Cryopreservation of Oocytes Retrieved from Ovarian Tissue to Optimize Fertility Preservation in Prepubertal Girls and Women

Published: October 23, 2020

doi:

Sophie Brouillet², Alice Ferrieres-Hoa, Alice Fournier, Guillaume Martinez, Julien Bessonnat, Aurore Gueniffey, Anna Gala, Vanessa Loup, Samir Hamamah²

¹Développement Embryonnaire Précoce Humain et Pluripotence,Univ Montpellier, INSERM 1203, ²Département de Biologie de la Reproduction, Biologie de la Reproduction/DPI et CECOS,CHU Montpellier, Univ Montpellier, ³Centre d’AMP-CECOS,CHU Grenoble-Alpes, Univ Grenoble-Alpes

Summary

We propose a protocol for fertility preservation in prepubertal girls and women at risk of premature ovarian insufficiency. It combines ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue. This strategy improves the safety and optimizes the reproductive potential of fertility preservation, maximizing the chance of childbirth.

Abstract

Human ovarian tissue cryopreservation (OTC) is increasingly used worldwide to preserve female fertility in prepubertal girls and women at risk of premature ovarian insufficiency (POI) in the context of urgent gonadotoxic treatments or ovarian surgery. Fertility preservation is challenging because there is no consensus regarding patient management, preservation fertility strategies, or even technical laboratory protocols, which implies that each procedure must be adapted to the characteristics of the patient profile and its own risk-benefit ratio. During OTC, mature/immature oocytes can be aspirated directly from large/small antral follicles within ovarian tissue samples and/or be released into culture media from growing follicles during ovarian tissue dissection in prepubertal girls and women. In this manuscript, we present a protocol that combines ovarian tissue freezing with the cryopreservation of mature/immature oocytes retrieved from ovarian tissue samples, improving the reproductive potential of fertility preservation. Appropriate collection, handling, and storage of ovarian tissue and oocytes before, during, and after the cryopreservation will be described. The subsequent use and safety of cryopreserved/thawed ovarian tissue samples and oocytes will also be discussed, as well as the optimal timing for in vitro maturation of immature oocytes. We recommend the systematic use of this protocol in fertility preservation of prepubertal girls and women as it increases the whole reproductive potential of fertility preservation (i.e., oocyte vitrification in addition of OTC) and also improves the safety and use of fertility preservation (i.e., thawing of oocytes versus ovarian graft), maximizing the chance of successful childbirth for the patients at risk of POI.

Introduction

The field of fertility preservation has grown over the last two decades due to the increasing number of patients at risk of premature ovarian insufficiency (POI)¹^,²^,³. The current available medical options to preserve fertility are ovarian tissue cryopreservation (OTC)⁴, oocyte/embryo freezing after ovarian stimulation⁵, administration of GnRH analogues⁶, or ovarian transposition⁷. OTC is a major advance for fertility preservation, particularly in prepubertal girls, where it is the only option currently available to preserve fertility and also in women who cannot delay the onset of their gonadotoxic treatment²^,⁴.

OTC allows the preservation of a high number of primordial follicles, which are located in the outer 1 mm of the ovarian cortex². Frozen/thawed ovarian tissue can be subsequently used by graft (orthotopic or heterotopic, autologous or donor) or cultured in vitro to obtain mature oocytes². The graft of frozen-thawed prepubertal ovarian tissue samples has been shown to induce puberty⁸^,⁹. In women, the reproductive outcomes after orthotopic autografting of frozen-thawed ovarian cortex are reassuring, with live birth rates reaching 57.5% after natural conceptions¹⁰ and between 30% to 70% after Assisted Reproductive Techniques (ART) conceptions¹¹. Since the first live birth from orthotopic transplantation of frozen/thawed human ovarian tissue in 2004¹², this technique allowed the birth of at least 130 children worldwide². The hormonal and reproductive functions of the graft tissue generally last for several years¹¹^,¹³^,¹⁴, confirming its long-term functionality.

However, auto-transplantation of ovarian tissue samples carries a theoretical risk of reintroducing viable malignant cells in some patients¹⁵^,¹⁶^,¹⁷^,¹⁸, particularly in leukemia survivors¹⁹. To date, no case of transmission of cancer via the graft of frozen/thawed ovarian cortex has been reported in healthy cancer survivors¹¹, suggesting that the fibrous avascular nature of the ovarian cortex could represent an inhospitable microenvironment for the dissemination of malignant cells. Nevertheless, the graft of ovarian tissue still represents an experimental and challenging technique, indicating that the use of oocytes should be currently considered as an easier and safer approach than ovarian tissue graft to restore fertility. Interestingly, immature oocytes could be easily retrieved from ovarian tissue during OTC in both prepubertal girls and women¹⁶, suggesting that it could represent a reliable source to maximize the fertility-restoring potential in addition to ovarian cortical tissue freezing. These oocytes could be aspirated manually ex vivo in the ART laboratory from visible antral follicles or be isolated from spent media after ovarian tissue dissection. Retrieved oocytes could then be directly vitrified at an immature stage or be matured before the vitrification step using in vitro maturation (IVM)²⁰^,²¹.

