Can 19-Nortestosterone Derivatives Be Aromatized in the Liver of Adult Humans? Are There Clinical Implications?

By Kuhl, H Wiegratz, I

ABSTRACT Context Previous studies in postmenopausal women have demonstrated that, after oral administration of norethisterone, a small proportion of the compound is rapidly converted into ethinylestradiol. The shape of the concentration-time curve suggested that this occurred in the liver. The results were confirmed by in vitro investigations with adult human liver tissue. In 2002, it was shown that, after oral treatment of women with tibolone, aromatization of the compound occurred, resulting in the formation of a potent estrogen, 7alpha-methyl-ethinylestradiol. The result has been called into question, because the adult human liver does not express cytochrome P450 aromatase, which is encoded by the CYP 19 gene. Moreover, it has been claimed that the serum level of 7alpha-methylethinylestradiol measured by gas chromatography/mass spectrometry was an artifact.

Reply Aromatization of steroids is a complex process of consecutive oxidation reactions which are catalyzed by cytochrome P450 enzymes. The conversion of the natural C19 steroids, testosterone and androstenedione, into estradiol-17beta and estrone is dependent on the oxidative elimination of the angular C19-methyl group. This complex key reaction is catalyzed by the cytochrome P450 aromatase, which is expressed in many tissues of the adult human (e.g. ovary, fat tissue), but not in the liver. However, 19- nortestosterone derivatives are characterized by the lack of the C19- methyl group. Therefore, for the aromatization of these synthetic steroids, the action of the cytochrome P450 aromatase is not necessary and the oxidative introduction of double bonds into the A- ring can be catalyzed by other hepatic cytochrome P450 enzymes. The final key process in the formation of a phenolic A-ring, both in natural androgens and 1 9-nortestosterone derivatives, is the enolization of a 3-keto group to the C2-C3-enol or the C3-C4-enol moiety, which occurs without the action of enzymes.

Conclusion 19-nortestosterone derivatives (norethisterone, norethynodrel, tibolone) can readily be aromatized in the adult human liver. This leads to the formation of the potent estrogens ethinylestradiol from norethisterone or norethynodrel and 7alpha- methylethinylestradiol from tibolone. This may have clinical consequences, e.g. the elevated risk of venous thromboembolic disease in premenopausal women treated with high doses of norethisterone for bleeding disorders, or the elevated risk of stroke or endometrial disease in postmenopausal women treated with tibolone.

Key words: 19-NORTESTOSTERONE DERIVATIVES, TIBOLONE, 7alpha- METHYL-ETHINYLESTRADIOL, AROMATIZATION, HUMAN LIVER, CLINICAL IMPLICATIONS

INTRODUCTION

Progestogens are an important component of oral contraceptives owing to their pronounced interference with the regulation of follicular maturation and ovulation, cervical mucus, endometrium and tubal function. In postmenopausal women treated with estrogens, the addition of progestogens is obligatory to protect against endometrial hyperplasia. There are many progestogens used in contraception and hormone therapy which differ considerably with respect to their hormonal activities. Owing to the structural relationship between the progesterone receptors and the receptors for androgens, glucocorticoids and mineralocorticoids, progestogens can bind to these receptors and exert various agonistic or antagonistic effects, depending on their chemical structure and origin. Except for dienogest which does not contain an ethinyl group and shows a certain antiandrogenic effect, the nortestosterone derivatives show some androgenic properties. They are potent antiestrogens, but there are two compounds which also exert some estrogenic activity, namely norethisterone and related prodrugs (norethynodrel, lynestrenol and ethynodiol) as well as the norethynodrel derivative tibolone. This effect is brought about not by binding of the drug to the estrogen receptor, but by an enzymatic transformation of a small proportion of the drug into an estrogen1.

