Preservation of Women's Fertility: Evaluation of Innovative Methods for Ovarian Tissue Cryopreservation

Recruiting

• Conditions: Infertility, Female

• Registration Number: NCT06724471
• Lead Sponsor: University Hospital, Clermont-Ferrand

Brief Summary

The recent innovations in cancer diagnosis and therapy have improved the five-year survival for patients. However, anticancer treatments may impair patient fertility; therefore fertility preservation is recommended before therapy initiation. The sole method for preserving young prepubescent girls' fertility, which can also be used for pubescent women, is ovarian tissue cryopreservation (OTC) with later auto-transplantation. Although 200 births have been reported worldwide after OTC and transplantation, significant improvements are required. Indeed, freezing and thawing protocols vary according to laboratories (media, cryoprotectants, freezing curve, etc...) and selection criteria are not justified. In addition, most of the laboratories use unsafe devices (e.g. screw cap cryovials) for OTC, exposing ovarian tissues to biological hazards during sample storage in nitrogen tanks. To eliminate these risks, novel "high security" devices have been commercialized (welded cryotubes). However, while thermal welding could alter tissue quality, the functionality of the human ovarian tissue frozen with these innovative devices has not yet been evaluated. The objectives of our study are i) to optimize the freezing and the thawing protocols for human OTC according to thermodynamic properties of the freezing medium and the type of device (welded or screwed cryotube) and ii) to determine if the type of cryotube influences the quality of human ovarian tissue.

This project will enable to reach a better understanding of the impact of freezing on ovarian tissue functionality, as well as the implementation of an optimal protocol for OTC within the ART laboratory of Clermont-Ferrand hospital to optimize patient care.

Detailed Description

The aim of this project is to determine whether the type of cryogenic tube (with screw caps or thermosoldered) influences the quality and functionality of ovarian tissue after cryopreservation. First, a characterization of the thermodynamic properties of the freezing medium using a differential scanning calorimeter (Diamond DSC, PerkinElmer) will be carried out to optimize freezing and thawing protocols. This analysis will define important parameters such as crystallization temperature (Tc), end-of-melting temperature (Tm), and transition temperatures (Tg'1). Once freezing and thawing protocols are optimized, we will use human ovarian cortex surrounding benign cysts, a model previously validated by our laboratory, to determine whether the type of cryotube influences the quality of human ovarian tissue. These ovarian cortical samples usually destroyed in clinics share similar characteristics with normal ovarian cortex. Ovarian tissue will be cut into 1mm3 fragments and divided into three groups: fresh ovarian cortex (group1, control), ovarian cortex cryopreserved in thermosoldered cryogenic tubes (group 2) or in screw cap (group 3). We will assess ovarian tissue quality immediately after resection for group 1 or after thawing for group 2 and 3 by analyzing follicle density and morphology (HES staining) and proliferation/apoptosis balance (Immunohistochemistry (IHC) for KI67 and cleaved caspase 3). To assess the functionality of the ovarian tissue after thawing, ovarian cortex fragments will be in vitro cultured. We will analyze i) follicle density, type and morphology (HES staining), ii) the proliferation/apoptosis balance (IHC for KI67 and cleaved caspase 3), iii) the expression levels of major folliculogenesis regulators (IHC for GDF-9) and iv) the levels of folliculogenesis-associated hormones (AMH, estrogen) secreted in culture medium.

Study Timeline

• Study Start: 2022-05-05
• Status Verified: 2024-03
• Study First Submitted: 2024-12-05
• Study First Submitted QC: 2024-12-05
• Last Update Submitted: 2024-12-05
• Study First Posted: 2024-12-09
• Last Update Posted: 2024-12-09
• Primary Completion: 2025-06-01
• Study Completion: 2025-12-31

Recruitment & Eligibility

• Status: RECRUITING
• Sex: Female
• Target Recruitment: 30

Inclusion Criteria

• Adult woman scheduled for benign ovarian cyst resection (dermoid, functional, mucinous or serous cysts)
• Below 37 years old and above 18 years old
• Capable of giving written informed consent to participate in the research study
• Affiliated to social welfare service

