant effect on the incidence of intrauterine growth
retardation. Recently, the results of Caritis et al
30
demonstrated that low-dose aspirin has no
beneficial effect on perinatal outcome in high-risk women. It may be that the lack of aspirin’s
efficacy can be partially explained by the high incidence of underlying thrombophilic disorders
in these women.
10
The management of patients with uteroplacental insufficiency in the obstetric history and
documented thrombophilia is still undefined.31;32 For patients with a history of severe fetal
growth restriction and/or severe early onset preeclampsia with documented thrombophilia lowmolecular-weight heparin may be the way forward. However, the results of the two
retrospective studies that have focussed on this special population
11;25
indicate the need to start
an adequately sized randomized study to prove benefit of treatment with low-molecular-weight
heparin. Women with inherited coagulation abnormalities and women with antiphospholipid
antibodies need to be analyzed separately because of the different cause and the difference in
recurrence rate of uteroplacental insufficiency. The multicenter approach is especially needed
since the retrospective study and also a retrospective study of van Pampus et al showed the low
number of pregnancies in women after severe early-onset preeclampsia which is considered as a
negative life event with a high anxiety for recurrence.33
11
Aim of the study
Does preventive treatment with low-molecular-weight heparin plus aspirin reduce the
recurrence of preeclampsia and/or small for gestational age infants before 34 weeks gestational
age in women with documented thrombophilia with a history of preeclampsia and/or small for
gestational age infants with birth before 34 weeks.
Primary endpoint is a reduction of the recurrence of preeclampsia.
The secondary endpoint is a reduction of the recurrence of small for gestational age infants.
Other endpoints are reduction of spontaneous abortion, reduction of preterm birth, maternal
admission to the hospital and NICU admission.
12
Definitions
Pregnancy-induced hypertension is defined as a diastolic blood pressure of 90 mmHg or more
and an increment of 20 mmHg or more as compared to first trimester diastolic blood pressure.
The absolute blood pressure levels or threshold increments in pressure have to be observed on
at least two occasions six or more hours apart.34;35
Preeclampsia is defined as a pregnancy-induced hypertension plus significant proteinuria (300
mg/24hr or more) after 20 weeks of gestational age.34;35.
Chronic hypertension is defined as a diastolic blood pressure of 90 mmHg or more in the first
trimester and six or more weeks post partum.34;35
HELLP syndrome is defined as the presence of (1) hemolysis, defined by increased LDH (>600
IU/L) and (2) elevated liver enzymes, defined as increased SGOT (>70 IU/L) and (3)
thrombocytopenia (<100.10 9/L).30
Severe early-onset preeclampsia is pragmatically defined as preeclampsia and/or HELLP
syndrome and/or eclampsia necessitating delivery of the baby before 34 weeks gestational
age.34;35
Small for gestational age is defined as a neonatal birth weight below the 10th percentile.36
Customised fetal growth and/or Customised birth weight: customised centiles, which adjust
individually for physiological variables like maternal booking weight, height, parity and ethnic
origin and fetal gender.37
13
Coagulationtests
Normal values of each center are tested, applying the International Thrombophilia External
Quality Assessment within the ECAT Foundation. The measurement procedures are
standardized for each laboratory.
Laboratory tests and normal values specific for Free University Hospital Amsterdam:
Antithrombin III deficiency is a AT III activity < 75%, tested 2x (Chromogenic substrate,
Chromogenix).9
Protein C deficiency is protein C activity < 70%, tested 2x (Protein C Coamate, Protein C
Chromogenix).9
Activated protein C resistance is activated protein sensitivity ratio < 2.0 measured once in case
of positive factor V Leiden mutation, measured twice in case of negative factor V Leiden
mutation. 9
Protein S deficiency is total protein S < 60%, 2x, Antigen < 64%, free protein S < 60%
0(enzyme-linked immunosorbent assay with the use of rabbit antihuman anti-protein S from
Dakopatts (DAKO).9
factor V Leiden mutation: an adenine-to-guanine-mutation at nucleotide 506 in the factor V
gene (polymerise chain reaction).11
factor II mutation: A guanine-to-adenine- mutation at nucleotide 20210 in the prothrombin gene
(polymerise chain reaction).10
These tests have to performed at least ten weeks post partum and without using oral
contraceptives.
