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

Summary

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.

Abstract

Women are born with a non-renewable pool of ovarian follicles, referred to as the ovarian reserve. This reserve consists of primordial follicles in the ovaries and can be affected by many factors, such as genetic and endocrine disorders, medical interventions, and endocrine disruptors. Fertility preservation is recommended when gonadotoxic treatments are necessary. The preferred options for women are oocyte and embryo cryopreservation. However, in very young, sexually immature patients, ovarian tissue cryopreservation is the only option. Knowing the follicular density of cryopreserved tissue samples is essential in fertility counseling for young patients. This protocol demonstrates the use of Z-scores for cortical follicle density as a tool to evaluate the quality of ovarian tissue in girls and young women aged 25 years and below who are undergoing fertility preservation. Follicle density in patient samples is compared to age-normalized reference standards, developed by Hassan et al. to estimate possible deviations from the standard. The follicle density is measured through histological quantification. For this, a small piece of ovarian cortical tissue (~2 mm x 2 mm x 2 mm) is fixed in either Bouin's or formaldehyde solution, embedded in paraffin, sectioned at 4 µm thickness, stained with hematoxylin and eosin, and digitized using a slide scanner. Follicular stages present in the cortex range from primordial to primary follicles. The cortical area was 1 mm from the surface epithelium on histological sections. Follicle density is calculated using a modified formula presented by Schmidt et al., and the Z-score is determined using the reference mean and standard deviation. The Z-score indicates how much the measured value deviates from the reference mean, determining reduced (<-1.7 Z-score) ovarian reserve. With this method, follicle densities can be used as a valuable measure of ovarian reserve for young patients requiring fertility preservation.

Introduction

The ovary is a dynamic, endocrinologically active organ, consisting of an outer cortex and an inner medulla. Heterogeneously distributed within the ovaries are follicles, which contain the gonadal oocyte surrounded by stromal granulosa cells¹. All follicles are already formed during fetal development, peaking at around 20 weeks of gestational age, followed by an exponential decline. At birth, 1-2 million primordial follicles remain within the cortex, and only a few hundred thousand follicles survive until puberty². The non-growing follicle pool, also referred to as the ovarian reserve, influences a woman's fertility,....

Protocol

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 acc.......

Discussion

Women are born with a finite pool of oocytes, which cannot be replenished. Ovarian reserve is a key determinant of a woman's reproductive potential. This article outlines a method for the histological quantification of follicle density and its comparison to reference standards using Z-scores of ovarian cortical follicle densities in young patients and women up to 25 years of age.

In this protocol, ovarian tissue is obtained from girls and women at high or very high risk of infertility unde.......

Materials

Name	Company	Catalog Number	Comments
1x Dulbecco's Phosphate Buffered Saline	Thermo Fisher	14190144
1x Phosphate Buffered Saline w/ CaCl2, MgCl2, 1 g/L D-glucose, 36 mg/L pyruvate	Thermo Fisher	14287-080
37% Hydrochloric acid	Sigma-Aldrich	1.09057.1000
70% ethanol	Histolabs	01370.01L
95% ethanol	Fisher scientific	10572143
Absolute ethanol	Histolabs	01399.01L
Bouin's solution	Sigma-Aldrich	HT10132-1L
Eosin Y Free acid	Sigma-Aldrich	E4009-5G
Formaldehyde	Thermo Fisher	28908
Haematoxylin solution according to Delafield	Sigma-Aldrich	03971-250ML
ImageJ
Microsoft Excel
Microtome
Pertex mounting solution	Histolabs	00840-05
Propofol	Baxter Holding B.V.	N01AX10	ATC-code provided instead of catalog number
QuPath			https://qupath.github.io/
Rocuronium bromide	B. Braun Melsungen AG	M03AC09	ATC-code provided instead of catalog number
Scott's tap water substitute concentrate	Sigma-Aldrich	S5134-6x100ml
Sevoflurane	Baxter Medical AB	N01AB08	ATC-code provided instead of catalog number
Slide scanner
Sufentanil	Hameln Pharma gmbh	N01AH03	ATC-code provided instead of catalog number
Sugammadex	Baxter Holding B.V.	V03AB35	ATC-code provided instead of catalog number
SuperFrost plus white	Histolabs	06400
SuperFrost Plus White microscope slides	Histolabs	06400
Xylene	Saveen Werner	131769.1611