Z-Scores for Assessing Ovarian Reserve in Young Patients Undergoing Fertility Preservation

All of the procedures conducted were approved by the Swedish Ethical Review Authority (Sveafertil patients, Dnr: 2019-03802) and by the Ethics Committee of Helsinki University Hospital (HCH patients, Dnr 340/13/03/03/2015). Written and oral information about the study were provided by the research nurses, and informed consent was obtained in accordance with the Declaration of Helsinki. For patients under 18 years old, informed consent was obtained from the legal guardians. All samples were pseudonymized and processed according to the European General Data Protection Regulation (GDPR). The details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Ovarian tissue collection and processing

NOTE: Identify all eligible patients for fertility preservation when there is an indication for a high or very high risk of infertility following local guidelines (e.g., Nordic, European, or international specific guidelines). In Europe, recruit underage patients only as part of fertility preservation research programs. Determine the risk of infertility using your country's guidelines for fertility preservation.

1. Perform the surgical procedure under general anesthesia. Administer intravenous propofol (2 mg/kg), sufentanil (0.5 microgram/kg), and rocuronium bromide (0.6 mg/kg) for the induction of anesthesia.
   1. Intubate and ventilate the patient with a tidal volume of 6-8 mL/kg and positive expiratory pressure of 6-8 cm of H₂O. Deliver sevoflurane 1% to 2% via the endotracheal tube to maintain inhalation anesthesia.
   2. Maintain neuromuscular blockade with rocuronium bromide (0.15 mg/kg) during the surgical procedure to provide adequate muscle relaxation and to allow laparoscopy at lower intra-abdominal insufflation pressures.
   3. Use bispectral index to guide anesthetic depth to facilitate rapid awakening with a reduced anesthetic dose. Antagonize residual neuromuscular block with sugammadex (2-4 mg/kg).
2. Using a three-port technique, insert a 10 mm trocar into the peritoneal cavity at the umbilicus to introduce an optical lens with a camera attached, and two 5 mm trocars at the left and right iliac regions (2 cm above and 2 cm medial to the respective anterior superior iliac spine) under direct vision for the surgical instrumentation and tissue retrieval. Inspect the pelvic organs, including both ovaries, to confirm normality.
   1. Identify the ovary that is easiest to access, then grasp it. Cut a volume that is approximately one-third of the ovary (or as indicated in your ethical permit), then use diathermy, if needed, to secure hemostasis.
      NOTE: Perform the biopsy only if there are no contraindications, such as risk factors for excessive intra- or post-operative hemorrhage or increased risk of infection. Specify clearly and follow the country's inclusion and exclusion criteria. The entire ovary can be taken if the risk of infertility is very high and/or the risk of cutting a part only is considered too high (e.g., due to bleeding). Avoid using diathermy before collecting the sample because it can damage the tissue.
3. While in the operating theater, place the tissue in a tube containing 5 mL of 1x Dulbecco's Phosphate Buffer Saline (PBS) with CaCl₂, MgCl₂, 1 g/L D-glucose, 36 mg/L pyruvate, either warmed to 37 °C if the sample is immediately processed in the laboratory (within 30 min) or kept at 4 °C during longer transports (up to 24 h).
   NOTE: Transportation solutions include an array of commercially available organ transportation/perfusion solutions as well as simple buffers such as PBS and MEM³⁴.
4. Cut a small piece of the ovarian tissue (2 mm x 2 mm x2 mm) from the surface of the ovary. This piece contains the cortex, which is the outer portion of the ovary where the ovarian follicles are found³⁵.
   CAUTION: Handle human tissues such as the ovary using Biosafety Level 2 practices and procedures.
   NOTE: A specialized reproductive medicine unit clinically cryopreserves the rest of the ovarian tissue for potential future use in fertility restoration of the patients.
5. Place the tissue in 350 µL Bouin's solution at room temperature for 2 h or in 350 µL of 4% formaldehyde at 4 °C overnight, ensuring that the tissue is fully submerged. After incubation, wash the tissue with 70% ethanol three times.
   1. For Bouin's solution, wash until the yellow color disappears. Embed the tissue in paraffin wax³⁶. Cut a minimum of 30 serial tissue sections with a thickness of 4 µm and place them on glass slides.
      CAUTION: Handle Bouin's solution and formaldehyde inside a chemical hood and with proper protective equipment.
      NOTE: Formaldehyde might lead to shrinkage-related artifacts in the tissue. Bouin's solution is therefore the preferred choice for preserving histology. However, Bouin's solution carries additional risks, leading several institutions to prefer the use of formaldehyde³⁷.

