We wanted to assess ovarian reserve, based on small ovarian samples in patients aged up to 25 years old that require fertility preservation through ovarian tissue cryopreservation. Using a modified formula that estimates a total number of follicles in ovarian samples and the model for normal ovarian volume, we establish a reference dataset for follicle densities in women aged zero to 25 years old. Assessing the normality of follicle density is difficult because of one, the natural decline of ovarian reserve with increasing age, and two, the heterogeneous distribution of follicles.
Comparison to reference values is also challenging because they're not standardized to age and biopsy size, so we're trying to bridge this gap. Our protocol describes the strategy for assessing the normality of follicle density, based on small ovarian samples. We directly quantify these follicles using histology, and compare the results to age-normalized standard references via Z-scores.
With this tool, meta-analysis become possible because we can compare data from multiple studies. It is important to know if the ovarian reserve is low. We focus on identifying patients with reduced ovarian reserve and suggest a cutoff value for that.
This is of great value for physicians in counseling their patients for auto-transplantation of their cryopreserved ovarian tissue, as well as for further treatment after fertility preservation. 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 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 end 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 eight 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 Okay.
Then click on Image, then adjust brightness contrast, and select Auto. Now, click on Image, then adjust threshold and select Auto. Click on the one tool, select the area, and then press Ctrl M.Input the area value into the follicle Z-score calculator Excel file 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.
