Hôpital Tenon, Hôpitaux Universitaires Est Parisien, Assistance Publique Hôpitaux de Paris, France.

Groupe de ThromboseEquipe de recherche ER2UPMCInteractions cellulaires tumorales et leur environnement et réponses aux agents anticancéreux.

Venous thromboembolism (VTE) figures among the most serious complications in women who participate in programmes of Assisted Reproductive Techniques (ART) with an incidence up to 0.11% of treatment cycles.

Ovarian hyperstimulation syndrome, an iatrogenic and potentially fatal IVF complication, occurs as a mild form in up to 33% of all of IVF cycles and 3%-8% of successful IVF cycles are complicated by moderate or severe OHSS. The incidence of VTE associated with OHSS is up to 2.4%.

The incidence of first trimester VTE in relation to FIV is 0.2%, representing a 10-fold increase as compared to background population.

Although routine use of thromboprophylaxis is in not recommended, the identification of women being in VTE risk and the use of appropriate thromboprophylaxis is expected to reduce vascular complications of ART.

Hormone treatment and ovarian stimulation figure among the major factors triggering blood hypercoagulability. The presence of intrinsic risk factors (i.e. co-morbidities such as autoimmune diseases, obesity, age etc) significantly contribute to the increase of VTE risk.

The association of clinical and biological criteria of hypercoagulability might contribute to the identification of patients at risk for vascular complications during FIV and pregnancy.

The routinely used blood coagulation assays, such as prothrombin time and activated partial thromboplastin time, are not mandatory for the detection of blood hypercoagulability.

New laboratory tools such as the Calibrated Automated Thombogram, allow global evaluation of the actors in plasma that participate in thrombosis.

The clinical relevance of these tests has already been assessed in pregnant women as well as in small series of patients participating in ART programmes. However, the evolution of thrombin generation in women receiving hormone treatment treatment for ART has not been investigated.

Aim of the study

Materials and MethodsIntroduction

Results

Conclusion

Impact of hormone treatment on thrombin generation in infertile women undergoing Artificial Reproductive Techniques.

Emmanuelle Mathieu d'Argent1, Marjorie Comtet1, Hela Ketatni2,3, Vassiliki Galea2,3, Severine Bouffard2,3, Jean-Marie Antoine1, Patrick Vandreden4, Aurelie Rousseau4, Ismail Elalamy2,3, Grigoris T Gerotziafas.2,3

1Department of Obstetrics and Gynecology, Hôpital Tenon, Hôpitaux Universitaires Est Parisien, Assistance Publique Hôpitaux de Paris, France.2Service d’Hématologie Biologique Hôpital Tenon, Hôpitaux Universitaires Est Parisien, Assistance Publique Hôpitaux de Paris, France.3ER2UPMC, Faculté de Médecine Pierre et Marie Curie, Université Paris VI, France.4Research and Development, Diagnostica Stago, Gennevilliers, France.

We conducted an observational prospective study of thrombin generation profile in women eligible for ART aiming to investigate the impact of hormone treatment on thrombin generation process.

Thrombin generation assay

Thrombin generation in platelet poor plasma (PPP) was measured by CAT assay (Diagnostica Stago) using PPP-reagent 5pM (Thrombinoscope b.v. Maastricht, Netherlands). Thrombin generation was triggered with a 20 µl solution containing CaCl2 (16.7 mM final concentration) and the fluorogenic substrate Z-Gly-Gly-Arg-AMC (417 pM final concentration). Each patient’s plasma was studied in duplicate. In a third well well, PPP reagent 5 pM was replaced with the same volume of Thrombin Calibrator (Thrombinoscope b.v. Maastricht, Netherlands). Fluorensence was measured using a Fluoroscan Ascent® fluorometer (ThermoLabsystems, Helsinki, Finland). Acquisition of thrombin generation parameters was done using the appropriate software (Calibrated Automated Thrombogram® b.v. Maastricht, The Netherlands).

The following parameters of thrombogram were analyzed:

Peak: the maximum concentration of thrombin

Endogenous Thrombin Potential (ETP) : it reflects the total amount of thrombin activity.

Mean Rate Index (MRI): it reflects the velocity of the propagation phase of thrombin generation and is calculated by the formula MRI=Peak/(ttPeak – lag-time).

The Upper Normal Limit (UNL) for each parameter of thrombogram was defined in the control group as follows : UNL= mean + 2SD.

Influence of gonadotrophin treatment on thrombin generation

Administration of hormone treamtnet had a marginal effect on thrombin generation profile of infertile women (Table 1 and Figure 1).

