Hemostatic changes associated with menopausal hormone therapy. Comparison of transdermal and oral administration

Introduction

The advent of the menopause is associated with an increased risk of cardiovascular disease. Women after menopause have higher levels of known risk factors for cardiovascular disease, such as fibrinogen and factor VII, compared with younger women [1]. Hormone therapy (HT) is a popularly recommended treatment for eliminating or alleviating the symptoms of menopause [2, 3]. It is established that combined hormone therapy containing estrogen and progestin is related with a small, but clinically significantly increased risk of arterial and venous thrombosis, may increase myocardial infarction and stroke in older women [4, 5]. During the treatment with oral hormone therapy (o-HT) the risk of venous thromboembolic complications increases 2- to 3-fold, particularly within the first

6 months of therapy [6, 7]. The hormonal treatment also affects the structure and metabolism of proteins involved in coagulation and fibrinolysis [8-10]. However, the effects of HT on hemostasis may depend on the dose and route of administration. In oral hormone therapy, a high dose of estrogens is necessary because oral formulations have bioavailability problems, such as intestinal and hepatic first-pass effects, and thus stimulate production of coagulation factors, increasing the risk of venous thrombosis (VTE). The transdermal route of administration has little influence on coagulation factors and consequently decreases the risk of VTE [11, 12]. The purpose of our study was to assess the effects of oral and transdermal hormone therapy on hemostasis determining such parameters as: fibrinogen concentration, plasma level or thrombin inhibitor of fibrinolysis (TAFI) and compare two other parameters: amidolytic activity of generated thrombin and plasmin in plasma. Our research was undertaken to confront the hemostatic effects of taking combined estrogen preparations containing two different progestogens in oral and transdermal HT in postmenopausal Polish women compared with non-users (controls) matched for age [12]. In the present study, the effect of HT on hemostatic factors was studied for 6 months.

Material and methods

Study participants



Fifty postmenopausal Polish women who visited the Department of Gynecology and Menopausal Disorders, Polish Mother’s Memorial Hospital – Research Institute in Lodz were examined. All of them were Caucasian living in the industrial area of central Poland. None of the women had a history of thrombosis, hypertension, diabetes or any metabolic disorders affecting the coagulation system. None of them smoked, used alcohol or had a thyroid or liver disease. They did not take any cholesterol-lowering drugs, antioxidant-vitamin supplements or any medication that might affect the metabolism of sex steroids before the present study. All patients were divided into three groups according to treatment: group I (mean age 55.6 years, n = 18): oral hormone therapy; 17b-estradiol (1 mg daily) and dydrogesterone (5 mg daily), group II (mean age 52.2 years, n = 14): transdermal hormone therapy; 17b-estra­diol (50 μg daily) 19-norethisterone (250 μg daily), group III (mean age 54.6 years, n = 18): controls, women without HT treatment. The study was approved by the Bioethics Committee at the Polish Mother’s Memorial Hospital – Research Institute in Lodz (no. RNN/57/06/KE). All subjects gave written informed consent.



Laboratory assays



Prior to the study, the following examinations were performed: gynecological examination with cervical cytological analysis, transvaginal ultrasound assessment of the reproductive organs, palpative breast examination and mammography. Blood samples were collected from an antecubital vein of the overnight-fasting subjects between 8.00 and 9.00 to avoid diurnal variation. All studies related to coagulation factors and fibrinolytic components were performed on platelet-free plasma obtained from centrifugation (2500xg, 15 min, 20°C) of 4.5 ml of blood mixed with 0.5 ml of 3.8% sodium citrate solution. Concentrations of glucose, fibrinogen (Fg) estimated by a method of functional fibrinogen determination using a Multifibren U kit (Dade-Behring Inc), platelet count (PLT) – using a Baker 810 platelet analyzer, activated partial thromboplastin time (APTT) – using a Pathromtin SL reagent, (Dade-Behring Inc) and triglyceride levels in blood of all patients were estimated in the Laboratory of the Polish Mother’s Memorial Hospital – Research Institute in Lodz.



