Hormone Replacement Therapy on the Postmenopausal Endometrium
Condition: Menopause
Study type: Interventional
Phase: Phase 4

Summary: 
      Healthy postmenopausal women, amenorrheic for 1-10 years, aged 45-60 years and in use of
      follicle stimulating hormone > 30 mille-International unit/milliliters (mIU/mL), will
      participate in the study.

      All women will be informed by a physician of the risks and possible benefits of Hormone
      Replacement Therapy (HRT).

      The continuous combined treatment with drospirenone 2 mg and 17β-estradiol 1 mg or tibolone
      1.25 mg will be administered for a 24-week period.

      Transvaginal ultrasound was performed to evaluate the thickness of the endometrium at
      baseline. At study end-point, an endometrial biopsy will be performed with a Pipelle
      endometrial sampler to examine histological and immunohistochemical parameters.

      Immunohistochemical detection of Bcl-2 protein, estrogen and progesterone receptors will be
      performed in stroma and glandular epithelium.
    

Detailed description: 
      Subjects

      Healthy postmenopausal women, amenorrheic for 1-10 years, aged 45-60 years and in use of
      follicle stimulating hormone > 30 mille-International unit/milliliters (mIU/mL), will
      participate in the study.

      All women will be informed about the trial and signed the informed consent documents,
      eligibility was determined based on medical history, and on general physical and
      gynecological examination. All women were informed by a physician of the risks and possible
      benefits of Hormone Replacement Therapy (HRT). Upon inclusion, Transvaginal ultrasound
      (TV-US) was performed to evaluate the thickness of the endometrium at baseline. At study
      end-point, an endometrial biopsy will be performed with a Pipelle endometrial sampler to
      examine histological and immunohistochemical parameters . Vital signs, blood pressure,
      weight, Pap smear, mammography, routine clinical chemistry assays (including, cholesterol,
      high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides) and
      hematology will also be evaluated at screening.

      Treatment Groups Of the 100 women initially evaluated, 32 did not meet the inclusion and
      exclusion criteria, while eight refused to participate, giving a final total of 60 patients
      for inclusion in the study. The sixty postmenopausal women were randomly allocated into three
      groups: Group I: control group comprising 20 patients (no hormonal treatment); Group II: also
      comprising 20 patients, to whom a continuous combined treatment with drospirenone 2 mg and
      17β-estradiol 1 mg (DRSP/E2) will be administered daily; and Group III: also comprising 20
      patients, to whom treatment with tibolone 1.25 mg (Tib) will be administered daily. All
      treatments will be administered for a 24-week period. DRSP/E2 was purchased from "Bayer
      Schering Pharma™", Brazil and Tib from "Biolab™", Brazil. The duration of the treatment will
      be 6 cycles, each lasting a period of 28 days (conducted for a total of 24 weeks).
      Randomization will be done with the aid of computer software in order to prevent bias in the
      selection process.

      Objective: A prospective randomized controlled trial will be conducted to evaluate the effect
      of low dose of tibolone and estradiol/drospirenone on the histology, expression of estrogen
      (ER) and progesterone receptors (PR) and Bcl-2 protein, in endometrium of postmenopausal
      women.

      The effects on endometrial thickness will be determined by Transvaginal Ultrasound (TV-USG)
      at study baseline and again after 6 cycles of treatment in all women participants. Scans were
      performed with a real-time scanner, "Hitachi 405"ultrasound system equipped with a
      5.0-megahertz (MHz) vaginal transducer, at the "Vale do Sapucai University". An endometrial
      thickness < 4 millimeters (mm) will be interpreted as not supporting a diagnosis of
      endometrial cancer.

      Endometrial biopsy and sampling will be performed at the end of the treatment course using a
      "Pipelle Curette" de Cornier, placed high into the upper fundus of the uterus. An aliquot of
      the endometrial sample will be pushed from the pipette into a polypropylene vial. The vial
      will be capped, immediately frozen, and then stored at -20º Celsius until shipping in dry ice
      to the Department of Pathology at the "Vale do Sapucai University", directed by one of the
      authors, for measurement of Progestogen Receptor (PR) and Estrogen Receptor (ER) receptors as
      well as Bcl-2 protein. The remaining endometrial tissue will fix in 10% neutral buffered
      formalin for 6-48 h and then process into paraffin blocks.