In a paper published by our group in 2002, it was demonstrated that, after oral administration of 2.5 mg tibolone (7a-methyl- norethynodrel) to premenopausal women, a small proportion of the dose was converted to the potent estrogen 7amethyl-ethinylestradiol (MEE)2. The peak serum level of MEE of 125 pg/ml, occurring 2 h after intake, suggested a potential clinical relevance. The findings were immediately questioned, using the argument that, in the adult human liver, the cytochrome P450 aromatase (CYP^sub arom^) is not expressed and, consequently, an aromatization of this 1 9- nortestosterone derivative could not be possible3. Our reply was the challenge to repeat our study, and the producer of tibolone initiated a clinical study with postmenopausal women in 2002. The results have, however, been kept secret, including the validation of the method.

Recently, it was claimed that our findings could not be the result of hepatic aromatization of tibolone, but were an artifact of the analytical method occurring during derivatization and gas chromatography4. This was based on a simple non-biological experiment that revealed an artificial conversion of 0.3% of tibolone during this analytical procedure. However, as the maximum amount of tibolone circulating in blood is only 1.5 ng/ml^sup 5^, the artifactual aromatization of this amount of tibolone present in the serum sample taken at the t^sub max^ of MEE, would reveal a maximum of 5 pg/ml. In face of the high peak level of 125 pg/ml MEE measured 2 h after oral administration of 2.5 mg tibolone, the true serum maximum of MEE after correction would be 120 pg/ml^sup 6^.

The questions remains whether or not the presence of CYP^sub arom^ is a prerequisite for the aromatization of 1 9- nortestosterone derivatives in the adult human liver and whether the formation of potent estrogens during treatment of women with norethisterone or tibolone would be of clinical relevance.

AROMATIZATION OF NATURAL ANDROGENS

The natural estrogens estradiol and estrone originate from the natural androgens testosterone and androstenedione. They are characterized by a phenolic ?-ring which implies that the angular C19-methyl group located between the ?-ring and B-ring of the androgen molecule must be eliminated. This conversion of the androstane to the estrane structure is brought about by a complex series of oxidative reactions catalyzed by so-called aromatase.

Aromatase is a cytochrome P450 enzyme complex consisting of the hemoprotein CYP^sub arom^, which is encoded by the CYP19 gene, and the NADPH-cytochrome P450 reductase. The CYP^sub arom^ converts androgens (Cl 9 steroids) into estrogens (Cl 8 steroids) containing a phenolic ?-ring. The reductase transfers reducing equivalents to CYP^sub arom^. The preferred substrate of CYPl 9 is androstenedione, which is converted into estrone by three successive oxidation steps7’8. It has been proposed that, by means of the first two oxidation reactions, the angular C19 methyl group is oxidized and, after a third oxidation step at C 1beta, the C 10-Cl 9 bond is cleaved by splitting off formic acid and accompanied by the formation of an intermediate double bond between Cl and ClO (Figure 1). Simultaneously, the 3-keto group is enolized, resulting in the delocalizing of electrons within a stable aromatic A-ring7.

In conclusion, the key reaction of the aromatization of natural androgens is the oxidative elimination of the angular Cl 9 methyl group. Moreover, enolization of the keto group at C3 to the C2-C3- enol moiety is a further prerequisite for the immediate formation of a phenolic A-ring. Therefore, compounds containing a hydroxyl group at C3 instead of a keto group, e.g. the tibolone metabolites 3alpha- OH-tibolone and 3beta-OH-tibolone, cannot be aromatized.

AROMATIZATION OF 19-NORTESTOSTERONE DERIVATIVES

The situation is completely different in the aromatization of 1 9- nortestosterone derivatives like norethynodrel or tibolone. The term ’19-nor’ represents the lack of an angular Cl 9 methyl group. Therefore, the action of the CYP^sub arom^, which eliminates the angular Cl 9 methyl group of natural androgens, is not needed for the aromatization of 1 9-nortestosterone derivatives (Figure 2).