Levels of thrombin inhibitor of fibrinolysis (TAFI)



The concentration of thrombin-activatable fibri­no­­lysis inhibitor (TAFI) was measured using ELISA kit Im­clone Tafi’s produced by American Diagnostica GmbH.

Measurement of thrombin generation by subsampling method

Plasma was mixed with fibrin polymerization in­hi­bitor Sigma® T1895 and incubated with rabbit throm­boplastin. Using S-2238 chromogenic substrate ami­dolytic activity of generated thrombin was measured according to a method described by Lau et al. [13].



Plasmin generation activity assays



Plasmin formation in plasma was determined by chromogenic substrate (Chromozym PL). For the activa­tion, plasma was diluted 10 times in 50-mM phosphate buffer, pH 7.4 and incubated with streptokinase for

30 minutes, at 37ºC. Assays were performed at 25ºC in 96 well-polystyrene flat-bottom plates, using the kinetic protocol in a microplate reader at 415nm (Bio-Rad Micro­plate Reader, model 550) [14].



Statistical analysis



All the values in this study were expressed as mean values ± SD using StatSoft Inc. “Statistica” v. 7.0. To check normality of sample distribution the data were analyzed with a Shapiro-Wilk test. When the distribution was normal, the statistical analysis of differences between the control plasma and plasma treated with o-HT and t-HT was done with a paired Student’s t-test using StatSoft Inc. “Statistica” v. 7.0. When the distribution was not normal, the non-parametric Mann-Whitney U test was used.

Results

The patients’ characteristics are presented in Table I.

There were no significant differences in terms of their mean age, years after menopause, body mass index (BMI) among the three groups (Table I). The clinical and biochemical characteristics of postmenopausal women who received oral and transdermal HT and patients did not take any hormones are summarized in Table II. We observed that after 6 months of HT, the level of fibrinogen was higher than in the control group (Fg 3.12 g/l vs. 4.14 g/l (o-HT); 3.6 g/l (t- HT); p < 0.001). There were no significant differences in platelet count, APTT and triglycerides between the three groups of patients. Variables of plas­min and thrombin activity are presented in Table III. The activity of generated thrombin was statistically higher in plasma of women after o-HT (72.6 ±8.5 mOD/min) than in patients with t-HT (53.7 ±10.1 mOD/min) and controls (51.2 ±10 mOD/min). There were no statistically significant differences between controls and women on t-HT treatment (p = 0.430). Amidolytic plasmin activity was the highest in controls (84.5 ±10.2 mOD/min). The value of plasmin activity in women after o-HT treatment was lower (61.9 ±7.9 mOD/min) compared to patients taking t-HT (77.7 ±14.5 mOD/min). There were no statistically significant differences between controls and women on t-HT treatment (p = 0.132). The concentration of TAFI

in patients’ plasma is shown in Table IV. The highest

level of TAFI was observed in patients after oral hormo­nes (80.38 ±8.23%); women on transdermal HT had

61.58 ±9.81% and the lowest concentration of TAFI was noted in the control group (44.70 ±10.16).

Discussion

Postmenopausal women are often prescribed estrogen-replacement therapy to treat menopausal symptoms and to prevent osteoporosis, but there is increasing recognition that it is also associated with important health risks [11, 15]. Harmful effects of HT include breast cancer and venous thromboembolism (VTE). Hormone treatment might also increase the risk of coronary heart disease and stroke [16]. In our study, HT with estrogen combination of two different progestogens, dydrogesterone and 19-norethisterone showed significant differences in hemostatic para­me­ters. We compared the effects of two preparations of

