Title of the study: Comparative Analysis of Stromal Fibrosis and Ovarian Reserve in Premenopausal and Postmenopausal Women Undergoing Oophorectomy: an observational  study. 

Novelty/ Innovation

3. Understanding the role of stromal fibrosis in ovarian aging could have implications for fertility preservation, hormone replacement therapy, and predicting ovarian function decline, especially in premenopausal women undergoing oophorectomy for benign conditions. 

 sample size. 

we used a study that had assessed the ovarian AFC and serum levels of AMH correlation with the ovarian primordial follicle number (1). They found that correlation between the ovarian primordial follicle count and AMH was 0.48.

Considering a correlation coefficient of 0.48, 5% significance level, 80% power, and two tailed test, the required sample size is 29 participants per group, calculated with the help of the following formula.

Methods:

1. Recruitment and Data Collection:

         Informed consent will be obtained from all participants

         Detailed medical history, including menstrual history, family history of ovarian diseases, and comorbid conditions, will be collected.

        Participants will undergo clinical assessment to confirm their perimenopausal status, including serum FSH, AMH, Estradiol and AFC and ovarian volume, ovarian stromal blood flow measurement via ultrasound.

2. Ovarian Tissue Collection

    Oophorectomy (unilateral or bilateral) will be performed under standard surgical protocols for indicated benign condition

    Ovarian tissues will be preserved for histopathological examination.

3. Histopathological Examination of Stromal Fibrosis:

     The primary pathological features of ovarian fibrosis are a thick capsule, increased mesenchymal connective tissue, and decreased or absent follicles (2)

     Representative tissue samples (entire cross-section of the ovary) from the included cases will be fixed in either 10% Formalin and will be processed as per standard protocol.

      Haematoxylin and eosin (H&E) stained slides and Masson trichrome stained slides of each case will be evaluated for the degree of stromal fibrosis using a semi-quantitative scale  (Grade 0: No fibrosis; Grade 1: Mild fibrosis; Grade 2: Moderate fibrosis; Grade 3: Severe fibrosis) depending upon the area of fibrosis (3, 4).

            Other histopathological findings will also be documented

    Next, only the cases that show grade 1 to 3 fibrosis, will be stained using a Picrosirius Red solution, which consists of Sirius Red F3B dye dissolved in a saturated aqueous solution of picric acid.

 After staining, the sections will be washed in acidified water (0.5% acetic acid) to remove excess stain.

      The stained slides will be analysed under bright-field microscopy, wherein the collagen will appear red against a yellow background.

       The stained slides will also be analysed under polarized light microscopy, wherein the birefringent properties of collagen will differentiate thick fibres (type I, appearing yellow-orange) from thin fibres (type III, appearing green).

         Semi-quantitative assessment of degree of type 1 fibres and type III fibres will be done (4).

         Fibre orientation, whether the collagen fibres are randomly arranged or aligned in a specific direction, will also be documented.

4. Biochemical Markers of Ovarian Reserve

Anti-Müllerian Hormone (AMH)

Follicle-Stimulating Hormone (FSH)

Antral Follicle Count (AFC) and Ovarian volume-