1 Department of Women and Child Health, Box 140, 2 Department of Laboratory Medicine/Coagulation Research, 3 Department of Laboratory Medicine/Coagulation Research, Karolinska Institutet, 171 76 Stockholm and 4 Department of Rheumatology,Sahlgren's Hospital, 413 45 Gothenburg, Sweden
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Abstract |
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Key words: APC resistance/menstrual cycle/ovarian stimulation
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Introduction |
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During normal pregnancy APC ratio decreases significantly. About 57% of healthy pregnant women who were non-carriers of the mutation developed APC resistance, most commonly observed in the third trimester (Cumming et al., 1996; Bokarewa et al., 1997
).
This change is compatible with the increased risk of thrombosis related to pregnancy, although the mechanisms responsible for the APC change are not yet understood. The variety of hormonal alterations throughout pregnancy could most likely play a role in the development of a transitory APC resistance.
During ovarian stimulation, oestradiol reaches extremely high concentrations as compared to the natural menstrual cycle, while the concentration of progesterone is maintained at a low level, due to administration of gonadotrophin-releasing hormone agonists (GnRHa). In this study, ovarian stimulation has been used as a model to study the response to APC under the influence of rapid oestradiol increase. We also studied APC during the physiological hormonal changes occurring in the menstrual cycle.
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Materials and methods |
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COH group
Twenty women, 2639 years of age (mean 33.6), undergoing in-vitro fertilization (IVF) treatment were included. Infertility work-up of the couples had shown tubal infertility in eight women and severe endometriosis in four women, male infertility in five men, and unexplained infertility in three couples. All women were previously healthy and were Arg506-Gln mutation negative. None had had a thrombotic event.
IVF treatment protocol
All women involved underwent ovarian stimulation after pituitary suppression with a GnRHa (Suprefact, Svenska Hoechst AB, Stockholm, Sweden). GnRHa was administered as nasal spray, 1200 µg per day, starting on day 21 of the menstrual cycle. This medication was continued for 14 days. Endometrium thickness <3 mm measured by ultrasound indicated sufficient down-regulation. A few women required another 1 or 2 weeks of GnRHa medication to fulfil this criterion. After down-regulation was verified, blood samples were drawn by venepuncture for serum oestradiol, progesterone and leukocyte genetic analysis of the Leiden mutation in the factor V gene, and for plasma normalized APC (nAPC) ratio.
Ovarian stimulation was performed with FSH (Gonal F, Serono Nordic AB, Sollentuna, Sweden or Puregon, Organon AB, Västra Frölunda, Sweden). Follicular development was monitored by vaginal ultrasound measurements of follicles combined with blood samples for oestradiol analysis. When the day for oocyte retrieval was decided, 5000 IU human chorionic gonadotrophin (HCG, Profasi, Serono Nordic AB) was given i.m. Approximately 35 h later, oocyte retrieval was performed by transvaginal ultrasound-guided follicle aspiration. In this group, blood sampling was performed when down-regulation was achieved, during stimulation and at oocyte retrieval.
Laboratory analyses
Serum oestradiol and progesterone
Standardized methods using competitive immunoassay with commercial kits from Diagnostic Products Inc. (Los Angeles, CA, USA) were used.
nAPC
For APC resistance samples, a buffered sodium citrate medium (9 parts of blood + 1 part of trisodium citrate; 0.129 mol/l; pH 7.4) was used. Plasma underwent a double centrifugation at 3000 g for 20 min and was stored at 70°C until testing. APC resistance was tested in duplicate by a plasma-coagulation surface induced clotting time (earlier activated partial thromboplastin time) based assay (Chromogenix, Mölndal, Sweden), using the Behnk coagulator (Behnk Electronika, Nordestedt, Germany) and calculated as the ratio of the recalcification clotting time registered with and without the addition of APC. The assay gives a 3-fold prolongation of the coagulation surface induced clotting time when APC has been added (range 3.263.36). The APC ratio of a sample was then compared to the APC ratio of pooled normal plasma (normalized APC ratio = nAPC ratio). nAPC ratio <0.75 was considered to represent APC resistance.
Factor V Leiden gene mutation
Genomic DNA preparations were obtained from 0.2 ml of peripheral blood leukocytes from EDTA whole blood samples, using a QIA amplification kit column (Qiagen, Chatsworth, CA, USA). About 1 µg of DNA from this preparation was used in the polymerase chain reaction enzyme digestion of an amplified gene fragment using the primers specified by Gandrille et al. (Gandrille et al., 1995).
Statistics
Friedman analysis of variance (ANOVA) was used to compare nAPC ratios within groups. A P-value < 0.05 was considered statistically significant.
Ethics
The study was approved by the Ethics Committee at the Karolinska Hospital, Stockholm.
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Results |
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When nAPC ratios in the ovarian stimulation group were compared with those in the normal menstrual cycle group, there were no significant differences during the menstrual cycle or the ovarian stimulation treatment.