17β-estradiol/progestogen, one oral and one trans­dermal, on markers of coagulation and fibrinolysis. Both preparations are low-dose continuous combined HT products, thus the data allowed us to estimate the impact of the route of administration. We found that the transdermal route of hormones administration appears to have a less marked effect overall on the hemostatic system compared to the oral form and non-users. Levels of fibrinogen did not change significantly in the transdermal group compared to controls, whereas a considerable increase was observed in the oral group. The unchanged fibrinogen concentration after transdermal HT has been also found in other studies [17, 18]. Apart from being an acute phase reactant, fibrinogen has been considered a major cardiovascular risk factor and its level seems to be important during HT treatment. Enhanced thrombin activity in Polish women on oral hormone therapy compared to the transdermal group and non-HT users has been observed by us and other groups of investigators assessing the use of estrogen therapy [19]. In most previous studies, investigators suggest that oral, but not transdermal hormones increase plasma concentrations of pro­throm­bin fragment 1+2, which is a marker for in-vivo thrombin generation. In postmenopausal HT treatment, estrogens are usually given orally, but such a delivery route has drawbacks, including intestinal and hepatic first-pass effects. Oral estrogen administration leads to high hormone levels in the liver and promotes hepatic protein synthesis [20]. A study of pharmacokinetics of oral and transdermal estradiol showed a dose-dependent increase in serum estradiol exposure. The route of administration significantly affected fibrinolysis in this study. TAFI (thrombin activatable fibrinolysis inhibitor) plays an important role in regulation of this process. TAFI is activated by thrombin and protects the fibrin clot against lysis [21]. We found increased TAFI antigen levels in oral HT patients and this could be associated with a long time of fibrinolysis. We showed that the oral route of hormone administration appears to have a marked effect overall on the fibrinolytic system compared to the transdermal form and non-users. In accordance with these results, much lower amidolytic plasmin activity was found on oral treatment in our study, compared with transdermal HT and controls. We choose TAFI and plasminogen to measure because both are expressed in the liver and changes in these proteins may also influence plasmin production. Our studies show a relationship between high activity of thrombin generated and the level of TAFI in the plasma of patients taking oral hormone preparations. High concentrations of thrombin are required for the activation of TAFI and contrast the small amounts of thrombin that are sufficient for fibrin formation [22]. Our data suggest that oral hormone therapy might impair the balance between procoagulant factors and antithrombotic mechanisms, whereas transdermal treatment seems to have a little or no effect on hemostasis. Transdermal administration has the benefit of avoiding first-pass effect in the liver where many of the coagulation factors are synthesized. In addition to the influence of delivery route on hemostatic changes in HT patients, the dose and composition of each treatment can impact results. The oral HT used in our study was low-dose (1 mg daily) estradiol preparation combined with dydrogesterone (5 mg daily). It may explain a little the differences between our results and those found in similar ravages using higher-dose HT. The influence of hormone therapy on hemostasis has been largely attributed to the estradiol part of the preparation, although progestogen can also affect observed changes. The effect we observed may in part be explained by the dose and type of progestogen. Recent studies suggest that progestogens in oral combined postmenopausal therapy have differential effects on the fibrinolytic system and can change the prothrombotic impact of estradiol [23]. The clinical significance of our studies might be important for women at high risk of VE who need short-term HT for severe menopausal symptoms. We propose that the goal of selecting HT for climacteric complaints with optimal hemostatic safety should be replaced by that of finding a preparation with minimal changes in coagulation and fibrinolysis markers. Both efficacy and safety can accordingly be evaluated in future experimental studies.

Acknowledgements

This work was supported by grant 545/230 from the University of Lodz.

References

1. Norris L, Joyce M, O’Keeffe N, et al. Haemostatic risk factors on healthy postmenopausal women taking hormone replacement therapy. Matu­ritas 2002; 43: 125-33.

2. Farquhar C, Marjoribanks J, Lethaby A, et al. Long term hormone therapy for perimenopausal and postmenopausal women. Cochrane Database Syst Rev 2009; 2: CD004143.

3. Wolfang J, El-Samalouti V, Gerlinger C, Schaefers M. Effects of menopausal hormone therapy on hemostatic parameters, blood pressure, and body weight: open-label comparison of randomized treatment with estradiol plus drospirenone versus estradiol plus norethisterone acetate. Eur

J Obstet Gynecol Reprod Biol 2009; 147: 195-200.

4. Sandset PM, Høibraaten E, Eilertsen AL, Dahm A. Mechanisms of throm­bosis related to  hormone therapy. Thromb Res 2009; 123: 70-3.