Tibolone, which is the 7a-methyl-derivative of norethynodrel (Figure 3) , contains a double bond between C5 and ClO and a keto group at C3. This 3-keto group can readily be enolized to a 3- hydroxy-4-ene group (Figure 1). Therefore, only one additional double bond between Cl and C2 must be oxidatively formed in order to allow the energetically favored formation of a phenolic ?-ring. This is initiated by the introduction of a hydroxy group at C2alpha catalyzed by a cytochrome P-450 monooxygenase (CYP450) (Figure 2). Among the various metabolites of tibolone, the 27agr;-OH-tibolone has been identified9. Only one further oxidation step at this position, catalyzed by CYP450 and splitting off one water molecule, leads to an intermediate double bond between Cl and C2. A simultaneous enolization of the 3-keto group results in a rapid formation of a stable aromatic ?-ring (Figure 2).

Figure 1 Mechanism of action of the CYP19 aromatase (CYParum) as exemplified by the conversion of testosterone into estradiol-1 7 ss (after Brueggemeier et al. 20057). The formation of a phenolic A- ring is mediated by several oxidation steps catalyzed by the CYP^sub arom^. The key reaction is the oxidative elimination of the angular C19 methyl group located between the A-ring and the B-ring. The final step is an enolization of the keto group at C3, resulting in the phenolic A-ring Concerning an aromatization of norethisterone, hydroxylation at C2 and a further oxidation step at this position may lead to an intermediate double bond between Cl and C2. A simultaneous enolization of the 3-keto group to the C3-C4-enol moiety results in a rapid formation of a phenolic A-ring.

After a single oral dose of 10 mg norethisterone acetate administered orally to postmenopausal women, a small proportion of norethisterone is rapidly aromatized to ethinylestradiol (EE), reaching a peak serum level of about 200 pg/ml after 3 h (Figure 4) . Similarly, in premenopausal women treated for 7 days with 2.5 mg tibolone per day, a peak level of 125 pg/ml MEE was measured2. The shape of the MEE concentration curve, with a peak serum level after 2 h, suggests that aromatization of tibolone occurs mainly in the intestinal tract and/or during the first liver passage (Figure 4)2. As CYP^sub arom^ is not expressed in the adult liver8, experiments on the interaction of this enzyme system with tibolone or its Delta4- isomer (7alpha-methyl-norethisterone, MeNET) cannot contribute towards clarifying the question. Neither the use of human recombinant CYP^sub arom^ nor of cancer cells containing high activity of CYP^sub arom^ can exclude that tibolone is converted to MEE by hepatic CYP45011,12. Human recombinant CYP^sub arom^ was also not able to aromatize norethisterone11, and, in a study with cancer cells, aromatization of norethisterone was not investigated12. In contrast to these results, norethisterone was demonstrated to be aromatized into EE in both women and human liver tissue10’13-17.

Figure 2 Putative mechanism of aromatization of 1 9- nortestosterone derivatives as exemplified by the conversion of tibolone into 7alpha-methyl-ethinylestradiol. Hydroxylation at C2alpha and a further oxidation step lead to the formation of a double bond between Cl and C2. The final step is an enolization of the keto group at C3 resulting in the phenolic A-ring

Figure 3 Structural formulae of the prodrugs norethynodrel and tibolone and their hormonally active metabolites (after Kuhl, 2005 1J

Figure 4 Aromatization of tibolone and norethisterone in women (after Kuhl, 20051). (a) Time course of the serum concentration of 7alpha-methyl-ethinylestradiol in premenopausal women after oral administration of 2.5 mg tibolone ; (b) time course of the serum concentration of ethinylestradiol in postmenopausal women after oral administration of 10 mg norethisterone acetate10

Moreover, the norethisterone-prodrug norethynodrel, the first progestogen contained in oral contraceptives, has previously been demonstrated in the Allen-Doisy test to display an estrogenic efficacy 100 times that of norethisterone18. This suggests that a structural peculiarity of norethynodrel, the 5(10) double bond, facilitates its aromatization of norethynodrel in vivo. The 3alpha- and 3beta-hydroxy-tibolone metabolites, which are claimed to be responsible for the pronounced estrogenic activity of tibolone on bone and hot flushes, showed a weaker estrogenic potency than tibolone and MeNET after oral administration to rats19. This also suggests that an estrogenic metabolite other than the 3 -hydroxy- metabolites of tibolone is responsible for most of the estrogenic activity of tibolone.

