Response to activated protein C during normal menstrual cycle and ovarian stimulation

M.L. Wramsby1,5, M.I. Bokarewa2,4, M. Blombäck3 and A.K. Bremme1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oestrogen has been pointed out as a pre-thrombotic factor. Protein C is a key enzyme in the down-regulation of blood coagulation. Recent data suggest that activated protein C (APC) resistance which is not due to the factor V:Q 506 Leiden mutation and appears to be acquired, is also a risk factor for thrombosis. In this study, we evaluated the endogenous oestradiol production and its possible influence on APC. Eighteen normally menstruating women were studied during one ovulatory cycle. Furthermore, 20 women undergoing controlled ovarian stimulation, and achieving extremely high oestradiol concentrations, were investigated. Normalized APC (nAPC) ratio (clotting time of tested sample/clotting time of pooled control plasma) was measured. Samples collected on menstrual cycle days 1–3, 6–8, 13–14, 20–24 corresponded to nAPC ratios 1.02 ± 0.19 (mean ± SD), 1.05 ± 0.15, 1.02 ± 0.16 and 1.03 ± 0.21 respectively. During ovarian stimulation, the nAPC ratios were 0.99 ± 0.12, 1.03 ± 0.18, 1.01 ± 0.16 and 0.97 ± 0.13 at oestradiol minimum, days 5–8 pre-oocyte retrieval, oestradiol maximum and at oocyte retrieval respectively. In spite of the great difference in the concentrations of oestradiol between women in normal menstrual cycle and women undergoing ovarian stimulation, no difference in nAPC ratios was observed.

Key words: APC resistance/menstrual cycle/ovarian stimulation


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activated protein C (APC) is a very important proteolytic factor, inactivating factor V and VIII. A decreased response to APC, APC resistance, has been recognized in families predisposed to thrombosis (Dahlbäck et al., 1993Go). APC resistance is generally related to a mutation Arg506-Gln in the factor V gene (Leiden mutation) and carriers may thus be homo- or heterozygous (Bertina et al., 1994Go).

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., 1996Go; Bokarewa et al., 1997Go).

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study groups
Menstrual cycle group
Eighteen normally menstruating women 21–35 years of age (mean 30.0) were studied during one ovulatory menstrual cycle, confirmed by elevated serum progesterone concentration (>17 nmol/l) at cycle day 20–24. Blood samples were collected during follicular phase cycle days 1–3, 6–8, mid-cycle phase days 13–14 and during luteal phase days 20–24.

COH group
Twenty women, 26–39 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 3.26–3.36). 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., 1995Go).

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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
During one ovulatory menstrual cycle 18 women were followed by blood sampling on cycle days 1–3, 6–8, 13–15, 20–24. Oestradiol concentrations (mean ± SD) during the menstrual cycle are shown in Table IGo.


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Table I. Normalized APC ratio, oestradiol and progesterone during menstrual cycle and ovarian stimulation (mean values ± SD)
 
All 20 women in the IVF treatment group underwent oocyte retrieval resulting in recovery of 4–14 oocytes (mean eight). Nineteen women underwent embryo replacement. A maximum of two pre-embryos was replaced. Four women became pregnant. The lowest oestradiol concentration (mean ± SD), 68 ± 40 pmol/l, rose to 1060 ± 621 pmol/l 5–8 days before oocyte retrieval and increasingly rose to a mean ± SD oestradiol maximum concentration of 5188 ± 2007 pmol/l, which was significantly higher as compared to the mean ± SD oestradiol concentration at oocyte retrieval (2762 ± 1218 pmol/l).

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.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two groups were investigated, normally menstruating women and women undergoing ovarian stimulation. Both groups showed an expected variation in their circulating concentrations of oestradiol and progesterone over time. Women undergoing ovarian stimulation had a rapid increase in oestradiol during ovarian stimulation and reached a value 10-fold higher than the highest value during the menstrual cycle. The progesterone concentration was kept low during ovarian stimulation due to GnRHa administration and was still low during the peak concentration of oestradiol. After injection of HCG, progesterone increased to concentrations above those seen in the luteal phase of normal menstrual cycle.

A transitory change leading to acquired APC resistance is considered a risk factor for thrombotic events (van der Bom et al., 1996Go; Sakata et al., 1996Go). Although previous reports have shown indications for a hormonal influence on APC (Henkens et al., 1995; Oliviero et al., 1995Go), 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., 1996Go; Bokarewa et al., 1997Go). 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., 1995aGo), 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., 1995bGo). Increased factor VIII activity has been observed during pregnancy (Bokarewa et al., 1997Go) and during ovarian stimulation (Aune et al., 1991Go; Bremme et al., 1994Go). During the menstrual cycle there is a considerable intra-individual variation in the level of factor VIII (Blombäck et al., 1992Go). 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.


    Acknowledgments
 
The authors acknowledge the co-operation of Laila Einarson, Ingrid Johansson and Sylvia Johansson at the Reproductive Medical Centre, Karolinska Hospital. We also wish to express our gratitude to the staff of the Research Laboratory of Women's Health, Karolinska Hospital. This study was supported by a grant from the Axelsson Johansson Foundation and from Baxter, Sweden. Kits for analysis of nAPC were kindly provided by Chromogenix, Möndal, Sweden.


    Notes
 
5 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aune, B., Høie, K.E. and Øian, P. (1991) Does ovarian stimulation for in vitro fertilization induce a hypercoagulate state? Hum. Reprod., 6, 925–927.[Abstract]

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Submitted on July 12, 1999; accepted on December 10, 1999.