Progesterone Action on Endometrium of PCOS
Condition: Polycystic Ovary; Progesterone Resistance
MeSH terms: Polycystic Ovary Syndrome
Study type: Interventional

Summary: 
      Objective: To study the influence of metabolic factors on the action of progesterone on the
      endometrium of women with PCOS.

      Design: A prospective open-label clinical trial. Setting: Participants were recruited from an
      outpatient reproductive endocrinology clinic at an academic tertiary medical care center from
      2014 to 2019.

      Subjects: To assess the sole effect of progesterone (P4) on the endometrium we studied
      amenorrheic (cycles> 90 days) women with PCOS (n=23), comparing them to healthy eumenorrheic
      control women ( n=13).

      Intervention(s): All subjects underwent an endometrial biopsy (EB) in the follicular phase of
      the first study cycle. They were then treated with micronized P4 (400 mg/day x 10 days
      intravaginally) from days 14-28 of the cycle, and a second EB was performed between days
      20-24 of the cycle (second phase).

      Main Outcome Measure(s): EB samples were analyzed under light microscopy for
      histomorphometric analysis.
    

Detailed description: 
      PATIENTS AND METHODS

      Subjects Inclusion criteria for PCOS women will be: a) ages 18 to 35 years, and b) PCOS was
      defined by the Rotterdam criteria, which includes two of the following three features: a)
      oligo-amenorrhea; b) clinical or biochemical signs of hyperandrogenism; and c) polycystic
      ovaries (6). These criteria yield four PCOS phenotypes (A thru D) (1-3). Controls were
      recruited from the family planning outpatient office. All participants will be healthy women
      ages 18 to 35 years with regular menstrual cycles, no evidence of hirsutism, no
      comorbidities, and not using hormone medications in the previous 3 months.

      Exclusion criteria are as follows: a) use of any hormone treatment in the previous 3 months;
      b) other causes of anovulation as evidenced by abnormal levels of prolactin, cortisol,
      dehydroepiandrosterone sulphate (DHEAS), 17-hydroxyprogesterone (17-OHP), androstenedione,
      thyroid-stimulating hormone (TSH), and free T4, in blood samples obtained between days 2 and
      5 of the cycle in cycling subjects or on any other day in amenorrheic subjects; c)
      gynecologic disorders or other associated diseases, such as endometriosis, hypertension, or
      diabetes mellitus; d) follicle-stimulating hormone (FSH) >15 UI/L (cycle days 2-5); or e) a
      positive β-subunit of human chorionic gonadotropin (β-HCG).

      Study Protocol

      This trial was previously approved by the Research Ethics Committee (#21253613.6.0000.0068)
      of the Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo (FMUSP), São
      Paulo, Brazil. The patients will be evaluated at Gynecology Outpatient office. The patients
      will be divided into two groups: a) PCOS and b) controls. All subjects will be cared for at
      the Endocrinology Gynecology Section and the Human Reproductive Center of the Gynecology
      Division, Hospital das Clínicas, FMUSP. After signing the informed consent will be written in
      accordance with Resolution 196/96 of the National Health Board (Resolução 196/96 do Conselho
      Nacional de Saúde), all subjects will be included in the study and evaluated as follows:

      Preselection: This visit includes the following: interview for medical history; overall
      physical and gynecologic examinations; weight measurement (scale Filizola, model PL-150, with
      a capacity for 150 kg); height measurement with a metal stadiometer attached to the scale;
      abdominal circumference (AC) measurement with a measuring tape at the narrowest region
      between the last costal arch and the iliac crest; hip circumference (HC) measured around the
      circumference of hips at the top of the iliac crest and buttocks, body mass index (BMI) and
      blood pressure (BP) measurement; hirsutism according to the modified Ferriman-Gallwey (mFG)
      scoring system with nine areas, with patients with a score higher than 8 score deemed
      hirsute.

      The menstrual cycle are considered as normal when the interval between periods was 21 to 35
      days, and the blood flow lasted no more than 7 days. The cycle was characterized as
      anovulatory when the span between blood flows was >35 days (1). Amenorrhea is defined as the
      absence of a menstrual flow for a period corresponding to the three previous cycles or for
      >90 days (1,20).

      Bloods will be collected and a transvaginal pelvic ultrasound will be performed on all study
      subjects.

      Study A: Six PCOS patients with amenorrhea (cycles >90 days) and negative β-HCG will be
      prescribed daily micronized P4 (400 mg) administrated via the vaginal route for 10 days. Two
      endometrial biopsies will be performed, one between the 5th and 9th day and the other between
      the 21st and 24th day (under the second treatment cycle with P4) of the menstrual cycle (day
      1 being the first day of vaginal bleeding). A third biopsy will be repeated on the 4th cycle
      of P4 treatment, between cycle days 21 and 24 (Fig. 2). This part of the study will be
      designed to assess whether P4 could transform proliferative into secretory endometrium,
      regardless of the number of treatment cycles.

      Study B:

      PCOS: Seventeen PCOS subjects underwent two endometrial biopsies after the first P4-treated
      cycle, the first between days 5-9 of the cycle and the other between days 20-22 (i.e., under
      P4 treatment) of the cycle.

      Controls: Thirteen ovulatory control women under two endometrial biopsies, will be performed
      after the first month of luteal P4 treatment, the first between days 5-9 and the second
      between days 20-22 of the cycle.

      Assays The oral glucose tolerance test (OGTT) consisted of 75 grams of glucose administered
      orally after three days of a carbohydrate-rich diet; blood samples for glucose and insulin
      measurements were drawn before administration of the glucose solution and 30, 60, 90, and 120
      minutes after taking the glucose solution.