Exclusion Criteria

• Women above 37 years old and below 18 years old
• Polycystic ovary syndrome
• Diminished ovarian reserve
• Severe endometriosis
• Malignant and endometrial cysts

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Primary Outcome Measures

Name	Time	Method
Post-thawing Ovarian Follicle morphology	24 months	Follicle morphology of fresh versus frozen (with thermosoldered or screwed cryovials)/thawed ovarian cortex will be assessed on serial sections stained with hematoxylin-eosin-saffron. Fragments will be fixed on the day of collection for group 1 and directly after thawing for groups 2 and 3. Morphology of follicles will be evaluated according to the parameters described by Keros et al. Hum Reprod. 2009.

Secondary Outcome Measures

Name	Time	Method
Post-thawing Ovarian Follicle density	24 months	Follicle density (=number of follicle/mm2) of fresh versus frozen (with thermosoldered or screwed cryovials)/thawed ovarian cortex will be assessed on serial sections stained with hematoxylin-eosin-saffron. Fragments will be fixed on the day of collection for group 1 and directly after thawing for groups 2 and 3.
Post-thawing Ovarian Follicle proliferation/apoptosis	24 months	the percentage of proliferative (oocyte or granulosa cells positive for KI-67) and apoptotic (oocyte or granulosa cells positive cleaved caspase 3) in fresh and frozen (with thermosoldered or screwed cryovials) /thawed ovarian cortex will be assessed on immunostained serial sections. Fragments will be fixed on the day of collection for group 1 and directly after thawing for groups 2 and 3.
Ovarian Follicle stage after organotypic culture	36 months	Follicle stages (% of primordial, primary, secondary and antral follicles) in fresh versus frozen (with thermosoldered or screwed cryovials)/thawed ovarian cortex will be assessed on serial sections stained with hematoxylin-eosin-saffron. Fragments will be fixed after 7 or 14 days of organotypic culture.
Ovarian Follicle morphology after organotypic culture	36 months	Follicle morphology of fresh versus frozen (with thermosoldered or screwed cryovials)/thawed ovarian cortex will be assessed on serial sections stained with hematoxylin-eosin-saffron. Fragments will be fixed after 7 or 14 days of organotypic culture. Morphology of follicles will be evaluated according to the parameters described by Keros et al. Hum Reprod. 2009.
GDF-9 expression after organotypic culture	36 months	the percentage of GDF9 positive follicles (i.e. with an oocyte positive for GDF-9) in fresh and frozen (with thermosoldered or screwed cryovials) /thawed ovarian cortex will be assessed on immunostained serial sections. Fragments will be fixed after 7 or 14 days of organotypic culture.
Proliferation/apoptosis after organotypic culture	36 months	the percentage of proliferative (oocyte or granulosa cells positive for KI-67) and apoptotic (oocyte or granulosa cells positive cleaved caspase 3) in fresh and frozen (with thermosoldered or screwed cryovials) /thawed ovarian cortex will be assessed on immunostained serial sections. Fragments will be fixed after 7 or 14 days of organotypic culture.
hormone secretion after organotypic culture	36 months	AMH and estrogen levels will be quantified in culture medium during organotypic culture of fresh and frozen (with thermosoldered or screwed cryovials) /thawed ovarian cortex. Culture media will be harvested every three days of culture for 14 days. AMH and estrogen will be quantified by electrochemiluminescence
Thermodynamic properties of freezing medium	12 months	A differential scanning calorimeter (DiamondDSC, PerkinElmer) will be used to determine the crystallization temperature (Tc), end-of-melting temperature (Tm), and transition temperature (Tg'1) of the freezing medium (Leibovitz with 4 mg/mL HSA, 1.5M DMSO and 0.1M sucrose).

Trial Locations

Locations (1)

University Hospital; 🇫🇷 Clermont-Ferrand, France