5. Miller VM, Jayachandran M, Heit JA, Owen WG. Estrogen therapy and thrombotic risk. Pharmacol Ther 2006; 111: 792-807.

6. Peruga J, Kopff A, Krzemińska-Pakuła M. Hormone replacement therapy. The cardiologist‘s point of view. Przegl Menopauz 2007; 1: 3-7.

7. Sumino H, Ichikawa S, Sawada Y, et al. Effects of hormone replacement therapy on blood coagulation and fibrinolysis in hypertensive and normotensive postmenopausal women. Thromb Res 2005; 115: 359-66.

8. Norris LA, Brosnan J, Bonnar J, et al. Inhibitors and activation markers of the haemostatic system during hormone therapy: a comparative study  of oral estradiol (2 mg)/ dydrogesterone and estradiol (2 mg)/tri­megestone. Thromb  Haemost 2008; 100: 253-60.

9. Simomcini T, Mannella P, Genazzani AR. When hormone therapy sneaks under your nose. Menopause 2008; 15: 215-6.

10. Callejon DR, Franceschini SA, Montes MB, Toloi MR. Hormone replacement therapy and hemostasis: Effects in Brazilian postmenopausal women. Maturitas 2005; 52: 249-55.

11. Scarabin PY, Oger E, Plu-Bureau G. Differential association of oral and transdermal oestrogen-replacement therapy with venous throm­bo­em­bolism risk. Lancet 2003; 362: 428-32.

12. Koh SCL, Dramusc V, Ratnam S. The effects of long-term hormone-replacement therapy on coagulation, fibrinolysis and inhibitors in post­menopausal women. Int J Gynaecol Obstet 1998; 62: 279-85.

13. Lau A, Berry LR, Mitchell LG, Chan AK. Effect of substrate and fibrin polymerization inhibitor on determination of plasma thrombin gene­ration in vitro. Thromb Res 2007; 119: 667-77.

14. Wohl RC, Summaria L, Robbins KC. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 degrees C. J Biol Chem 1980; 255: 2005-13.

15. Peverill R. Hormone therapy and venous thromboembolism. Best Pract Res Clin Endocrinol Metab 2003; 17: 149-64.

16. Writing Group for the Women’s Health Initiative Investigators. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the Women’s Health Initiative randomized con­trolled trial. JAMA 2002; 288: 321-33.

17. Brosnan JF, Sheppard BL, Norris LA. Haemostatic activation in post-menopausal women taking low-dose hormone therapy: Less effect with transdermal administration? Thromb Haemost 2007; 97: 558-65.

18. Stevenson JC, Oladipo A, Manassiev N, et al. Randomized trial of effect of transdermal continuous combined hormone therapy on cardiovascular risk markers. Br J Haematol 2004; 124: 802-8.

19. Koh SCL, McCarthy TG, Dramusic V. Randomized comparative cross-over study between two hormonal preparations for hormone-replacement therapy; Their effects on the coagulation, fibrinolysis and inhibitor mechanisms. Singapore J Obstet Gynaecol 1998; 62: 279-85.

20. De Lignieres B, Basdevant A, Thomas G, et al. Biological effects of estradiol-17 beta in postmenopausal women: oral versus percutaneous administration. Clin Endocrinol Metab 1986; 62: 536-41.

21. Bouma BN, Mosnier LO. Thrombin activatable fibrinolysis inhibitor (TAFI) at the interface between coagulation and fibrinolysis. Pathophysiol Haemost Thromb.2003/2004 33: 375-81.

22. Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombin-activable fibrinolysis inhibitor (TAFI) and activated TAFI com­pared with respect to glycosylation, thrombin/thrombomodulin-depen­dent activation, thermal stability, and enzymatic properties. Biol Chem 1998; 273: 2127-35.

23. Rosendaal FR, Van Hylckama Vlieg A, Tanis BC, Helmerhorst FM. Estrogens, progestogens and thrombosis. J Thromb Haemost 2003; 1: 1371-80.