Progesterone Action on Endometrium of PCOS

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.