Antiphospholipid antibodies: positive tests for anticardiolipin antibodies and/or lupus
anticoagulant (LAC)
Anticardiolipin antibodies: -:  10 GPL and/or MPL, +/-: 10 to 15 GPL and/or MPL, +: 15 to
20 GPL and/or MPL, ++: > 20 GPL and or MPL. One MPL or GPL is equivalent to 1 µg of
affinity-purified ACA, either IgM or IgG assays is positive.38
14
Lupus anticoagulant: The presence of lupus anticoagulant is examined if
the partial
thromboplastin time is prolonged and a mixing activated partial-thromboplastin time test using
1:1 patient and pooled normal plasma is abnormal. Lupus anticoagulant is considered to be
present when one of the two assays is positive. For confirmation, the thromboplastin dilution
test using Thromborel S reagent and the platelet neutralisation procedure are used. 9
Hyperhomocysteinemia: Measured by a methionine loading test: a blood sample for
determination of the fasting homocysteine concentration is drawn at 8 AM after an overnight
fast. Subsequently, an oral dose of L-methionine (0.1g/kg body weight) is administered in
orange-juice. The patients use a standardized low-methionine breakfast and lunch. Fasting
normal values: 6-15 µmol/L and postloading normal values 18-51µmol/L (2.5-97.5
percentile).13
During at least six months prior to the test no supplemention with folic acid, vitamin B6/B12
should be used. The test is performed 14-18 days after the last menstrual period.
15
Study design
Trial:
A randomized open two-armed study.
A double blind study is not an option because at the injection sites it is easy to see the small
hematomas due to low-molecular-weight heparin. Furthermore it is ethical not justifiable to ask
the control group to inject physiological salt subcutaneously on a daily basis throughout
pregnancy.
Setting:
Multicenter study:
University Hospital Vrije Universiteit Amsterdam
Academic Medical Center Amsterdam
Dijkzigt Hospital Rotterdam
Academic Hospital Maastricht
Isala Hospital Zwolle, locatie Sophia
Leiden University Medical Center
Academic Hospital Utrecht
Radbout Hospital Nijmegen
Sint Joseph Hospital Veldhoven
Medical Center Alkmaar
Medical Spectrum Twente, Enschede
Academic Hospital Groningen
North Western Adelaide Health Service, University of Adelaide, Australia
Patients:
Patients with a subsequent pregnancy after an index pregnancy complicated by preeclampsia or
small for gestational age infants and delivery before 34 weeksand documented thrombophilia
will be randomized. Women who already received low-molecular-weight heparin in a previous
pregnancy are excluded from the trial.
At the booking visit, patients will be asked if they want to participate in the trial. Patients will
be fully informed about the aim of the study, the methods used and the potential positive and
negative effects. All patients will receive written information about the trial and will get one to
two days time to consider participation to the study. Eligible patients will be included in the
study after obtaining written informed consent.
16
Inclusion criteria trial:
1. Patients with a history of preeclampsia and/or small for gestational age infants before 34
weeks gestation and documented thrombophilia restricted to
a. Protein C deficiency
b. Activated protein C resistance
c. Activated protein C resistance and f V Leiden mutation
d. Protein S deficiency
e. Factor II mutation
f.
Antiphospholipid
antibodies
(Anticardiolipin
antibodies
with
or
without
lupus
anticoagulant)
2. Age > 18 years
3. Informed consent
Exclusion criteria trial:
1. Antithrombin deficiency
2. Diabetes mellitus
3. Known malignancy
4. Gastro-duodenic ulcer
5. Severe renal or hepatic insufficiency
6. Thrombo-embolism in history
7. Hemorrhagic diathesis
8. Idiopathic thrombocytopenia
Intervention of the trial:
Patients will be included in the study before 12 weeks of gestational age. Patients with
preexisiting hypertension have a higher risk on uteroplacental insufficiency. Therefore patients
will be stratified for presence or absence of chronic hypertension. Patients are also stratified for
center. Patients will be stratified for presence or absence of antiphospholipid antibodies. After
After stratification patients will be randomized. Stratification and randomization will be
performed by the pharmacist of the University Hospital Vrije Universiteit. A supervising
committee, which consist of a gynecologist, hematologist and epidemiologist will control the
medical and ethical standards according to the declaration of Helsinki. The continuation of the
study is reported to the committee.
