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NCT Number: NCT06724471

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

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.

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Key information

Conditions

Infertility, Female Female Urogenital Diseases Female Urogenital Diseases and Pregnancy Complications Genital Diseases Genital Diseases, Female Infertility Urogenital Diseases

Age range

18 year–37 year

Sex eligibility

Female

Study type

Observational

Primary location

University Hospital

Clermont-Ferrand, 63100, France

About this study

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.

Who can participate

Healthy volunteers accepted: No

Only the study team can determine whether someone qualifies for participation.

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

Treatment and study plan

Human ovarian tissue cryopreservation using thermosoldered or screwed cryovials

Device

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).

Primary outcomes

  1. Post-thawing Ovarian Follicle morphology

    Time frame: 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 outcomes

  1. 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.

  2. 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.

  3. 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.

  4. Ovarian Follicle stage after organotypic culture

    Time frame: 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Estrogen secretion after organotypic culture

    Time frame: 36 months

    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

  11. Thermodynamic properties of freezing medium

    Time frame: 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).

Sponsors and collaborators

Lead sponsor

University Hospital, Clermont-Ferrand

Other

Collaborators

  • CryoBioSystem
  • IMoST UMR 1240 Inserm-Université Clermont Auvergne

Registry information

Acronym: FERTIOVO

Important dates

Study start
2022
Primary completion
2025
Study completion
2025
First posted
Dec 9, 2024
Registry last updated
Jun 29, 2025

OpenTrials presents study information sourced from ClinicalTrials.gov. The official registry record should be consulted for the latest information.

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This listing is for discovery and informational purposes only. It is not medical advice, does not guarantee that a study is recruiting, and does not determine eligibility. Contact the study team and a qualified healthcare professional when considering participation.

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