2. Hematoxylin and Eosin (H&E) staining

1. Weigh 5 g of eosin Y powder into a 100 mL autoclaved glass bottle. Add 800 mL of 95% ethanol and 200 mL of deionized water to make a 0.5% eosin stock solution. Prepare a 0.25% eosin working solution by diluting 200 mL of the 0.5% eosin stock solution with 160 mL of 100% ethanol, 1 mL of acetic acid, and 40 mL of deionized water.
   NOTE: The powder may not dissolve completely. Filter the solution before use to reduce precipitate.
2. Place slides in racks. Immerse the rack with slides into the corresponding containers in the following order: 15 min in xylene, 5 min in 100% ethanol, dip 10 times in 70% ethanol, dip 10 times in 50% ethanol, and then dip 10 times in deionized water.
   1. Immerse the rack with slides in hematoxylin solution for 3 min, then transfer the rack with slides to a container with distilled tap water for 30 s to wash away excess hematoxylin stain.
   2. Place the rack with slides into a container with differentiation solution (1 mL of 37% HCl added to 100 mL of 70% ethanol) for 1 min, then transfer the rack with slides to a container with distilled tap water for 30 s.
      NOTE: Ensure that the tissue sections are submerged in the liquid. Add more reagent to the container if needed. Change xylene after every 5 uses when staining large amounts. Prepare 50% ethanol by diluting 70% ethanol (e.g., add 35 mL of 70% ethanol to 15 mL of DPBS to make 50 mL of 50% ethanol).
      CAUTION: Handle xylene inside a hood and with proper protective equipment.
3. Place the rack with slides in Scott's tap water (also known as bluing solution) for 30-60 s, then transfer the rack with slides to a container with distilled tap water for 30 s. Immerse the rack with slides in the eosin working solution for 1 min.
   1. Dehydrate the slides by placing them in the following reagents: 100% ethanol for 30 s, a second container with 100% ethanol for 30 s, and then dip twice in xylene. After removing the rack from the xylene solution, let it dry in a glass container to prevent xylene from dripping inside the hood.
   2. Mount the slide with 2 drops of mounting solution. Carefully place the cover slides to avoid bubbles and let the mounting solution dry.
      CAUTION: Handle xylene inside a hood and with proper protective equipment.
4. Digitize slides by scanning them using slide scanners if possible. If this option is not available, use a regular inverted microscope at 20-40x magnification to directly count follicles. Ensure to take an image of the entire biopsy to facilitate area measurement in image analysis software.

