female patients diagnosed with cancer who wish to preserve their fertility by vitrifying their oocytes

freezing of the patient's retrieved oocytes via vitrification (rapid freezing)

oocyte vitrification

Avner Hershlag, MD

The purpose of this study is to preserve the fertility potential in females diagnosed with cancer by vitrifying (rapidly freezing) their oocytes (eggs) before undergoing treatment for their cancer.

Fertility Preservation in Female Cancer Patients

Vitrification of Oocytes From Female Cancer Patients to Preserve Their Fertility Potential

the primary objective of this study is the establishment of a successful pregnancy, following a disease-free interval after treatment for her cancer; this interval will be patient-dependent and will undoubtedly vary between participants.

patients will be followed for five years following cancer treatment to assess the risks associated with ovarian stimulation and the recurrence of cancer.

Northwell Health

Ovarian tissue will be vitrified and warmed by Ova kit type M protocol.

there are 3 types of experiments in each group, including Follicle survival evaluation by Neutral red staining, Histological evaluation by Hematoxylin and Eosin staining, 2.4. Evaluation GDF-9 and Caspase-3 gene expression by real-time PCR.

evaluation of the effectiveness of vitrification technique on ovarian tissue quality

Lan N Nguyen, MSC

Dang Q Vinh, MD, MCE

Lan Nguyen, MSC

Vinh Dang, MD,MCE

Tuong M Ho, MD, MCE

Ovarian tissue cryopreservation (OTC) is a new approach on fertility preservation for young female patients with cancer. It is now used by many IVF centers in the world. However, this is the first study which OTC by vitrification is carried on and assessed the effectiveness of the technique.

Ovaries from all less than 45 year-old breast cancer patients who undergo oophorectomy by laparoscopy after treated with chemotherapy will be enrolled to the study. Pieces of cortex of each patient will divide into two groups. Group 1 is fresh fragments (control group) and group 2 is vitrified group. Ovarian tissues in group 2 will be vitrified or warmed with medium and devices of Ova-kit type M (Kitazato, Japan).

Cortex fragments of each group will have:

* Follicle survival evaluation by Neutral red staining
* Histological evaluation by Hematoxylin and Eosin staining
* Evaluation relative Growth Differentiation Factor 9 (GDF-9) and Caspase-3 gene expression by real-time Polymerase chain reaction (PCR).

Comparison of data collected from 2 groups will be carried on to evaluate the effectiveness of the technique before applying this technique for cancer patients in Vietnam.

Assessing the Technique of Ovarian Tissue Cryopreservation by Vitrification in Vietnam

Vitrified Ovarian Tissue in Cancer Patients: Assessing the Technique in Vietnam

Percentage of survivable follicles per counted follicles in one fragment

Healthy follicles of primordial, primary, secondary follicles will be counted in one slide

Follicles in ovarian tissue will be collected, complementary deoxyribonucleic acid (cDNA) synthesis after messenger ribonucleic acid (mRNA) purification, relative quantification PCR for detecting gene expression

Mỹ Đức Hospital

Pharmaceutical Solutions

All Drugs and Chemicals

Protective Agents

oocytes will be frozen by slow freeze/ rapid thaw

oocytes will be frozen using rapid freezing/rapid thaw

Eggs will be placed in a cryo-protected solution and frozen slowly in a programable freezer

slow freeze

Eggs will be placed in a high concentration of cryo-protectant and frozen very quickly by plunging in liquid nitrogen

Vitrification

Richard T Scott, MD, HCLD

Kathleen Ferry, BS

This study seeks to evaluate whether the vitrification technique, as compared to the more traditional slow-cooling technique, leads to higher rates of successful thawing, fertilization, implantation and delivery.

Cryopreservation (freezing) of human gametes (unfertilized egg) provides a great potential to preserve or extend fertility in the face of disease and social circumstances (cancer diagnosis, delaying childbirth, single women, etc.). There are two methods for storage of oocytes (unfertilized eggs): slow freezing or vitrification (uses higher concentrations of cryoprotectant and faster cooling rates). Slow freezing is the standard method and has been successful for embryos since 1983 and more recently for oocytes. Recent reports indicate that vitrification may be more successful than slow freezing. The aim of this study is to examine the rate at which frozen eggs survive freezing and thawing, the rate at which the frozen/thawed eggs fertilize with sperm and the pregnancy outcomes of oocyte (egg) vitrification cycles to determine whether the outcomes are similar or better than standard oocyte (egg) cryopreservation (freezing) cycles.

Oocyte Cryopreservation by Slow Freezing and Vitrification

Longitudinal Evaluation of Vitrification of Human Oocytes

Whether each oocyte survives the thawing process

Whether each oocyte is successfully fertilized

Reproductive Medicine Associates of New Jersey

Immature oocytes were vitrified using closed system vitrification. After warming, they were cultured during 36 hours in IVM medium and fixed for cellular analysis

Immature oocytes were cultured in vitro in IVM medium during 36 hours. After IVM, they were vitrified. After warming, they were fixed for cellular analysis.