The results of an investigation on the effect of derivatization by trimethylsilylation at 100[degrees]C revealed that only norethynodrel was converted to EE, but not norethisterone20. Similarly, tibolone, which is the 7alpha-methyl derivative of norethynodrel, is, to a small extent, aromatized during the derivatization/gas chromatography procedure, whereas MeNET is not. Obviously, 1 9-nortestosterone derivatives (e.g. tibolone and norethynodrel), containing a 3-keto group and a double bond between C5 and ClO, seem to be more prone to both an artifactual and enzymatic aromatization than 1 9-nortestosterone derivatives with a Delta4-3-keto (synonym 3-keto-4-ene) group like norethisterone or MeNET (Figure 3).

IS THE FORMATION OF ETHINYLESTRADIOL FROM NORETHISTERONE OR 7alpha-METHYL-ETHINYLESTRADIOL FROM TIBOLONE CLINICALLY RELEVANT?

Cardiovascular disease

Due to a rapid aromatization of a small proportion of norethisterone during the first liver passage, a peak level of about 200 pg/ml EE was measured in the blood of postmenopausal women 3 h after oral administration of 10 mg norethisterone acetate (NETA) (Figure 4) . This corresponds to plasma concentrations of EE during the use of high-dose oral contraceptives containing 100 pg EE and may explain the 5-6-fold increase in the risk of thromboembolic disease in patients treated for bleeding disorders or benign breast disease with high-dose NETA . Contrary to this, long-term daily treatment with 10 mg Chlormadinone acetate of patients with thrombophilia did not increase the risk of venous thrombosis22.

Table 1 Health risks in postmenopausal women during treatment with tibolone

The low EE levels observed in postmenopausal women during hormone replacement therapy (HRT) with estradiol and 1 mg or less NETA are without clinical relevance .

The peak levels of 125 pg/ml MEE observed 2 h after administration of 2.5 mg tibolone (Figure 4) may explain the increased risk of several diseases in postmenopausal women treated with tibolone (Table 1), because MEE has been demonstrated to exert potent estrogenic activity similar to EE .

In February 2006, the randomized placebocontrolled LIFT study was prematurely discontinued because an intermediate analysis revealed a relative risk of stroke of 2.59 in 4538 postmenopausal women during treatment with tibolone 4. This is remarkable, as, in this study on the prevention of bone fractures, the daily dose of tibolone was only 1.25 mg, i.e. half the dose used in HRT. Even though the mean age of the women was relatively high (68 years), the relative risk is much higher than that observed in the WHI study during treatment of older postmenopausal women with 0.625 mg conjugated estrogens and 2.5 mg medroxyprogesterone acetate (MPA). The hazard ratio was 1.35 in the age group 60-69 years, and 1.26 in the age group 70-79 years25.

Endometrial disease

Another clinically relevant effect which can be explained by the formation of the potent estrogen MEE is the significantly increased risk in endometrial cancer (relative risk 2.02) in current users of tibolone26. Even though the Million Women Study has been criticized for methodologie shortcomings (e.g. selection bias) concerning the method of breast cancer screening, this could not have affected the results on endometrial cancer, which is usually diagnosed in women with bleeding disorders. Moreover, the observation that the relative risk of endometrial cancer is not increased by tibolone in obese postmenopausal women corresponds to the findings in many large epidemiological studies which found no effect of HRT on the elevated breast cancer risk in overweight women27. In another nested case- control study, a significantly higher risk in endometrial cancer by 54% was observed during treatment with tibolone as compared to sequential HRT28.

A recent report suggests that the dose reduction of tibolone from 5 mg to 2.5 mg in a study on the effect of tibolone on bone was the consequence of bleeding disorders, which were caused by overt endometrial stimulation by the high tibolone dose . This was confirmed in animal experiments showing that higher doses of tibolone clearly have a proliferative effect in the uterus29. This effect is to a certain degree comparable with the effects of 10 mg norethisterone on the human uterus30.