The nAPC ratios during ovarian stimulation did not differ statistically from those seen during normal menstrual cycle. No relationship was found between nAPC ratio and the concentrations of oestradiol and progesterone respectively.
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Discussion |
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A transitory change leading to acquired APC resistance is considered a risk factor for thrombotic events (van der Bom et al., 1996; Sakata et al., 1996
). Although previous reports have shown indications for a hormonal influence on APC (Henkens et al., 1995; Oliviero et al., 1995
), we found no significant change in the nAPC ratio during the menstrual cycle or during ovarian stimulation. Neither did we observe a difference in the nAPC ratio between the two conditions or a relationship between nAPC ratio and oestradiol or progesterone.
In some studies, nAPC ratios were found to decrease significantly during normal pregnancy and the nAPC ratios returned to normal after delivery (Cumming et al., 1996; Bokarewa et al., 1997
). The stage of early pregnancy when nAPC ratio starts to decline has not been determined.
In the present study there was a rapid increase in oestradiol concentration, shortly followed by a decline. This might indicate that the time of increased oestradiol is of importance for the decreased response to APC seen during pregnancy. A hormonal connection was also indicated in a previous study (Henkens et al., 1995a), which showed that women taking oral combined contraceptive pills (OC) had lower response to APC than women without OC who, however, had a lower response than men.
In a laboratory study, decreased APC ratio was related to increased factor VIII clotting activity (Henkens et al., 1995b). Increased factor VIII activity has been observed during pregnancy (Bokarewa et al., 1997
) and during ovarian stimulation (Aune et al., 1991
; Bremme et al., 1994
). During the menstrual cycle there is a considerable intra-individual variation in the level of factor VIII (Blombäck et al., 1992
). In the present study, we have not measured the factor VIII level but if its concentration was increased during ovarian stimulation as in previous studies, this has not been enough to alter the ratio of nAPC.
In conclusion, our observations of the response to APC during normal menstrual cycle and during ovarian stimulation suggest that the increasing oestradiol per se or the rapid increase in progesterone during the luteal phase in the menstrual cycle and at the time for OPU do not seem to have an impact on nAPC alteration.
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Acknowledgments |
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Notes |
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References |
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Bertina, R.M., Koeleman, B.P.C., Koster, F. et al. (1994) Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature, 369, 6467.[ISI][Medline]
Blombäck, M., Eneroth, P., Landgren, B.-M. et al. (1992) On the intraindividual and gender variability of haemostatic components. Thromb. Haemost., 67, 7075.[ISI][Medline]
Bokarewa, M., Wramsby, M., Bremme, K. et al. (1997) Variability of the response to activated protein C during normal pregnancy. Blood Coag. Fibrin., 8, 239244.[ISI][Medline]
van der Bom, J.G., Bots, M.L., Haverkare, P. et al. (1996) Reduced response to activated protein C is associated with increased risk for cerebrovascular disease. Ann. Int. Med., 72, 255260.
Bremme, K., Wramsby, H., Andersson, O. et al. (1994) Do lowered factor VII levels at extremely high endogenous estradiol levels protect against thrombin formation? Blood Coag. Fibrin., 5, 205210.[ISI][Medline]
Cumming, A., Tait, R.C., Fildes, S. et al. (1996) Diagnosis of APC resistance during pregnancy. Br. J. Haematol., 92, 10261027.[ISI][Medline]
Dahlbäck, B., Carlsson, M. and Svensson, P.J. (1993) Familial thrombophilia due to a previously unrecognised mechanism characterised by poor response to activated protein C. Prediction of a cofactor to activated protein C. Proc. Natl Acad. Sci. USA, 90, 10041008.[Abstract]
Gandrille, S., Alhenc-Gelas, M. and Aiach, M. (1995) A rapid screening method for the factor V Arg506-Gln mutation. Blood Coag. Fibrin., 6, 245248.[ISI][Medline]
Henkens, C.M.A., Bom, A.J., Seinen, J. et al. (1995a) Sensitivity to activated protein C; influence of oral contraceptives and sex. Thromb. Haemost., 73, 402404.[ISI][Medline]
Henkens C.M.A., Bom, A.J. and van der Meer, J. (1995b) Lowered APC sensitivity ratio related to increased VIII-clotting activity. Thromb. Haemost., 74, 11981199.[ISI][Medline]
Oliviero, O., Friso, S., Manzato, F. et al. (1995) Resistance to activated protein C in healthy women taking oral contraceptives. Br. J. Haematol., 91, 465470.[ISI][Medline]
Sakata, T., Kario, K., Katayama, Y. et al. (1996) Clinical significance of acquired protein C resistance as a potential marker for hypercoagulable state. Thromb. Res., 82, 235244.[ISI][Medline]
Submitted on July 12, 1999; accepted on December 10, 1999.