After gonadotropins administration (T1) thrombin generation profile did not significantly modify as compared to that observed in T0.

After HCG administration (T2) thrombin generation tended to decrease as compared to T0.

28 infertile women eligible for ART were included in the study.

Inclusion criteria : Women had to be planed for IVF according to established selection criteria and having hemogramme platelet count, basic haemostasis profile (PT, aPTT, Fibrinogen), renal and liver function within the normal range.

Exclusion criteria: Women were excluded if they responded to at least one of the following criteria

Timing of blood sampling

Blood samples were obtained

at the inclusion (T0)

the 5th to 8th day of gonadotrophins administration (T1)

on the day of HCG administration (T2).

The control group consisted of 30 healthy age matched women who had at least one uneventful pregnancy.

Patients and Methods

Control(n=30)

UNL ART-T0(n=28)

ART-T1(n=28)

ART-T2(n=28)

Peak (nM)

345 ± 61 468 381 ± 96 342 ± 108 375 ± 60$

ETP (nM.min)

1670 ± 246 2161

2085 ± 679** 1812 ± 602 1997 ± 574*

MRI (nM/min)

150 ± 42 234 146 ±42 136 ± 55 157 ±45&

Table 1. Thrombogram parameters in the control group and in the ART group before and after ovarian stimulation.** T0 vs control; p=0.049 *T1 vs T2; p<0.001 $T1 vs T2; p=0.006 &T1 vs T2; p=0.04  

Baseline thrombin generation profile in infertile women eligible for ART

At inclusion (T0) infertile women showed significantly increased thrombin generation which was characterised by a marked increase of the ETP as compared to the control group.

The other parameters of thrombogram were not significantly different as compared to the control group (Table 1).

At T0, 13 out of 28 women (46%) showed ETP values higher than the UNL as defined in the control group (Figure 1).

Influence of gonadotrophin treatment on thrombin generation

Administration of hormone treamtnet slightly modified thrombin generation profile of infertile women. The two types of treatment had dinstict effect (Table 1 and Figure 1).

After gonadotropin administration (T1) thrombin generation profile did not significantly modify as compared to that observed in T0.

After HCG administration (T2) thrombin generation tended to decrease as compared to T0 but the difference was not statisticall significant.

Hormone treatment and Inter-individual variablity of thrombin generation

The analysis of individual profile of thrombin generation after hormone therapy showed that

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Figure 2. Individual variation of endogenous thrombin potential after gonadotropin administration in infertil women undergoing ART.

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Figure 3. Individual variation of endogenous thrombin potential after HCG administration in infertil women undergoing ART.

The present pilot study analysed the profile of thrombin generation in infertile women eligible for ART and investigated the influence of hormone treatment with gonadotropins and HCG on thrombin generation.

Hypercoagulability, in terms of increased endogenous thrombin potential is present in 46% of inferile women eligible for ART.

These women remain in an hypercoagulable state throughout the entire period of hormone treatment.

Administration of HCG has a significantly higher impact on thrombin generation as compared to the treatment with gonadotropins.

However, neither of the two treatments induces a hypercoagulable state.

The present study indicates that hypercoagulability is a common biological phenomenon in infertile women eligible for ART. Hormone therapy has no amplifying effect on thrombin generation.

personal or family history of venous thromboembolism or hemorragic syndrome

known hereditary or acquired thrombophilia

known systematic or chronic disease (autoimmune syndrome, cancer heart disease, severe or non equlibritatedthyroide disease

ongoing antithrombotic treatment with VKA or UFH or LMWH or clopidogrel or aspirin. Patients who received antithrombotic treatment for at least 30 days before inclusion in the study were also excluded.

treatment with non steroide intinflammatory drugs during the last 10 days before inclusion in the study

recent hospitalisation during 30 days before inclusion in the study

ovarian insufficiency (FSH>9 IU/ml and/or number of follicular antaraux <8) or SPOK (defined according to the Rotterdam criters).

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Figure 1. Thrombin generation after administration of gonadotropins and HCG.

The administration of gonadotropins (T1) resulted in increase of thrombin generation in 4 out of 28 women (14%) as compared to T0 (Figure 2)

Women who had ETP values lower than the UNL at T0 continued to have normal values at T1.

Administration of HCG (T2) resulted in significant increase of thrombin generation as compared to T1 (Table 1).

At T2 thrombin generation was increased in 18 out of 28 womens (64%).

Women who had ETP values lower than the UNL at T1 continued to have normal values at T2 (Figure 3).

In 9 out of 13 women (69%) who showed high ETP values at T0 the profile of hypercoagulability persisted until the end of the period of hormone treatment (T2).