A histological and immunohistological evaluation of the postmenopausal endometrium after 3 weeks of treatment with daily 2.5 mg tibolone or 2 mg estradiol, with or without the addition of 5 mg MPA, revealed that, in the glandular cells, the estrogen-dependent expression of the progesterone receptor was significantly increased in women treated both with tibolone and estradiolonly, whereas MPA clearly inhibited the estrogen effect . Estradiol alone stimulated largely the mitotic activity in the glandular cells and stroma, as measured by Ki-67. The glandular proliferation was significantly suppressed by the addition of MPA, whereas, in stroma, the mitotic activity was reduced, but still remained elevated. In glandular cells, tibolone increased non-significantly Ki-67 staining by 70%, while, in stroma, the mitotic activity was significantly elevated by 250%. The anti-apoptotic marker Bcl-2 increased in glandular cells during treatment both with estradiol-only and tibolone, whereas it was significantly reduced by the estradiol/MPA combination31. The results clearly show that, during treatment with tibolone, the antiproliferative activity of MeNET does not sufficiently suppress the estrogenic action of MEE on the endometrium.

Table 2 Hormonal activities of norethynodrel, tibolone and some of their metabolites

Finally, a 3-year prospective study on the treatment of postmenopausal women with 2.5 mg tibolone revealed a development of endometrial polyps in 33.4% of the women, as compared to a rate of 10.8% in patients treated continuously with a combination of 2 mg estradiol and 1 mg NETA . Although the majority of polyps in the tibolone group were atrophic, the findings confirm a proliferative effect of tibolone on the uterus.

It is known that continuous combined HRT reduces the risk of endometrial cancer in postmenopausal women, owing to the permanent presence of a potent progestogen. Therefore, it must be surprising that the continuous influence of the progestogenic tibolone metabolite MeNET does not protect against the development of endometrial cancer. However, MeNET has been demonstrated to exert a relatively weak progestogenic activity of only 13% of that of NET, whereas its strong androgenic activity is similar to that of testosterone (Table 2)33. Obviously, in the presence of the potent estrogen MEE, the effect of the weak progestogen MeNET might be insufficient to prevent an estrogen-induced endometrial proliferation in all women (Table 2). Due to the large interindividual variations in the pharmacokinetics of sex steroids, in some women the ratio between the serum levels of MEE and MeNET might be too high. Concerning the effects of norethisterone on the endometrium, the aromatization of norethisterone may explain why the dose-dependently enhanced suppression of nuclear estrogen receptors, as well as the increase in the activity of estradiol- 17beta- dehydrogenase, in the endometrium of postmenopausal women by norethisterone at doses between 1 mg and 5 mg, was partly reversed after treatment with 10 mg norethisterone30. Accordingly, the suppression of the estrogen-induced endometrial mitosis rate by the addition of 1 mg and 2.5 mg norethisterone was attenuated at the dose of 10 mg norethisterone30.

Similarly, the progestogenic effect of MeNET was diminished at higher doses, probably due to the relatively strong estrogenic activity of a metabolite of MeNET that was formed after oral administration . Moreover, in the Allen-Doisy test, MeNET was about 50 times more estrogenic than norethisterone33, even though norethisterone is aromatized to EE.

Breast disease

The high androgenic potency of MeNET may explain the weak effect of tibolone on the breast, since relatively low levels of testosterone have been shown to prevent proliferation of mammary epithelium induced by estradiol and progesterone34,35. However, extrapolation to malignant tissue of the weak proliferative effect of steroids on healthy breast epithelium must be taken with caution. The significantly elevated risk of breast cancer (relative risk 1.45) observed in the Million Women Study36 might be questionable because of the shortcomings of the study, and the significant increase in the relative breast cancer risk during current use of tibolone, as found in a Danish study, may be called into question due to the low number of cases37.