17
The patients will be divided in to two groups:
-Group A will receive dalteparin (Fragmin) subcutaneously 1 x 5000 IU AXA per day after
ultrasound confirmation of a viable intrauterine pregnancy. Fragmin will be continued until six
weeks post partum. Acetyl salicyl acid (ASA 80 mg) will be started at 6-12 weeks gestation and
given till 36 weeks.
-Group B will receive ASA and no low-molecular-weight heparin.
All patients will be treated according to the standard hospital protocol. Minimal care contains
ultrasound examination, blood (hemoglobin, platelets, liver enzymes, creatinine) and urine
(proteinuria) examination at booking, at 22-24 weeks (plus additional fetal growth and Doppler
flow of the umbilical and uterine arteries) and 28-30 weeks (fetal growth and umbilical artery
Doppler flow). After the delivery all patients whether or not they received low-molecular
weight heparin during pregnancy, will be treated with low-molecular weight heparin during six
weeks post partum.
Diastolic blood pressure will be measured by using Korotkoff V.39;40. If a patient has chronic
hypertension at time of randomization, oral hypertensive drugs can be started, except for
ketanserin because of its intrinsic effect on platelets.41
There is no need to adjust the dose of low-molecular-weight heparin during pregnancy. Women
with a bodyweight of less than 50 kg are likely to have satisfactory heparin concentrations on
low doses (2500 IU dalteparin), whereas obese women (>80 kg) require 7500 IU.42
If patients who receive low-molecular-weight heparin have an allergic reaction, platelet count
will be performed to look for heparin-induced thrombocytopenia. If the platelet count falls
below 50% of the baseline value the patient is tested for heparin-induced thrombocytopenia by
a standard ELISA-test.43;44 In patients who receive low-molecular-weight heparin anti-Fxa
measurements will be performed at 20 and 30 week’s gestation.
Positive and negative effects for patients:
In the University Hospital Vrije Universiteit Amsterdam and several other hospitals treatment
during pregnancy with low-molecular-weight heparins has been used since 1993. Patients who
receive ASA and low-molecular-weight heparin can be informed of the knowledge that lowmolecular weight heparins have unfrequent side-effects. This is supported by systematic
investigations in pregnant women treated with low-molecular-weight heparin during pregnancy
and post partum
11;45
and supported by the experience of two other Dutch hospitals. Women
being adequately instructed in the procedure of subcutaneous injections reported high
18
acceptance and a minority reported minor complaints such as hematomas, pain, swelling and
itching.
The patients will be fully informed about all these potential negative side effects. Concerning
the potential positive effects the patients will be informed that although the theoretical basis for
low-molecular-weight heparin appears to be sound there is no current evidence that the use of
low-molecular-weight heparin in addition to low-dose ASA is associated with improved
maternal and/or perinatal outcome.
Duration of the study:
The financial support for patient intake is guaranteed for a period of two years.
Endpoints
The primary endpoint of the study is a reduction of preeclampsia before 34 weeks gestational
age.
The secondary endpoint is a reduction of small for gestational age infants.
Other endpoints of the study are: percentage of abortion, difference in gestational age, birth
weight, days of admission to the hospital, NICU admission.
19
Size
Inherited coagulation abnormalities: the recurrence rate of preeclampsia in a population with a
prior pregnancy with severe early onset preeclampsia (before 34 weeks gestation) being treated
with or without low-molecular-weight heparin and aspirin in women with coagulation
abnormalities is 35%. To detect a 50% reduction from 35% recurrence rate of preeclampsia 262
patients will be randomized, aiming for 131 + 131 patients in the 2 arms of the study (2-tailed,
an alpha of 0.05 and a power of 80%).
Antiphospholipid antibodies: The recurrence rate of uteroplacental insufficiency in a population
with a prior pregnancy with severe early onset preeclampsia and or small for gestational age
infants (before 34 weeks gestation) and anticardiolipin antibodies with an or without inherited
coagulation abnormalities, not being treated with low-molecular- weight heparin and aspirin is
60%.