      Histological and immunohistochemical assessment Histology classification will be carried out
      by two pathologists. Both pathologists will do a blind reading of different tissue sections
      of the same block-sample. After staining with hematoxylin/eosin and Schiff Periodic Acid
      (PAS), each specimen was classified into one of the following categories: tissue insufficient
      for diagnosis, atrophic and/or inactive endometrium, proliferative or weakly proliferative
      endometrium, and progestogenic .

      Immunohistochemical detection of Bcl-2 protein will be performed by two pathologists. After
      antigen retrieval and blocking of non-specific sites with normal serum using the
      Avidin/Biotin Blocking kit (Vector Laboratories, Inc, California, USA), the sections will be
      incubated overnight at 4º Celsius with anti-Bcl-2 mouse monoclonal antibody (1:100, Clone
      124, DAKO, Denmark). The tissue-bound antibody will be incubated for 30 min at room
      temperature with the Envision System. Staining will be visualized by use of Envision (Kit
      N1587, DAKO, Denmark). The cellular staining pattern for this antibody is cytoplasmic. The
      presence of ER and PR will be detected as described previously. The cellular staining
      patterns for these antibodies are nuclear. A breast cancer specimen will be used as a
      positive control for the steroid receptors.

      Examinations and photography will performed using an "Axioskop 40 - Zeiss" light microscope
      equipped with a Canon™ D30 digital camera (Tokyo, Japan) and video monitor (LG 14" LCD)
      according to the procedures of the Pathology Department from Santa Casa of Sao Paulo Medical
      School, Sao Paulo, Brazil.

      The morphometric technique for immunohistochemistry quantification will be done with the aid
      of a Neubauer graduated chamber. This consists of a glass slide, thicker than a regular
      slide, each having a grid design dividing it into squares with known dimensions, visualized
      only through an optical microscope. The Neubauer counting chamber is divided into 9 large
      squares, each with 1 mm sides, by means of triple lines (thus area of a large square= 1 mm²).
      Each large square contains 16 (4 x 4) medium-size squares. Each medium size square is further
      divided by triple lines into 25 (5 x 5) smaller squares, each with a side length of 1/5 of a
      mm. Thus, the area of an individual small square is 0.05 x 0.05 mm= 0.0025 mm² and 0.1 mm
      deep .

      The image observed through the microscope lenses will be captured by the video camera and
      displayed on the chosen monitor. The Neubauer chamber will be then placed under the optical
      microscope, using an objective which magnifies the original size of the object by 400x or by
      any other magnification elected by the researcher. Finally, the area under the high
      magnification field (400x magnification) is estimated in mm² by counting the total number of
      individual small 0.0025 mm² squares of the chamber within the magnification area chosen. The
      total area under high magnification corresponded to 0.094 mm².

      Using this approach, each section prepared by the immunohistochemical method will be
      evaluated under the light microscope. The number of cells in the study will be counted at
      400x magnification. A 10-field count will be performed in the different situations evaluated.
      The mean number of cells present in this 10-field count represented an area of 0.094 mm². By
      means of a simple rule of three, the number of cells will be converted into 1 mm² counts. The
      cell count will be expressed as numerical density, i.e. number of positive cells per square
      millimeter of epithelium.

      The number of Bcl-2-positive cells and immunoreactivity for PR and ER expression will
      quantified manually by two trained observers, blind to the experiment, and will evaluate
      separately for glandular and stromal cells. The immunohistochemical staining index (numerical
      density) will be defined as the mean number of positive immunoexpression cells per mm²,
      calculate in 10 different fields.

      Statistical analysis Statistical analysis will be performed using the "StatistiXL Package™"
      (Statistical Power for Microsoft Excel version 1.8, 2007). Differences in demographic
      parameters, immunohistochemical staining index of ER, PR and Bcl-2 protein expression
      (numerical density), as well as baseline and treatment end-point endometrial thickness, will
      be analyzed by the paired Student's t-test in the different groups. The relationships between
      ER, PR and Bcl-2 protein expression in stroma and glandular epithelium will be analyzed using
      Spearman's test.

      Statistical significance will set at p < 0.05. Data will express as mean + standard deviation
      (SD).
    

Intervention name(s): Endometrial Biopsy; Transvaginal Ultrasound; Endometrial Immunohistochemical
Intervention type(s): Procedure; Device; Procedure
Intervention description(s): Histological assessment; Endometrial Thickness made by ultrasound; Immunoexpression of estrogen (ER) and progesterone receptors (PR) and Bcl-2 protein
Other intervention name(s): Endometrial Thickness
Allocation: Randomized
Intervention model: Single Group Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Treatment
Keywords: menopause; drospirenone; tibolone; Bcl-2; steroid receptors