Immature oocytes were cultured in vitro in IVM medium during 36 hours and subsequently, fixed for cellular analysis.

Immature oocytes were collected from patients (≤37 years old, without endometriosis, Polycystic syndrome or other ovulatory desease) who underwent to ICSI. In addition, immature oocytes collected from patient diagnosed with cancer will be included in this study. Oocytes were matured in vitro with IVM medium and vitrified in closed system (Vitrolife). Kinetic of maturation were analyzed by Primovision (Vitrolife) and actin, and spindle organization were studied by microscopy, immunostaining techniques and quantitive analysis.

Oocyte vitrification

Patrick LACARIN

Florence BRUGNON

Aïcha METCHAT

Human oocyte cryopreservation is routinely used for fertility preservation of women who will be exposed to gonadotoxic effect of cancer treatment. After ovarian stimulation, matured oocytes are vitrified. However, this strategy cannot always be used, particularly for hormone-sensitive cancer or when ovarian stimulation is not possible. An approach including immature oocytes and in vitro maturation (IVM) could be considered in these cases. While some qualitative analysis of oocytes vitrified before or after IVM suggest that vitrification should be performed after IVM, little is known about vitrification effects on actin and tubulin cytoskeleton and kinetic of maturation of human ovocytes.

To answer to this question, Investigator performed quantitive analyses comparing matured oocytes from three different groups: vitrified before IVM or after IVM and non-vitrified oocytes. Non-vitrified matured oocytes were used as a control. Different parameters have been analysed during maturation and in matured oocytes.

Immature oocytes were collected from patients (≤37 years old, without endometriosis, Polycystic syndrome or other ovulatory desease) who underwent to ICSI. In addition, immature oocytes collected from patient diagnosed with cancer will be included in this study. Oocytes were matured in vitro with IVM medium and vitrified in closed system ("Vitrolife"). Kinetic of maturation were analyzed by Primovision ("Vitrolife") and actin, and spindle organization were studied by microscopy, immunostaining techniques and quantitive analysis.

Comparison of Vitrification Effect Before or After In Vitro Maturation

Characterization of a Method of Fertility Preservation for Patients Diagnosed for a Cancer

After oocytes retrieval, immature oocytes will be vitrified (Rapid Vit Ovocyte®, Vitrolife) in a closed system (Rapid-I®, Vitrolife) and thawed for in vitro maturation few days after. The meiotic process will be analyzed by time lapse technology (Primovision®, Vitrolife). This will permit to score the time of maturation from resumption to polar body extrusion of ovocytes.

Metaphase-II stage oocytes from both protocols will be used for Immuno-Fluorescence experiments to stain actin, tubulin and chromosomes. Oocytes will be imaged using confocal microscope to perform high resolution imaging and quantitative image analysis. The length, position and orientation of the second meiotic spindle will be quantifying. The actin network and chromosomes will be analyzed quantitatively. All measurements will be compared with fresh Metaphase-II oocytes used as a control group.

The first asymmetric division is highly error prone. To investigate whether chromosomes are segregated accurately after vitrification, we will perform Fluorescence In Situ Hybridization to paint each chromosome after chromosome spread from Metaphase-II stage oocytes from both conditions

A staining with Lectin will be used to mark cortical granules of matured oocytes from both protocols to observe whether the vitrification does modify their spatial distribution. To analyze this staining, we will use quantitative image analysis method.

Maternal factors stored in the oocyte cytoplasm during oogenesis as proteins and transcripts are essential for the early embryonic development. To know if the stock of maternal factors is diminished by the vitrification procedure, we will perform Reverse Transcription combined with Real Time PCR to quantify transcript amounts of candidates genes selected from human oocytes databases.

Study title	NCT ID	Conditions	Interventions	Eligibility	Organization
Fertility Preservation in Female Cancer Patients	NCT01268592	Fertility	oocyte vitrific...	14 Years - 42 Years	Northwell Health
Assessing the Technique of Ovarian Tissue Cryopreservation by Vitrification in Vietnam	NCT04666376	Ovarian Tissue ... Vitrification	evaluation of t...	- 45 Years	Mỹ Đức Hospital
Oocyte Cryopreservation by Slow Freezing and Vitrification	NCT01223105	Women Diagnosed... Egg Donors Single Women Failed Sperm Pr...	slow freeze Vitrification	21 Years - 50 Years	Reproductive Medicine Associates of New Jersey
Comparison of Vitrification Effect Before or After In Vitro Maturation	NCT03416400	Oocytes Vitrifi... Oocytes Matured...	Oocyte vitrific...	18 Years - 37 Years	University Hospital, Clermont-Ferrand
Oocyte Cryopreservation by Slow Freezing and Vitrification	NCT01223105	Women Diagnosed... Egg Donors Single Women Failed Sperm Pr...	slow freeze Vitrification	21 Years - 50 Years	Reproductive Medicine Associates of New Jersey

Spots Global Cancer Trial Database for vitrification