Tibolone, a tissue-selective compound with a combination of estrogenic, progestogenic and androgenic properties, is used as an alternative for estrogen or estrogen plus progesterone hormone therapy for the treatment of symptoms associated with menopause and osteoporosis. The current study compares endometrial histology, biochemistry (hormone levels) and gene-expression profiles after short-term (21-days) treatment with tibolone, to the findings after treatment with estradiol-only (E2) and E2+Medroxyprogesterone Acetate (MPA) in healthy postmenopausal women undergoing hysterectomy for endometrial prolaps.

Since short-term tibolone use results in increased spotting and bleeding but long-term treatment with tibolone has been shown to lead to an atrophic endometrium our hypothesis is that tibolone first displays a more estrogenic mode of action, which over time, is counterbalanced by tibolone's progestagenic properties

Effects of estrogen deficiency in women are climacteric symptoms and bone loss, which can efficiently be treated by substituting with estrogens (1). Unopposed estrogen treatment, however, increases the risk for endometrial hyperplasia and subsequent carcinoma. For example in the HOPE study (Women's Health, Osteoporosis, Progestin, Estrogen study (2, 3)), after two years of treatment with conjugated equine estrogens (0.625mg/day CEE) the rate of hyperplasia went up to 27,7%. When the treatment was combined with MPA (2.5 mg) the incidence of hyperplasia completely disappeared. Thus, the addition of progestagens is necessary for endometrial safety. Progestagen addition (in continuous combined estrogen plus progestin therapy), however, also causes negative side effects like withdrawal bleeding and an increased breast cancer incidence that was recently documented in the Women's Health Initiative (4) and Million Women Study (5).

Tibolone is a tissue-specific compound used for treatment of climacteric complaints and prevention of osteoporosis and does not stimulate the endometrium like estrogens (6). It is converted by steroid metabolizing enzymes in the liver and intestine into three active metabolites: two hydroxy-metabolites: 3a-hydroxy-tibolone and 3b-hydroxy-tibolone (exerting estrogenic effects and in vivo present in significant amounts) and the delta-4-isomer (exerting progestagenic and androgenic effects, and in vivo present in circulation for a relatively short period of time (7)). The estrogenic metabolites are responsible for the effects on hot flushes, vagina and bone.

Tibolone's effects on the endometrium have been studied by a number of investigators (8-14) using transvaginal ultrasound (TVUS) and histology in biopsies after long-term treatment. With ultrasound it was observed that after 6 years of treatment endometrial thickness was slightly increased (p < 0.05, (8)) in tibolone-users compared to controls. Dőren et al. (9) confirmed that tibolone treatment results in a slight increase in endometrial thickness, which was not different from estrogen + norethisterone acetate treatment (E2+NETA). Histology data after tibolone use for more than 3 months, show no change in 90% of the subjects and only in a few cases an endometrial profile was shown which was comparable to the early follicular phase of a normal cycle (15). Even after two years of tibolone use hyperplasia rarely occurs (10).

In a double-blind, randomized controlled trial, Hammar (16) showed that the bleeding and/or spotting episodes in tibolone users in the first months of use were significantly lower than in E2+NETA users, and the authors hypothesized that this was due to a higher progestagenic activity of tibolone on the endometrium compared to the combined treatment. Our in vitro studies show indeed that tibolone can exert a progestagenic effect and that the balance between the progestagenic properties and the estrogenic properties of tibolone takes effect through a different set of genes acting in the same genetic network (17, 18).

In order to investigate whether our cell line data on tibolone action could be confirmed in vivo, and because relatively short-term effects of tibolone and other hormonal treatments in patients have never been described, postmenopausal women were treated for 21 days with tibolone, estrogen-only, or estrogen together with progestagen (estrogen+progestagen). From these women sera were obtained and endometria were removed in order to assess the histological and immunohistochemical, biochemical (hormone levels) and molecular (gene expression) effects of relatively short-term tibolone use (21-days). The overall results may help us to obtain insight in the mechanism of action of tibolone on the human endometrium compared to reference preparations.

Subjects:

This study was designed as a controlled clinical trial. Patients who visited our clinics (Amphia Hospital Breda, Albert Schweitser Hospital Dordrecht, Erasmus University Medical Center Rotterdam, The Netherlands) to undergo vaginal hysterectomy for treatment of prolapse, were eligible to participate in this study. A description of the inclusion and exclusion criteria of the current experiments were documented earlier (19).

The trial was performed in the period before the scheduled surgery. After informed consent, the patients were sequentially assigned to one of the following treatment groups: Control-group (no hormonal treatment); Tibolone-group (2.5 mg tibolone (Livial, N.V. Organon, Oss, The Netherlands) administered orally every day, starting 21 days prior to surgery); E2 group (2 mg of estradiol administered orally every day, starting 21 days prior to surgery); E2+MPA-group (2 mg estradiol + 5 mg MPA administered orally every day, starting 21 days prior to surgery). The investigators were kept blinded to the patient treatments during the analysis. The study protocol was approved by the local ethics committees of the participating hospitals.

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Inclusion Criteria:

Healthy postmenopausal women with a uterus. "Postmenopausal'' was defined as amenorrhoeic for at least one year prior to screening, or amenorrhoeic for at least six months prior to screening with a serum E2 concentration of < 20 pg/ml and a serum FSH concentration of > 40 IU/L at screening. If the patient used any kind of steroid hormone therapy prior to the study, a washout period of 6 months (for intra-uterine progesterone and oral estrogen+progestagen combination therapy) or 12 months (for progesterone implants or injections and injected estrogen+progestagen combination therapy) was applied.

Exclusion Criteria:

1. Histological diagnosis by a local pathologist of an endometrial biopsy (with the Pipelle suction curette) taken before treatment, as proliferative, secretory or menstrual type endometrium, endometrial metaplasia, endometrial or endocervical polyp(s), endometrial hyperplasia, cancer or any other histological abnormality (leiomyoma(ta), stromal nodules or mesenchymal or (endo)cervical neoplasia(s)).
2. Double-layer endometrium thickness > 4 mm as assessed by transvaginal ultrasound, immediately before endometrial biopsy.
3. History or presence of any malignancy, except successfully treated non-melanoma skin cancers.
4. Any unexpected vaginal bleeding following the menopause.
5. Liver disease, except cholecystectomy.
6. Abnormal cervical Pap smear test result, or abnormal mammography result obtained within one year prior to the start of the trial
7. Deep vein thrombosis, thrombophlebitis, thromboembolic disease, or suspicions of having hereditary predisposition for developing venous thromboembolic disease.
8. Use of one or more of the following drugs within the last two months: hepatic microsomal enzyme-inducing anticonvulsant drugs known to affect or interfere with the pharmacokinetics of steroids (e.g. hydantoins, barbiturates such as Phenobarbital (alone or in combinations, such as Bellergal) rifampicin, griseofulvin, primidone or carbamazepine).