To detect a 50% reduction from 60% recurrence rate of preeclampsia 84 patients will be
randomized, aiming for 42+ 42 patients in the 2 arms of the study (2-tailed, an alpha of 0.05
and a power of 80%).
In the Netherlands it is estimated that about 1% (2.000) of all pregnancies per year (200.000)
are complicated with severe utero-placental insufficiency. In this group coagulation
abnormalities exist in approximately 15-20%. That means 300-400 women per year. The
combined effort of these Dutch and South Australian hospitals should result in an adequate
recruitment.
Drop-outs
The expected number of drop-outs due to the use of low-molecular-weight heparin is low.
The use of long lasting heparins is associated with osteoporosis. However, bone density
measurements in women treated with low-molecular-weight heparin showed similar bone
density loss compared with untreated pregnant women.46 The rare occurring side-effect on
reduction of platelets is checked carefully and if it occurs it is a reason to stop low-molecularweight heparin and the platelets increase spontaneously.
In a review article of Sanson et al47, 486 pregnant women received low-molecular-weight
heparin; there were 3 (0.6%) thromboembolic complications, in 3 (0.6%) women there were
allergic reactions. Heparin-induced thrombocytopenia was not observed. Clinically important
hemorrhagic complications did not occur.
Furthermore, any patient is free to end participating in the investigation at any time, for
whatever reason. Causes for drop-outs will be documented in a case record form and presented
in the results of the study.
20
Statistical analysis:
Analysis will take place based on the intention-to-treat principle.
Differences between the two groups will be assessed, accounting for prognostic factors, with
the Mantel-Haenszel method or with logistic regression. Quantitative analysis will be assessed
with linear regression.
Consequence:
If there is a significant reduction in the incidence of preeclampsia or small for gestational age
infants after treatment with low-molecular weight heparins, we can introduce this new
treatment modality in daily practice.
Publication:
The research is of evident international importance and will be published in international
journals.
21
Protocol overview
6-12 weeks gestation:
Informed consent, inclusion, entry form, randomisation
examination: blood pressure, maternal weight and height
lab: hemoglobin, platelets, creatinine, uric acid, liver enzymes
24 hr urine protein
ultrasound (confirmation gestational age)
22-24 weeks:
examination: blood pressure, maternal weight
lab: hemoglobin, platelets, creatinine, uric acid, liver enzymes
ultrasound: fetal growth, PI umbilical artery, RI uterine artery
20 and 30 weeks:
lab:anti-Fxa activity in patients who receive LMWH
28-30 weeks:
examination: blood pressure, maternal weight
lab: hemoglobin, platelets, creatinine, uric acid, liver enzymes
ultrasound: fetal growth, PI umbilical artery.
34 weeks:
examination: blood pressure, maternal weight
lab: hemoglobin, platelets, creatinine, uric acid, liver enzymes
Admission to the hospital:
reason, gestational age, laboratory, ultrasound, therapy.
For more details and delivery, post partum period, maternal and neonatal parameters see case
record form.
22
Product information
Fragmin (Dalteparin), produced by Pharmacia and Upjohn, is a low-molecular-weight heparin
produced by depolymerization of sodium heparin by nitrous acid degradation with a mean
molecular weight of 4,000-6,000 daltons. Its specific anticoagulant activities are about 160
U/ml in an anti-Fxa assay and 40 U/mg by APTT assay, with reference to the first international
standard for LMW heparin.46;48;49 The low-molecular-weight heparins do not cross the placental
barrier
46
but offer several advantages compared to unfractionated heparin. For years low-
molecular weight heparins have been used as thrombophylaxis during pregnancy. 47 It has a
longer half-life, better bioavailability and less effect on platelets than unfractionated heparin.
Furthermore, it is considered to have a better thrombophylactic effect and a lower risk on
bleeding complications. The systemic absorption from subcutaneous injection is better and antiXa activity is longer than unfractionated heparin.
In several studies
46;50
the authors concluded that the use of low-molecular-weight heparin
appears to be relatively safe and well tolerated during pregnancy, delivery and the immediate
postpartum period. As the long term use of heparin is associated with the development of
osteoporosis, bone density measurements performed by pregnant women treated with Fragmin
and untreated pregnant women showed similar bone density loss in both groups.51 Furthermore,
heparin-induced osteoporosis is reversible when heparin is stopped.45
In a study where low-molecular-weight heparin (5000 IU dalteparin once daily), was given to
22 pregnant women there were no thromboembolic recurrences or severe bleeding
complications. Three women had hematomas larger than 5 centimeters. Pain, swelling and
itching were occasionally reported. Postpartum in 18 women a scan of the vertebrae was made.
