To begin, collect the ovarian tissue from the operating theater after biopsy in a tube containing five milliliters of supplemented 1X DPBS. Using a sterile scalpel, cut a small piece of ovarian tissue of about two-millimeter cubes from the surface of the ovary. Then submerge the tissue in 350 microliters of Bouin's solution at room temperature for two hours.
After incubation, wash the tissue with 70%ethanol three times. After embedding and sectioning the tissues, perform hematoxylin and eosin staining and acquire digital images for analysis. Open the files using appropriate software such as QuPath.
Drag the file onto QuPath's main window and set the image type to Brightfield H&E. Define one millimeter from the surface epithelium using the scale bar. Click on the ellipse tool and annotate the follicles.
Count the number of follicles within this one-millimeter area from the primordial to the primary maturation stage. Input the sample ID, age, number of follicles for each slide, section thickness, and N section counted into the sheet named Follicle in millimeter cube in the Follicle Z-Score Calculator Excel file. After identifying the largest 10%of the follicles in each counted section, click on the line tool and draw two perpendicular lines through the oocyte membrane.
Select Measure, then click on Show annotation measurements to determine the mean diameter of these oocytes. Record the measurements under the column labeled length in micrometers. Input the mean oocyte diameter of the largest 10%follicles in micrometers for each slide in the Follicle Z-Score Calculator Excel file.
Place the image in the center of the screen in QuPath and click on the ImageJ icon. Then select Send snapshot to ImageJ. In ImageJ, demarcate one millimeter from the border using the brush tool and click on Image, then Type, and select 8-bit.
Next, click on the line tool and draw a line on the scale bar. Select Analyze, then Set Scale, input the length indicated on the scale bar in the Known distance field and click OK.Then click on Image, then adjust Brightness/Contrast, and select Auto. Now click on Image, then adjust Threshold and select Auto.
Click on the wand tool, select the area, and then press Ctrl M.Input the area value into the Follicle Z-Score Calculator Excel file. Once all data are tabulated in the Follicle Z-Score Calculator Excel file, Go to the sheet named Z-Score Follicle Per Millimeter Cube in the same Excel file to determine if there is reduced ovarian reserve. The surface epithelium of a child's ovary was clearly visible, with the ovarian reserve residing beneath it after the staining procedure.
Follicle density was used to calculate the ovarian reserve Z-scores, with a Z-score less than negative 1.7 indicating reduced ovarian reserve.
