Z-Scores for Assessing Ovarian Reserve in Young Patients Undergoing Fertility Preservation
October 25th, 2024
The present protocol describes a method for assessing ovarian reserve in patients under 25 years old who require fertility preservation through ovarian tissue cryopreservation. This method involves: (1) histological assessment of ovarian reserve in cortical samples, (2) comparison to a reference dataset, and (3) calculation of Z-scores.
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.
Overview
This protocol outlines a method for evaluating ovarian reserve in patients under 25 years old requiring fertility preservation through ovarian tissue cryopreservation. It includes histological assessment of ovarian samples and comparison to a reference dataset to calculate Z-scores.
Key Study Components
Area of Science
- Reproductive biology
- Fertility preservation
- Histological assessment
Background
- Ovarian reserve assessment is crucial for fertility preservation.
- Follicle density varies with age and is difficult to standardize.
- Existing reference values are not age-standardized.
- This study aims to bridge the gap in assessing follicle density.
Purpose of Study
- To assess ovarian reserve in young patients.
- To establish a reference dataset for follicle densities.
- To improve the accuracy of ovarian reserve assessments.
Methods Used
- Histological quantification of follicles in ovarian samples.
- Comparison of results to age-normalized standard references.
- Calculation of Z-scores to assess normality of follicle density.
- Use of a modified formula for estimating total follicle numbers.
Main Results
- Establishment of a reference dataset for women aged 0-25.
- Successful quantification of follicles in small ovarian samples.
- Identification of challenges in standardizing assessments.
- Demonstration of the method's effectiveness in assessing ovarian reserve.
Conclusions
- The protocol provides a reliable method for assessing ovarian reserve.
- It addresses the need for standardized reference values.
- This approach can aid in fertility preservation strategies for young patients.
Frequently Asked Questions
Executive Industry Relevance
Quantitative assessment of ovarian reserve in young patients is critical for fertility preservation decisions, especially when only small tissue samples are available. The use of Z-scores for follicle density enables standardized, age-adjusted evaluation, supporting predictive confidence in tissue quality for future reproductive options. This approach enhances portfolio-level decision-making for fertility preservation programs and translational research in reproductive biology.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables objective interrogation of ovarian reserve status using histological quantification and statistical normalization.
- Supports biological de-risking by identifying reduced ovarian reserve through validated Z-score thresholds.
- Facilitates predictive confidence in tissue selection for fertility preservation interventions.
Screening & Assay Development
- Standardizes follicle density measurement across small, heterogeneous ovarian samples.
- Provides reproducible, quantitative outputs for cross-study and cross-cohort comparisons.
- Enables assay readiness for integration into broader fertility preservation workflows.
Translational & Preclinical Research
- Aligns histological outputs with clinically relevant thresholds for ovarian reserve assessment.
- Supports continuity from tissue collection through preclinical validation of preservation strategies.
- Reduces mechanistic ambiguity in evaluating tissue suitability for auto-transplantation or further treatment.
Pipeline & Workflow Integration
This Z-score-based method positions ovarian reserve assessment at the intersection of discovery biology, screening, and translational research for fertility preservation in young patients.
- Discovery Biology: Provides a framework for hypothesis testing regarding ovarian reserve status and age-related decline.
- Screening: Delivers standardized, quantitative follicle density outputs for reliable sample evaluation.
- Analytics: Enables statistical comparison of patient samples to age-normalized reference datasets.
- Translational Research: Bridges discovery findings with clinical counseling and tissue selection for fertility preservation.
- Enterprise Reuse: Establishes a reusable, reference-based capability for ovarian tissue assessment across studies and cohorts.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces biological risk in fertility preservation decisions.
- Operational Value: Promotes standardization, reproducibility, and scalability of ovarian reserve assessment.
- Strategic Value: Informs go/no-go decisions for tissue cryopreservation and transplantation, optimizing resource allocation.
- Portfolio Impact: Supports risk-adjusted prioritization of patients and tissue samples for fertility preservation programs.
Implementation Considerations
- Requires expertise in histological analysis and digital image quantification.
- Depends on access to slide scanning, image analysis software, and validated reference datasets.
- Necessitates cross-team standardization of tissue processing and data entry protocols.
- Adaptation may be needed for different age groups or tissue types based on reference availability.
- Accuracy is influenced by sample size, sectioning consistency, and adherence to measurement protocols.