Four women had osteopenia in the lumbar vertebrae compared with young healthy adults.52
Rai describes a trial with aspirin and aspirin with low-molecular weight heparin in pregnant
women with recurrent miscarriages associated with phospholipid antibodies. There were no
signs of thrombocytopenia. The median loss in lumbar bone density was 5.4%, which is
equivalent to that loss after 6 months lactation without the use of low-molecular-weight
heparin.14
Appendix:
Product
information
Pharmacia
&
Upjohn.
23
Reference List
1. Duley L. Maternal mortality associated with hypertensive disorders of pregnancy in Africa, Asia,
Latin America and the Caribbean [see comments]. Br.J.Obstet.Gynaecol. 1992; 99:
547-553.
2. Williams DJ, de Swiet M. The pathophysiology of pre-eclampsia. Intensive.Care Med. 1997;
23: 620-629.
3. vd Post JAM., Steegers EAP. hypertensie in de zwangerschap. Tijdschrift voor verloskundigen.
1997; 6-11.
4. Schuitemaker N, van Roosmalen J, Dekker G, van Dongen P, van Geijn HP, Bennebroek GJ.
Confidential enquiry into maternal deaths in The Netherlands 1983-1992.
Eur.J.Obstet.Gynecol.Reprod.Biol. 1998; 79: 57-62.
5. Barker DJ, Gluckman PD, Godfrey KM, Harding JE, Owens JA, Robinson JS. Fetal nutrition and
cardiovascular disease in adult life [see comments]. Lancet 1993; 341: 938-941.
6. Barker DJ. Fetal nutrition and cardiovascular disease in later life. Br.Med.Bull. 1997; 53: 96108.
7. van Beek E, Peeters LL. Pathogenesis of preeclampsia: a comprehensive model.
Obstet.Gynecol.Surv. 1998; 53: 233-239.
8. Dekker GA, de Vries JIP, Doelitzsch PM, et al. Underlying disorders associated with severe earlyonset preeclampsia. Am.J.Obstet.Gynecol. 1995; 173: 1042-1048.
9. de Vries JIP, Dekker GA, Huijgens PC, Jakobs C, Blomberg BM, van Geijn HP .
Hyperhomocysteinaemia and protein S deficiency in complicated pregnancies.
Br.J.Obstet.Gynaecol. 1997; 104: 1248-1254.
10. Kupferminc MJ, Eldor A, Steinman N, et al. Increased frequency of genetic thrombophilia in
women with complications of pregnancy [see comments]. N.Engl.J.Med. 1999; 340: 913.
11. Riyazi N, Leeda M, de Vries JIP, Huijgens PC, van Geijn HP, Dekker GA. Low-molecular-weight
heparin combined with aspirin in pregnant women with thrombophilia and a history of
preeclampsia or fetal growth restriction: a preliminary study.
Eur.J.Obstet.Gynecol.Reprod.Biol. 1998; 80: 49-54.
12. van Pampus MG, Wolf H, Buller HR, Huijgens PC, jacobs, Dekker G. Underlying disorders
associated with severe preeclampsia and HELLP syndrome. Am.J.Obstet.Gynecol.
1997; 176: s26
13. Leeda M, Riyazi N, de Vries JIP, Jakobs C, van Geijn HP, Dekker GA. Effects of folic acid and
vitamin B6 supplementation on women with hyperhomocysteinemia and a history of
preeclampsia or fetal growth restriction. Am.J.Obstet.Gynecol. 1998; 179: 135-139.
14. 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) [see comments]. BMJ. 1997; 314: 253-257.
15. Tait RC, Walker ID, Perry DJ, et al. Prevalence of antithrombin deficiency in the healthy
population. Br.J.Haematol. 1994; 87: 106-112.
24
16. Miletich J, Sherman L, Broze GJ. Absence of thrombosis in subjects with heterozygous protein C
deficiency. N.Engl.J.Med. 1987; 317: 991-996.