Surrogate parameters

Due to the strong androgenic potency of the Delta4-isomer (MeNET), which is comparable to that of testosterone, the effect of treatment with tibolone on various hepatic surrogate parameters indicates a predominance of the androgenic activity of MeNET over the estrogenic activity of MEE. This was exemplified by the pronounced reduction in the serum levels of high density lipoprotein (HDL) cholesterol and sex hormone binding globulin (SHBG)38,39. Such an effect has previously been observed in women treated with oral contraceptives containing low-dose EE and high-dose levonorgestrel40, although the latter exerted only 12% of the androgenic potency of testosterone41.

The potent estrogenic activity of MEE on the liver may, however, explain the beneficial effect of tibolone in postmenopausal women with type III hyperlipoproteinemia. This remnant removal disease leads to extremely high plasma levels of cholesterol and triglycerides, because the receptor-mediated clearance of chylomicron and very low density lipoprotein (VLDL) remnants by the liver is strongly impaired due to a dysfunctional apolipoprotein E. Treatment with 2.5 mg tibolone daily for 8 weeks led to a profound reduction in the cholesterol levels from 6.8 to 2.5 mmol/1 and in the triglyceride levels from 13.5 to 6.6 mmol/1 . This disease develops in women primarily after the menopause, and daily treatment with 50 [mu]g ethinylestradiol for 3 weeks has been demonstrated to cause a rapid fall in the cholesterol level from 13.0 to 6.5 mmol/1 and in the triglyceride level from 21.7 to 8.0 mmol/143. It remains an open question, how far the strong androgenic activity of the tibolone metabolite MeNET might have contributed to the reduction in the triglyceride levels42. Androgens are known to reduce the hepatic production of triglycerides and VLDL, but do not influence the postprandial formation of chylomicrons. Moreover, an androgen- induced suppression of the triglyceride synthesis is generally accompanied by a similar reduction in HDL cholesterol. In this study, however, the levels of triglycerides were reduced by 64% and those of HDL cholesterol non-significantly by only 16%42.

CONCLUSIONS

The conversion of the natural androgens testosterone and androstenedione into estradiol and estrone, which is dependent on the oxidative elimination of the C19-methyl group, is catalyzed by the classical cytochrome P450 aromatase system. Contrary to this, 1 9-nortestosterone derivatives can be aromatized by other hepatic cytochrome P450 enzymes after simple oxidative introduction of double bonds. This mechanism allows the rapid conversion of a small proportion of norethisterone into ethinylestradiol and of tibolone into 7alpha-methyl-ethinylestradiol, which occurs rapidly in the liver after oral administration. As the estrogenic potency of 7alpha- methyl-ethinylestradiol is similar to that of ethinylestradiol, treatment of postmenopausal women with tibolone is associated with various health risks, e.g. an elevated risk of stroke and endometrial cancer. Even though tibolone is also converted in the liver into a metabolite with progestogenic activity (7alpha-methyl- norethisterone), the relatively low progestogenic potency of this compound (only 13% of that of norethisterone) does not sufficiently counteract the proliferative effect of 7alpha-methyl- ethinylestradiol in the endometrium. This explains the elevated risk of endometrial cancer in postmenopausal women during treatment with tibolone. The high androgenic potency of 7alpha-methyl- norethisterone, comparable to that of testosterone, may explain the pronounced reduction in some surrogate parameters, e.g. the serum levels of HDL cholesterol and total triglycerides, and the low proliferative effect of tibolone on the mammary epithelium.

Conflict of interest Nil.

Source of funding Nil.

Received 17-12-06

Revised 15-02-07

Accepted 22-02-07

References

1. Kuhl H. Pharmacology of estrogens and progestogens: influence of different routes of administration. Climacteric 2005;8(Suppl l):3- 63

2. Wiegratz I, Sanger N, Kuhl H. Formation of 7alpha-methyl- ethinyl estradiol during treatment with tibolone. Menopause 2002;4:293-5

3. Davis SR, Simpson ER. Letter to the Editor. Kuhl H, Wiegratz I. In reply. Menopause 2003;10:105-7

4. Zacharia LC, Jackson EK, Kloosterboer HJ, Imthurn B, Dubey RK. Conversion of tibolone to 7a-methyl-ethinyl estradiol using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry: interpretation and clinical implications. Menopause 2006;13:92634