      Plasma glucose concentration was determined by the glucose oxidase method. All lipid
      measurements were taken directly in the plasma samples. Total cholesterol and triglycerides
      were assessed using enzyme methods (Roche Laboratories). The HDL-C was quantified by the same
      method and LDL-C was estimated with the Friedwald formula. The glycated hemoglobin were
      measured using high-performance liquid chromatography (with the use of the Tosoh A1c 2.2 Plus
      Glycohemoglobin Analyzer method in 2003-2004 and the Tosoh G7 method in 2007-2008, Tosoh
      Corp).

      For hormone analyses, the blood samples were processed in a centrifuge. Progesterone was
      measured by the immunofluorometric assay (Wallac, Finland) using Auto DELFIA kits;
      androstenedione, PRL, LH, and FSH, by the immunofluorometric assay; dehydroepiandrosterone
      sulfate (DHEAS), by radioimmunoassay (Cisbio International, France and DSL, Texas, USA); and
      insulin and 17-OHP, by radioimmunoassay using DSL kits. The testosterone and SHBG levels were
      measured using the electrochemiluminescent immunoassay (Modular, Roche). The free
      testosterone index was calculated based on the followed formula: total testosterone/SHBG*100.
      Free testosterone was calculated by Vermeulen's formula. All analyses were performed twice
      and the intra-assay and interassay coefficients of variation did not exceed 10% and 15%,
      respectively.

      Endometrial biopsies Endometrial samples will be obtained using the Pipelle de Cornier
      catheter (Laboratoire CCD, Paris, France). Part of the material was embedded in paraffin for
      histomorphometric and immunohistochemical analyses, and the remainder was preserved in RNA
      for later molecular analyses.

      Histochemistry Endometrial samples will be fixed for 24 hrs. in 10% formaldehyde, dehydrated
      in increasing concentrations of ethyl alcohol, cleared in xylene, and then embedded in
      paraffin. The paraffin-embedded samples will be then cut into ten 3-μm sections per subject.
      Eight of these sections were stained for Vascular Endothelial Growth Factor C (VEGF-C) and
      for immunohistochemistry (see below), and the two remaining sections will be stained with
      hematoxylin and eosin (H&E) to confirm the menstrual cycle phase using the criteria suggested
      by Noyes et al. (1953) and morphometry (see below).

      Sections willl be dewaxed conventionally; 0.3% H2O2 will be used to inactivate endogenous
      peroxidase at room temperature for 10 min, followed by washing with distilled water three
      times. The sections were immersed in 0.01 M citrate buffer (pH 6.0) and then placed in a
      microwave oven for heating up to the boiling point. After 10 min, this process will be
      repeated twice; then, the sections were cooled at room temperature and washed twice with
      phosphate-buffered saline (PBS) (pH 7.4). A 5% bovine serum albumin (BSA) blocking solution
      was added at room temperature for 20, min and the excess liquid was discarded. Rabbit
      anti-human VEGF-C polyclonal antibodies (Cell Signaling, Danvers, MA, USA) (VEGF-C: diluted
      at 1:100) were added to the incubation at 37°C for 24 h, and the sections were then washed
      for 5 min. thrice. A biotinylated goat anti-rabbit IgG (Cell Signaling, Danvers, MA, USA)
      working solution will be added at 1:1000 dilution for incubation at 37°C for 20 min. After
      washing, SABC will be added to the incubation at 37°C for 20 min, followed by washing and
      color development via diaminobenzidine (DAB) (DAB color development kit, Dako, Dinamarca) at
      room temperature for 5 to 30 min., followed by washing with distilled water, haematoxylin
      re-staining dehydration via graded ethanol, transparency via xylene, sealing via neutral gum,
      and inspection under light microscope. The primary antibody was replaced with PBS as a
      negative control. The positive sections that stained satisfactorily in the pre-experiment
      were used as positive controls.

      Morphological and histomorphometric analyses To quantify the parameters of interest, images
      were captured using a high-resolution camera (AxioCam-MCR, Carl Zeiss) adapted to a light
      microscope (Axiolab, Carl Zeiss) and adjusted with 40× objective lenses. The images were
      transmitted to a computer with AxioVision Rel 4.2 software (Carl Zeiss). For assessment of
      glandular and surface epithelial thickness, cell count, and counting newly formed blood
      vessels, ten images of each endometrial sample were made for each patient. For VEGF-C
      determination, the numerical density of blood vessels per mm2 will be established.

      Statistical analyses The minimum sample size needed for each group will be determined from a
      pilot study with 10 women per group after pilot study. Thus, in calculating the sample size,
      the correlation between the group measurements will be considered. For the results of the
      number of endometrial glands, a type I error of 5% and a type II error of 20% (sample with
      0.8% power) were adopted for the detection of >20% difference between groups. Commensurate
      with homogeneity, the quantitative data will be expressed as mean ± standard deviation. In
      conformity with data distribution, the Student t test or the Mann-Whitney U test will be
      used. The significance level was set at 95% (α ≤ 5%). Moreover, ANOVA or the Kruskal-Wallis
      test will be used for histologic and immunohistochemical comparisons, when the additional
      group (PCOS during the proliferative phase) was included. Then, in line with data
      distribution, either the Tukey test or the Dunn test will be applied. When possible, the
      subjects of Study A and B were combined for analysis.
    

Intervention name(s): Progesterone 400 Mg Vaginal Suppository on PCOS
Intervention type(s): Drug
Intervention description(s): The same drug will be used in control group
Other intervention name(s): Progesterone 400 Mg Vaginal Suppository on control group
Intervention MeSH terms: Progesterone
Intervention model: Single Group Assignment
Intervention model description: A prospective open-label clinical trial
Masking: None (Open Label)
Primary purpose: Diagnostic
Keywords: PCOS; Progesterone; endometrium