In this manuscript, we propose a protocol that combines the cryopreservation of ovarian tissue with the isolation and cryopreservation of mature (MII-stage oocytes) and/or immature oocytes (i.e., Germinal Vesicle (GV) and Metaphase I (MI)-stage oocytes) retrieved from ovarian tissue. This protocol outlines all the specific steps required to maximize the fertility preservation potential in both prepubertal girls and women.

Protocol

All women (over 18 years of age) as well as all minor girls and their parents signed an informed consent form to preserve the fertility of the patient at risk of POI. This protocol is considered as a routine ART procedure in our center for fertility preservation. It follows the guidelines of our institutions human research ethics committee. 1. Quality control Include female age ≤ 38 years, containing circulating anti-mullerian hormone concentration >1 ng/mL and showing th…

Representative Results

A total of 81 OTC from 81 female patients have been performed between 2007 and 2020, including 43 prepubertal girls and 38 women. The mean age of female patients (prepubertal girls and women) was 14.21 ± 9.61 years (mean ± standard error). The youngest patient was 5 months old and the oldest was 33.6 years old (Table 1). The mean age of prepubertal girls and women was 6.84 ± 4.81 years old (min–max: 0.5–15.1) and 22.76 ± 5.92 years old (min–max: 14.2–33.6), res…

Discussion

The current manuscript provides a protocol combining ovarian tissue freezing and cryopreservation of oocytes retrieved from ovarian tissue, increasing the potential of fertility in prepubertal girls and women at risk of POI. We strongly recommend performing this protocol before the start of any gonadotoxic therapy in order to optimize the quantity (i.e., number of viable oocytes) as well as the quality (i.e., DNA integrity and cytoplasm competence) of the preserved oocytes, optimizing their safety for clinical use<sup cl…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

We thank all members of our centers involved in the activity of fertility preservation (Gynecologists, Biologists, Oncologists, and Anatomopathologists). The study was conducted as part of the routine procedures for fertility preservation. No funding was received.

Materials

1 ml disposable syringe	CDD	1323101/7002655	Other material and sizes may also be suitable
21-gauge syringe needle	Merck	Z192481	Other material and sizes may also be suitable
35 mm IVF Petri Dish	Nunc	150255	Other material and sizes may also be suitable
60 mm IVF Petri Dish	Nunc	150270	Other material and sizes may also be suitable
90 mm IVF Petri Dish	Nunc	150360	Other material and sizes may also be suitable
Atraumatic forceps	Medlane	PI 299 04	Other material and sizes may also be suitable
Continuous Single Culture Complete with HSA	Irvine Scientific	90165	IVF culture medium for follicular fluid collection, COCs incubation, oocyte denudation and oocyte incubation until the vitrification step.
Cryotube	Thermo Scientific	368632	Other products may also be suitable
Dimethylsulfoxide (DMSO)	MILTENYI BIOTEC SAS	170-076-303	CryoMACS DMSO 10 (EP)
GT40	Air Liquide	1,13,517	Storage tank
HSV High Security Vitrification Straw	Irvine Scientific	25251	Vitrification straws
Human serum albumin	Vitrolife	10064	Other products may also be suitable
Leibovitz L15 medium	Eurobio	CM1L15000U	Culture medium for ovarian tissue collection, transport and tissue dissection
Leibovitz L15 medium	Eurobio	CM1L15000U	Culture medium for freezing solution
Mars-IVF Class II Workstation/L126 IVF Dual	CooperSurgical	WM1500/6-133-911-121	Workstation
Programmable freezer	Planner KRYO 500	Kryo 560-16	Other equipements may also be suitable
Scissors with sharp straight blades and finely sharpened points	Medlane	CI 034 03	Other material and sizes may also be suitable
Stripper	CooperSurgical	MXL3-STR-CGR	Other products may also be suitable
Tips (150µm) for Stripper	CooperSurgical	MXL3-150	Other products may also be suitable
Vitrification Kit	Irvine Scientific	90133	Protocols are available at http://www.irvinesci.com/products/90133-so-vitrification-freeze-solutions. Other products may also be suitable

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