17. Koster T, Rosendaal FR, Briet E, et al. Protein C deficiency in a controlled series of unselected
outpatients: an infrequent but clear risk factor for venous thrombosis (Leiden
Thrombophilia Study) [see comments]. Blood 1995; 85: 2756-2761.
18. Tait RC, Walker ID, Islam SI. Protien S deficiency in healthy blooddonors. Br.J.Haematol.
1994; 86: 118
19. Ferraresi P, Marchetti G, Legnani C, et al. The heterozygous 20210 G/A prothrombin genotype is
associated with early venous thrombosis in inherited thrombophilias and is not increased
in frequency in artery disease. Arterioscler.Thromb.Vasc.Biol. 1997; 17: 2418-2422.
20. Gris JC, Ripart-Neveu S, Maugard C, et al. Respective evaluation of the prevalence of
haemostasis abnormalities in unexplained primary early recurrent miscarriages. The Nimes
Obstetricians and Haematologists (NOHA) Study. Thromb.Haemost. 1997; 77: 1096-
1103.
21. Steegers-Theunissen RP, Boers GH, Trijbels FJ, et al. Maternal hyperhomocysteinemia: a risk
factor for neural-tube defects? Metabolism 1994; 43: 1475-1480.
22. Wouters MG, Boers GH, Blom HJ, et al. Hyperhomocysteinemia: a risk factor in women with
unexplained recurrent early pregnancy loss. Fertil.Steril. 1993; 60: 820-825.
23. Pattison NS, Birdsall MA, Chamley LW, Lubbe WF. Recurrent fetal loss and the antiphospholipid
syndrome. Recent Adv Obstet Gynaecol 1994; 18: 23-50.
24. Cowchock S. The role of antiphospholipid antibodies in obstetric medicine. cu rrent obstetric
medicin 1991; 1: 229-247.
25. Kalk JJ, Huisjes AJ, de Vries JIP. low-molecular weight heparin in women with a history of severe
early preeclampsia or small for gestational age infants and preeclampsia. Lancet 1999;
26. Sibai BM, Mercer B, Sarinoglu C. Severe preeclampsia in the second trimester: recurrence risk and
long- term prognosis. Am.J.Obstet.Gynecol. 1991; 165: 1408-1412.
27. Sullivan CA, Magann EF, Perry KGJ, Roberts WE, Blake PG, Martin JNJ. The recurrence risk of
the syndrome of hemolysis, elevated liver enzymes, and low platelets (HELLP) in
subsequent gestations. Am.J.Obstet.Gynecol. 1994; 171: 940-943.
28. Wallenburg HC, Dekker GA, Makovitz JW, Rotmans P. Low-dose aspirin prevents pregnancyinduced hypertension and pre- eclampsia in angiotensin-sensitive primigravidae. Lancet
1986; 1: 1-3.
29. AnonymousCLASP: a randomised trial of low-dose aspirin for the prevention and treatment of preeclampsia among 9364 pregnant women. CLASP (Collaborative Low-dose Aspirin Study
in Pregnancy) Collaborative Group [see comments]. Lancet 1994; 343: 619-629.
30. Caritis S, Sibai B, Hauth J, et al. Low-dose aspirin to prevent preeclampsia in women at high risk.
National Institute of Child Health and Human Development Network of Maternal-Fetal
Medicine Units [see comments]. N.Engl.J.Med. 1998; 338: 701-705.
31. Dekker GA, van Geijn HP. Endothelial dysfunction in preeclampsia. Part I: Primary prevention.
Therapeutic perspectives. J.Perinat.Med. 1996; 24: 99-117.
25
32. Dekker GA, van Geijn HP. Endothelial dysfunction in preeclampsia. Part II: Reducing the adverse
consequences of endothelial cell dysfunction in preeclampsia; therapeutic perspectives.
J.Perinat.Med. 1996; 24: 119-139.
33. van Pampus MG, Wolf H, Westenberg SM, van der Post JA, Bonsel GJ, Treffers PE. Maternal
and perinatal outcome after expectant management of the HELLP syndrome compared
with pre-eclampsia without HELLP syndrome. Eur.J.Obstet.Gynecol.Reprod.Biol.
1998; 76: 31-36.