5. Timmer CJ, Verheul HAM, Doorstam DP. Pharmacokinetics of tibolone in early and late postmenopausal women. Br J Clin Pharmacol 2002;54:101-6

6. Kuhl H, Wiegratz I. Letter to the Editor. Menopause 2007;14:331-4

7. Brueggemeier RW, Hackett JC, Diaz-Cruz ES. Aromatase inhibitors in the treatment of breast cancer. Endocr Rev 2005;26:331- 45

8. Simpson ER, Clyne C, Rubin G, et al. Aromatase – a brief overview. Annu Rev Physiol 2002;64:93-127

9. Vos RME, Krebbers SFM, Verhoeven CHJ, et al. The in vivo human metabolism of tibolone. Drug Metab Dispos 2002;30:106-12

10. Kuhnz W, Heuner A, Humpel M, et al. In vivo conversion of norethisterone and norethisterone acetate to ethinyl estradiol in postmenopausal women. Contraception 1997;56:379-85

11. de Gooyer ME, Oppers-Tiemissen HM, Leysen D, et al. Tibolone is not converted by human aromatase to 7alpha-methyl-17alpha- ethynylestradiol (7alpha-MEE): analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS. Steroids 2003;68:235-43

12. Raobaikady B, Parsons MFC, Reed MJ, et al. Lack of aromatization of the 3-keto-4-ene metabolite of tibolone to an estrogenic derivative. Steroids 2006;71:639-46

13. Yamamoto T, Yoshiji S, Yasuda J, et al. Aromatization of norethindrone to ethynylestradiol in human adult liver. Endocrinol Jpn 1986;33:527-31

14. Urabe M, Yamamoto T, Yoshiji S, et al. Aromatization of norethindrone to ethynylestradiol in human adult liver. Steroids 1987;50: 607-8

15. Yamamoto T, Kitawaki J, Shiroshita K, et al. The confirmation of norethindrone aromatization in primary human hepatocytes by the vitafiber-II cell culture system. Acta Obstet Gynaec Jpn 1988;40:87- 9

16. Reed MJ, Ross MS, Lai LC, et al. In vivo conversion of norethisterone to ethynyloestradiol in perimenopausal women. J Steroid Biochem Molec Biol 1990;37:301-3

17. Klehr-Bathmann I, Kuhl H. Formation of ethinylestradiol in postmenopausal women during continuous treatment with a combination of estradiol, estriol and norethisterone acetate. Maturitas 1995;21:245-50

18. Beier S, Dusterberg B, El Etreby MF, et al. Toxicology of hormonal fertility-regulating agents. In Benagiano G, Diczfalusy E, eds. Endocrine Mechanisms in Fertility Regulation. New York: Raven Press, 1983:261-346

19. Kloosterboer HJ. Tibolone and its metabolites: pharmacology, tissue specificity and effects in animal model of tumors. Gynecol Endocrinol 1997;ll(Suppl l):63-8

20. Thompson RM, Horning EC. Aromatization of the ?-ring of norethynodrel, a steroidal oral contraceptive, during trimethylsilylation. Steroids Lipids Res 1973;4:135-42

21. Gomes MPV, Deitcher SR. Risk of venous thromboembolic disease associated with hormonal contraceptives and hormone replacement therapy. Arch Intern Med 2004;164:1965-76

22. Conard J, Plu-Bureau G, Bahi N, et al. Progestogen-only contraception in women at high risk of venous thromboembolism. Contraception 2004;70:437-41

23. Bodine PVN, Harris HA, Lyttle R, et al. Estrogenic effects of 7a-methyl-ethinylestradiol: a newly discovered tibolone metabolite. Steroids 2002;67:681-6 24. Cummings SR. LIFT study is discontinued. BMJ 2006;332:667

25. Wassertheil-Smoller S, Hendrix SL, Limacher M, et al. Effect of estrogen plus progestin on stroke in postmenopausal women. JAMA 2003; 289:2673-84

