Preservation of Women's Fertility: Evaluation of Innovative Methods for Ovarian Tissue Cryopreservation (FERTIOVO)
June 25, 2025 updated by: University Hospital, Clermont-Ferrand
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 this 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.
Study Overview
Status
Active, not recruiting
Conditions
Intervention / Treatment
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, the investigators 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).
The investigators 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.
The investigators 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 Type
Observational
Enrollment (Actual)
30
Contacts and Locations
This section provides the contact details for those conducting the study, and information on where this study is being conducted.
Study Locations
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Clermont-Ferrand, France, 63100
- University Hospital
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Participation Criteria
Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.
Eligibility Criteria
Ages Eligible for Study
- Adult
Accepts Healthy Volunteers
No
Sampling Method
Non-Probability Sample
Study Population
Adult woman scheduled for benign ovarian cyst resection in the University Hospital Center of Clermont-Ferrand.
Description
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 Plan
This section provides details of the study plan, including how the study is designed and what the study is measuring.
How is the study designed?
Design Details
Cohorts and Interventions
Group / Cohort |
Intervention / Treatment |
|---|---|
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Fresh ovarian cortex fragments
Pericystic ovarian cortex will be cut into 1mm3 fragments and either directly analyzed on the day of collection (D0) or after organotypic culture (D7 and D14).
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Fragments of ovarian cortex frozen in thermosoldered cryovials.
Pericystic ovarian cortex will be cut into 1mm3 fragments, frozen using a slow programmable freezing device (Nano Digitcool, Cryo Bio System) in thermosoldered cryovials and stored in liquid nitrogen (-196°C).
Then, fragments will be thawed before being analyzed on the day of thawing (D'0) and after 7 and 14 days (D7 and D14) of organotypic culture.
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Human ovarian tissue Cryopreservation: immediately after cyst resection, pericystic ovarian cortex will be cut into 1mm3 fragments and cryopreserved in screwed or thermosoldered cryovials using a slow programmable freezing device (Nano Digitcool, Cryo Bio System).
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Fragments of ovarian cortex frozen in screwed cryovials.
Pericystic ovarian cortex will be cut into 1mm3 fragments, frozen using a slow programmable freezing device (Nano Digitcool, Cryo Bio System) in screwed cryovials and stored in liquid nitrogen (-196°C).
Then fragments will be thawed before being analyzed on the day of thawing (D'0) and after 7 and 14 days (D7 and D14) of organotypic culture.
|
Human ovarian tissue Cryopreservation: immediately after cyst resection, pericystic ovarian cortex will be cut into 1mm3 fragments and cryopreserved in screwed or thermosoldered cryovials using a slow programmable freezing device (Nano Digitcool, Cryo Bio System).
|
What is the study measuring?
Primary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
|---|---|---|
|
Post-thawing Ovarian Follicle morphology
Time Frame: 24 months
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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.
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24 months
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Secondary Outcome Measures
Outcome Measure |
Measure Description |
Time Frame |
|---|---|---|
|
Post-thawing Ovarian Follicle density
Time Frame: 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.
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24 months
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Ovarian Follicle stage after organotypic culture
Time Frame: 36 months
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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.
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36 months
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Ovarian Follicle morphology after organotypic culture
Time Frame: 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.
|
36 months
|
|
GDF-9 expression after organotypic culture
Time Frame: 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.
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36 months
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Thermodynamic properties of freezing medium
Time Frame: 12 months
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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).
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12 months
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Post-thawing Ovarian Follicle proliferation
Time Frame: 24 months
|
the percentage of proliferative (oocyte or granulosa cells positive for KI-67 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.
|
24 months
|
|
Post-thawing Ovarian Follicle apoptosis
Time Frame: 24 months
|
the percentage of 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.
|
24 months
|
|
Proliferation after organotypic culture
Time Frame: 36 months
|
the percentage of proliferative (oocyte or granulosa cells positive for KI-67) 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.
|
36 months
|
|
Apoptosis after organotypic culture
Time Frame: 36 months
|
the percentage of 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.
|
36 months
|
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AMH secretion after organotypic culture
Time Frame: 36 months
|
AMH 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 will be quantified by electrochemiluminescence
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36 months
|
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Estrogen secretion after organotypic culture
Time Frame: 36 months
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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.
Estrogen will be quantified by electrochemiluminescence
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36 months
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Collaborators and Investigators
This is where you will find people and organizations involved with this study.
Investigators
- Principal Investigator: Florence BRUGNON, MD, PhD, University Hospital, Clermont-Ferrand
Study record dates
These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.
Study Major Dates
Study Start (Actual)
May 5, 2022
Primary Completion (Actual)
June 1, 2025
Study Completion (Estimated)
December 31, 2025
Study Registration Dates
First Submitted
December 5, 2024
First Submitted That Met QC Criteria
December 5, 2024
First Posted (Actual)
December 9, 2024
Study Record Updates
Last Update Posted (Actual)
June 29, 2025
Last Update Submitted That Met QC Criteria
June 25, 2025
Last Verified
June 1, 2025
More Information
Terms related to this study
Keywords
Additional Relevant MeSH Terms
Other Study ID Numbers
- 2022_Brugnon_FERTIOVO
Drug and device information, study documents
Studies a U.S. FDA-regulated drug product
No
Studies a U.S. FDA-regulated device product
No
product manufactured in and exported from the U.S.
No
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