34. AnonymousNational High Blood Pressure Education Program Working Group Report on High
Blood Pressure in Pregnancy. Am.J.Obstet Gynecol. 1990; 163: 1689-1712.
35. Davey DA, MacGillivray I. The classification and definition of the hypertensive disorders of
pregnancy [see comments]. Am.J.Obstet Gynecol. 1988; 158: 892-898.
36. Bakketeig LS. Current growth standards, definitions, diagnosis and classification of fetal growth
retardation. Eur J Clin Nutr. 1998; 52 Suppl 1:S1-4: S1-S4
37. de Jong CL, Gardosi J, Dekker GA, Colenbrander GJ, van Geijn HP. Application of a customised
birthweight standard in the assessment of perinatal outcome in a high risk population.
Br.J.Obstet.Gynaecol. 1998; 105: 531-535.
38. Harris EN. Special report. The Second International Anti-cardiolipin Standardization
Workshop/the Kingston Anti-Phospholipid Antibody Study (KAPS) group.
Am.J.Clin.Pathol. 1990; 94: 476-484.
39. Shennan A, Gupta M, Halligan A, Taylor DJ, de Swiet M. Lack of reproducibility in pregnancy of
Korotkoff phase IV as measured by mercury sphygmomanometry [see comments] .
Lancet 1996; 347: 139-142.
40. Franx A, Evers IM, van der Pant KA, van der Post JA, Bruinse HW, Visser GH. The fourth sound
of Korotkoff in pregnancy: a myth. Eur.J.Obstet Gynecol.Reprod.Biol. 1998; 76: 5359.
41. Steyn DW, Odendaal HJ. Randomised controlled trial of ketanserin and aspirin in prevention of
pre-eclampsia. Lancet 1997; 350: 1267-1271.
42. Greer IA. Thrombosis in pregnancy: maternal and fetal issues [In Process Citation]. Lancet 1999;
353: 1258-1265.
43. Smoot EC, Marx A, Weiman D, Deitcher SR . Recognition, diagnosis, and management of
heparin-induced thrombocytopenia and thrombosis. Plast.Reconstr.Surg. 1999; 103:
559-565.
44. Warkentin TE, Levine MN, Hirsh J, et al. Heparin-induced thrombocytopenia in patients treated
with low-molecular- weight heparin or unfractionated heparin [see comments].
N.Engl.J.Med. 1995; 332: 1330-1335.
45. Dahlman TC. Osteoporotic fractures and the recurrence of thromboembolism during pregnancy and
the puerperium in 184 women undergoing thromboprophylaxis with heparin.
Am.J.Obstet.Gynecol. 1993; 168: 1265-1270.
46. Melissari E, Parker CJ, Wilson NV, et al. Use of low molecular weight heparin in pregnancy.
Thromb.Haemost. 1992; 68: 652-656.
47. Sanson BJ, Lensing AW, Prins MH, et al. Safety of low-molecular-weight heparin in pregnancy: a
systematic review [In Process Citation]. Thromb.Haemost. 1999; 81: 668-672.
26
48. Forestier F, Daffos F, Rainaut M, Toulemonde F. Low molecular weight heparin (CY 216) does
not cross the placenta during the third trimester of pregnancy [letter] . Thromb.Haemost.
1987; 57: 234
49. Harenberg J, Schneider D, Heilmann L, Wolf H. Lack of anti-factor Xa activity in umbilical cord
vein samples after subcutaneous administration of heparin or low molecular mass heparin
in pregnant women. Haemostasis 1993; 23: 314-320.
50. Omri A, Delaloye JF, Andersen H, Bachmann F. Low molecular weight heparin Novo (LHN-1)
does not cross the placenta during the second trimester of pregnancy. Thromb.Haemost.
1989; 61: 55-56.
51. Shefras J, Farquharson RG. Bone density studies in pregnant women receiving heparin.
Eur.J.Obstet.Gynecol.Reprod.Biol. 1996; 65: 171-174.
52. Blomback M, Bremme K, Hellgren M, Siegbahn A, Lindberg H. Thromboprophylaxis with low
molecular mass heparin, 'Fragmin' (dalteparin), during pregnancy--a longitudinal safety
study. Blood Coagul.Fibrinolysis 1998; 9: 1-9.
27