Methods for Fertility Preservation: Impact of Vitrification on in Vitro Matured Oocytes

Study Overview
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Brief Summary
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During the last decades, there was an improvement of the cancer treatments of the woman and the teenagers. Therefore higher survival rate is described. However, cancer treatments can alter the reproduction functions and reduce considerably the window of the fertility to the adulthood. Therefore, it is recommended to proceed to a fertility preservation by oocytes vitrification when it is possible. The vitrification is a freezing technique allowing high survival rate and similar results by assisted reproductive technologies compared with the use of fresh oocytes. An innovative method of automated vitrification was recently developed. The usual protocol consist to vitrify mature oocytes. However, this strategy cannot be used for hormone -sensitive cancer or when ovarian stimulation is not possible. In these situations, immature oocytes can be collected. It is also necessary to realize an in vitro maturation step for a use by assisted reproductive technology. According to the recent data of the literature, it remains unclear whether the vitrification of ovocytes must be performed before or after in vitro maturation (IVM). Therefore the aim of this study is to study the impact on structure and functions of ovocytes when vitrification is performed before or after IVM. The vitrification will be performed by a semi-automatic method which is an innovative method.

Detailed Description
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To perform this study, investigator will compare three groups. Group 1: immature ovocytes vitrified before IVM; Group 2: immature oocytes vitrified after IVM; Group3: fresh immature oocytes treated by IVM (without vitrification, control group). The immature oocytes provide from ICSI patients. In routine these oocytes (germinal vesicle) are normally destroyed because they cannot be used for injection. The women will give an informed and written consent. Inclusion criteria are women less 37 years without dysovulation. The vitrification will be performed with the semi-automatic method (Gavi, Merck). The kinetic and maturation rate will be analysed by time lapse (Primovision, Vitrolife) In the mature oocytes, the actin and tubulin cytoskeleton, the spindle organization and the cortical granules will be studied by immunofluorescence and 3D confocal microscopy. The expression of maternal factors transcription will be analyzed by RT-PCR. The ploidy will be analysed by multiFISH and/or CGH array.

Official Title
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Methods for Fertility Preservation: Impact of Vitrification on in Vitro Matured Oocytes

Conditions
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Fertility, Cancer

Intervention / Treatment
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* Other: Gavi , Merck® (automated vitrification instrument)
* Other: Vitrification


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: ICSI treatment Immature oocytes Without ovulation pathologies Exclusion Criteria: Polykistic 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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No

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 will be vitrified using closed system vitrification with a semiautomatic method. After warming, a maturation culture will be performed during 36 hours | Other: Gavi , Merck® (automated vitrification instrument)<br>* Gavi, Merck ® permits semi-automated vitrification with closed system.<br>Other: Vitrification<br>* Group 1: immature ovocytes vitrified before IVM; Group 2: immature oocytes vitrified after IVM; Group3: fresh immature oocytes treated by IVM (without vitrification, control group).<br>|
| Immature oocytes vitrified after in vitro maturation<br>A maturation culture of immature oocytes will be performed in vitro during 36 hours. After IVM, mature oocytes will be vitrify in closed system by a semi-automatic method. | Other: Gavi , Merck® (automated vitrification instrument)<br>* Gavi, Merck ® permits semi-automated vitrification with closed system.<br>Other: Vitrification<br>* Group 1: immature ovocytes vitrified before IVM; Group 2: immature oocytes vitrified after IVM; Group3: fresh immature oocytes treated by IVM (without vitrification, control group).<br>|
| Fresh oocytes<br>The culture of immature oocytes will be performed during 36 hours | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The embryonic development kinetics | from the records obtained with Time Lapse Primovision, investigator will be able to determine the precise times of embryonic development after in vitro maturation. | 6 days after ICSI and through study completion | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Analysis of actin and tubulin cytoskeleton and spindle organization in mature ovocytes (Metaphase II) | Metaphase-II stage oocytes 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 analysed. The actin network and chromosomes will be analysed quantitatively. All measurements will be compared with fresh matured (Metaphase-II) oocytes used as a control group. | 01/01/2019 - 31/12/2019 | 
| Analysis of chromosome segregation during the first meiotic division | A multi Fluorescence in Situ Hybridization and/or a CGH array will be performed to measure the chromosome segregation after vitrification | 01/01/2019 - 31/12/2019 | 
| Analysis of cortical granules distribution in mature (Metaphase II) oocytes. | A staining with Lectin will be used to mark cortical granules of matured oocytes to observe whether the protocol has an impact on their spatial distribution. To analyse this staining, investigator will use quantitative image analysis method. | 01/01/2019 - 31/12/2019 | 
| 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. Investigator will perform Reverse Transcription combined with Real Time PCR to quantify transcript amounts of candidates genes selected from human oocytes databases. | 01/01/2019 - 31/12/2019 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Vitrification, In vitro maturation, time lapse, High resolution imaging, quantitative molecular analysis