Comparison of Vitrification Effect Before or After In Vitro Maturation

Study Overview
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Brief Summary
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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.

Detailed Description
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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.

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

Conditions
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Oocytes Vitrified at Prophase-I Stage, Oocytes Matured in Vitro

Intervention / Treatment
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* Other: Oocyte vitrification


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: - ICSI treatment Immature oocytes < 37 years old Exclusion Criteria: - Polyckistic Ovarian Syndrome Endometriosis Ovulatory disease

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 37 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Immature oocytes vitrified before In Vitro Maturation<br>Immature oocytes were vitrified using closed system vitrification. After warming, they were cultured during 36 hours in IVM medium and fixed for cellular analysis | Other: Oocyte vitrification<br>* 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.<br>|
| Immature oocytes cultured in vitro before vitrification<br>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. | Other: Oocyte vitrification<br>* 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.<br>|
| Fresh oocytes<br>Immature oocytes were cultured in vitro in IVM medium during 36 hours and subsequently, fixed for cellular analysis. | Other: Oocyte vitrification<br>* 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.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Analysis of maturation kinetic of oocytes vitrified at Prophase-I stage compared to fresh oocytes | 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. | at day 1 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Analysis of actin and tubulin cytoskeleton at Metaphase-II stage | 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. | at day 1 | 
| Analysis of chromosome segregation during the first meiotic division. | 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 | at day 1 | 
| Analysis of cortical granules distribution in Metaphase-II stage oocytes. | 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. | at day 1 | 
| Analysis of maternal factor stabilities. | 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. | at day 1 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Vitrification, In vitro maturation, Cytoskeleton, chromosomes, High resolution imaging