26. Million Women Study Collaborators. Endometrial cancer and hormone-replacement therapy in the Million Women Study. Lancet 2005;365: 1543-51

27. Kuhl H, Stevenson J. The effect of medroxyprogesterone acetate on estrogen-dependent risks and benefits – an attempt to interpret the Women’s Health Initiative results. Gynecol Endocrinol 2006;22:303-17

28. de Vries CS, Bromley SE, Thomas H, et al. Tibolone and endometrial cancer. A cohort and nested case-control study in the UK. Drug Safety 2005;28:241-9

29. Bjarnason NH. Endometrial cancer and hormone-replacement therapy. Lancet 2005;366: 200-1

30. Whitehead MI, Townsend PT, Pryse-Davies J, et al. Effects of various types and dosages of progestogens on the postmenopausal endometrium. J Reprod Med 1982;27:539-48

31. Klaassens AHA, van Wijk FH, HanifiMoghaddam P, et al. Histological and immunohistochemical evaluation of postmenopausal endometrium after 3 weeks of treatment with tibolone, estrogen only, or estrogen plus progestogen. Fertil Steril 2006;86:352-61

32. Perez-Medina T, Bajo-Arenas J, Haya J, et al. Tibolone and risk of endometrial polyps: a prospective, comparative study with hormone therapy. Menopause 2003;10:534-7

33. Schoonen WGEJ, Deckers GH, de Gooijer ME, et al. Hormonal properties of norethisterone, 7alpha-methyl-norethisterone and their derivatives. J Steroid Biocbem MoI Biol 2000;74:213-22

34. Dimitrakis C, Zhou J, Wang J, et al. A physiologic role for testosterone in limiting estrogenic stimulation of the breast. Menopause 2003;10:292-8

35. Dimitrakis C, Jones RA, Liu A, Bondy CA. Breast cancer incidence in postmenopausal women using testosterone in addition to usual hormone therapy. Menopause 2004;11:531-5

36. Million Women Study Collaborators. Breast cancer and hormone- replacement therapy in the Million Women Study. Lancet 2003;362: 419- 27

37. Stahlberg C, Tonnes Pedersen A, Lynge E, et al. Increased risk of breast cancer following different regimens of hormone replacement therapy frequently used in Europe. Br J Cancer 2004; 109:721-7

38. Farish E, Barnes JF, Fletcher CD, et al. Effects of tibolone on serum lipoprotein and apolipoprotein levels compared with a cyclic estrogen/ progestogen regimen. Menopause 1999;6: 98-104

39. Milner M, Sinnott M, Gasparo D, et al. Climacteric symptoms, gonadotrophins, sex steroids, and binding proteins with conjugated equine estrogen-progestin and tibolone over two years. Menopause 1996;3:208-13

40. Larsson-Cohn U, Fahraeus L, Wallentin L, et al. Effects of the estrogenicity of levonorgestrel/ ethinylestradiol combinations on the lipoprotein status. Acta Obstet Gynecol Scand Suppl 1982;105:37-40

41. Phillips A, Hahn DW, Klimek S, et al. A comparison of the potencies and activities of progestogens used in contraceptives. Contraception 1987;36:181-92

42. de Beer F, Smelt AHM, van Vark LC, et al. The effect of tibolone on the lipoprotein profile of postmenopausal women with type III hyperlipoproteinemia. J Intern Med 2002;251:148-55

43. Kushwaha RS, Hazzard WR, Gagne C, et al. Type III hyperlipoproteinemia: paradoxical hypolipidemic response to estrogen. Ann Intern Med 1977;87:517-25

H. Kuhl and I. Wiegratz

Department of Obstetrics & Gynecology, J. W. Goethe University Frankfurt, Frankfurt am Main,

Germany

Correspondence: Professor H. Kuhl, Department of Obstetrics & Gynecology J.W. Goethe University Frankfurt, TheodorStern-Kai 7, Frankfurt am Main, D-60590 Germany

Copyright Taylor & Francis Ltd. Aug 2007

(c) 2007 Climacteric. Provided by ProQuest Information and Learning. All rights Reserved.