3. Follicle counting

1. If slides were digitized, open the files using appropriate software (e.g., open-source software QuPath³⁸). If not digitized, perform the analysis directly using an inverted microscope and its respective software.
   1. Drag the file onto QuPath's main window. Set the image type to Brightfield H&E (see Figure 1A). Define 1 mm from the surface epithelium (a single layer of cuboidal cells lining the surface) using the scale bar. Click on the ellipse tool and annotate the follicles (see Figure 1B).
   2. Count the number of follicles within this 1 mm area, from the primordial to primary stage. Identify the stage of each follicle (primordial – a layer of flattened granulosa cells surrounding the oocyte; intermediary stage – a mixture of flattened and cuboidal granulosa cells; primary – one full layer of cuboidal granulosa cells surrounding the oocyte).
   3. Input the sample ID, age, number of follicles for each slide, section thickness, and n^th section counted into the sheet named Follicle in mm³ on the Follicle Z-score calculator Excel file (see Supplementary File 1 and Figure 2A).
      NOTE: Due to the diameter of follicles (smallest follicles >35 µm), the same follicle might appear in consecutive sections. Exercise extra caution when counting sections where the distance between sections is less than the follicle diameter. Preferably, count follicles from every 10^th section (e.g., section #10, #20, #30). The surface epithelium may detach during tissue processing and might only be partially present in the slides. Count only follicles with visible cytoplasm. For quality control, have two people independently count the follicles and determine how well their counts align. Calculate inter-observer concordance using a simple correlation function between the follicle counts of each observer for the same sample.
2. Identify the largest 10% of the follicles in each counted section by eyeballing. Measure the mean diameter of these oocytes by clicking on the line tool, drawing two perpendicular lines through the oocyte membrane, and selecting Measure, then Show annotation measurements (see Figure 1C).
   1. Record the measurements under the column Length µm (see Figure 1D). Input the mean diameter of all oocytes into the Mean oocyte diameter (µm) field for each slide in the Follicle Z-score calculator Excel file (Follicle in mm³, see Supplementary File 1 and Figure 2A).
3. Place the image in the center of the screen in QuPath, then click on the ImageJ icon. Select Send snapshot to ImageJ (see Figure 3A). In ImageJ, demarcate 1 mm from the border using the brush tool. Click on the Image, then Type, and select 8-bit (see Figure 3B).
   1. Click on the line tool, draw a line on the scale bar at the lower left of the screen (see Figure 3C). Click on Analyze, then Set Scale, input the length indicated in the scale bar in the Known Distance field, and click on OK (see Figure 3C). Click on Image, then Adjust, Brightness/Contrast, and select Auto (see Figure 3D).
   2. Click on Image, then Adjust, Threshold, and select Auto (see Figure 3E). Click on the wand tool, select the area, then press Ctrl + M (see Figure 3F). Input the area value into the Follicle Z-score calculator Excel file (Follicle in mm³, see Supplementary File 1 and Figure 2A).

4. Follicle density, cortical volume, and Z-score calculations

1. Use the Follicle Z-score calculator Excel file provided to automatically perform the calculations outlined below. The results will appear in the sheet "Z-score (follicle per mm³)" in the Excel calculator (see Supplementary File 1), provided that the details indicated in section 3 are entered into the sheet "follicles in mm³."
2. To calculate follicle densities in these sections, use the modified formula presented by Schmidt et al.³². First, apply the correction factor α to account for double counting of the same follicle. The modified formula is as follows:
   
   
   NOTE: In this formula, t is the section thickness, d is the diameter of the oocyte, and q is the quotient of the follicle diameter divided by section thickness.
3. Extrapolate the total number of follicles (n _follicles) in the cortex from the follicle count in the section using the following formula³³:
   
   NOTE: This formula also accounts for all of the non-calculated sections. Here γ represents the number of sections that are not calculated (e.g., γ = 10 if every 10^th section is counted), α is the correction factor calculated in 4.1, N is the number of sections counted, and n _i is the total number of follicles in the counted sections.
   1. Extrapolate the whole cortical volume (V) from the counted areas using the following formula³³:
      
      ​NOTE: Here, A_i is the area of the section, and t_i is the tissue thickness. Please check step 4.2 for γ and N.
   2. Use the following formula³³ to calculate the follicle density:
      
4. Determine if there is reduced (<-1.7 Z-score) ovarian reserve by calculating the Z-score using the formula³³:
   
   NOTE: The Z-score is automatically calculated in the sheet "Z-score (follicle per mm³)" in the Excel calculator (see Supplementary File 1) based on information provided in Section 3 (Follicle Counting) (Figure 2B). In this formula, Z represents the Z-score, x is the calculated follicle density (follicles/cm³) of the study sample (step 4.4), M is the mean follicle density of the respective age group, and SD is the standard deviation of the respective age group. Reference values for women aged 0-25 years have been established in Hassan et al.³³. Means and standard deviations of follicle densities of the respective age groups can be found in the Follicle Z-score calculator Excel file (Supplementary File 1) and used for the Z-score calculations.