Effect of GM-CSF on CAPA-IVM System Success Rates in PCOS

Study Overview
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Brief Summary
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In-vitro maturation (IVM) of human oocytes obtained from minimally stimulated or unstimulated ovaries offers a more patient friendly treatment option than the conventional Assisted Reproductive Technology (ART) treatment with controlled ovarian hyperstimulation (COH). However, maturation rate and the total blastocyst yield in oocytes undergoing in vitro maturation are still limited. This pilot study aims to evaluate the addition of an important growth factor known as Granulocyte macrophage colony stimulating factor (GM-CSF). The investigators hypothesize that the addition of GM-CSF to human IVM culture media will increase pregnancy rates to comparable levels to that of IVF, making it a viable clinical option for couples undergoing assisted reproductive treatment.

Detailed Description
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In-vitro maturation (IVM) of human oocytes obtained from minimally stimulated or unstimulated ovaries offers a more patient friendly treatment option than the conventional Assisted Reproductive Technology (ART) treatment with controlled ovarian hyperstimulation (COH). Typically, IVM will be offered to women with polycystic ovaries (PCO/PCOS), or to patients with an excellent ovarian reserve, i.e. a high antral follicle count. IVM treatment is characterized by minimal administration of FSH or HMG and NO HCG ovulation trigger. The IVM approach is less disruptive to patients' daily life through the reduced need for hormonal and ultrasound monitoring, avoids a range of minor and major complications, such as ovarian hyperstimulation syndrome, and aims to reduce the total cost of infertility treatment for the patient and for the health care budget. Human oocytes retrieved from small antral follicles are able to resume meiosis by undergoing germinal vesicle breakdown and extrusion of the first polar body, if they have reached meiotic competence. These oocytes can be fertilized although only a proportion (less than 50%) of them can develop further into viable embryos. It has been hypothesized that failure of embryonic development may, at least in part, be due to an immature oocyte cytoplasm. The investigators have developed a novel human in vitro maturation (IVM) culture system (named CAPACITATION-IVM , hereafter named CAPA) using 1°) natural compounds known to influence cGMP and cAMP levels within the cumulus-oocyte-complex and 2°) compounds that are crucial for the oocyte-cumulus cross-talk. Keeping cyclic AMP high after retrieval in the GV oocyte prevents occurrence of nuclear maturation, enabling increased communication between the oocyte and the cumulus cells. This allows for the improvement in the synchronization of nuclear and cytoplasmic maturation processes in the oocyte, to the benefit of embryo quality. The clinical outcomes of using CAPA-IVM in PCOS instead of fully stimulated GnRH-antagonist HP-HMG stimulated IVF-ICSI treatments showed equivalent live-birth rates after a first embryo transfer cycle done in artificial endometrial preparation in the two arms. Nevertheless, the CAPA-IVM cumulative live birth at 12 months was 44%, while in IVF it was 62,6. (Vuong et al., 2020). Hence the attrition rate of embryos in CAPA-IVM is larger than in conventional ART. In order to correct for this difference in good embryo number there is a need to refine the culture conditions. This pilot study aims to evaluate the addition of an important growth factor known as Granulocyte macrophage colony stimulating factor (GM-CSF). In a recent breakthrough it has been shown that adding GM-CSF to IVM media can improve preimplantation embryo development in pigs, cattle and mice (unpublished results from Prof Mark Nottle, Adelaide University, Australia). Moreover, in mice, these improvements lead to an increased in implantation rate (+62%) and tended to increase birth rate (+25%) following the transfer of vitrified/warmed blastocysts compared with standard IVM. Based on these findings, this pilot study aims to determine the beneficial effect of using GM-CSF during the biphasic CAPA-IVM. The investigators hypothesize that the addition of GM-CSF to human IVM culture media will increase pregnancy rates to comparable levels to that of IVF, making it a viable clinical option for couples undergoing assisted reproductive treatment. GM-CSF (Leukine in EmbryoGene medium, Medicult) has previously been used as an additive in embryo culture in order to increase the implantation rate of exposed embryos. This study in more than 1000 patients showed no advantage of adding GMCSF routinely. However in a subset of patients, those with poor implantation, the addition of this factor improved clinical results (Ziebe et al., 2013). In this study no safety issues were mentioned by exposing human oocytes during fertilization and early embryo formation. In the intended study there will be only exposure of GM-CSF during the oocyte maturation steps, and the embryos will never be in direct contact with the investigational compound.

Official Title
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Effect of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) on Human in Vitro Oocyte Maturation (CAPA-IVM System) Success Rates in PCOS

Conditions
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IVF, PCOS, IVM

Intervention / Treatment
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* Other: GM-CSF group
* Other: Control group


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Diagnosed as PCOS using Rotterdam criteria (AFC=25 or the ovarian volume >10ml) Agree to participate in the study Exclusion Criteria: No major uterine or ovarian abnormalities Endometriosis grade 2 or upper Severe OAT (concentration <1 million/ml, motility <10%), surgical sperm

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 36 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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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: GM-CSF group<br>Patients will be randomly allocated to intervention group with GM-CSF added to CAPA and maturation medium. Following the 24h CAPA period, the evaluation of maturation (MII, GVBD, GV) will be done after 30 hrs IVM step. Mature eggs are fertilized using Intracytoplasmic sperm injection (ICSI). Fertilized oocytes will be placed in a time-lapse incubator (ASTEC) and their development until the Day 5/6 (blastocyst formation) will be followed. | Other: GM-CSF group<br>* GM-CSF added to CAPA and maturation medium<br>|
| Active Comparator: Control group<br>Patients will be randomly allocated to control group without the addition of GM-CSF to CAPA and maturation medium. Following the 24h CAPA period, the evaluation of maturation (MII, GVBD, GV) will be done after 30 hrs IVM step. Mature eggs are fertilized using Intracytoplasmic sperm injection (ICSI). Fertilized oocytes will be placed in a time-lapse incubator (ASTEC) and their development until the Day 5/6 (blastocyst formation) will be followed. | Other: Control group<br>* GM-CSF will not be added to CAPA and maturation medium<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Blastulation rate | Percentage of blastocyst by 2 types of IVM culture with or without GM-CSF | At least seven days after oocyte pick up | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Embryology data (maturation rate, fertilization rate, grade 1 and grade 2 blastocyst rate) | Maturation rate: percentage of mature oocyte over oocytes pick-up by 2 types of IVM culture Fertilization rate: percentage of 2PN over oocytes injected by 2 types of IVM culture Grade 1 and 2 blastocyst rate: percentage of grade 1 and 2 blastocyst over total number of blastocysts by 2 types of IVM culture | Maturation rate: 2 days after OPU, Fertilization rate: 3 days after OPU, grade 1 and 2 blastocyst: at least 7 days after OPU | 
| Ongoing Pregnancy Rate after the first transfer of the started treatment cycle. | Ongoing pregnancy is defined as pregnancy with detectable heart rate at 12 weeks' gestation or beyond, after the completion of the first transfer. | At 12 weeks' gestation | 
| Cumulative ongoing pregnancy rate at 6 months after randomization | Ongoing pregnancy is defined as pregnancy with detectable heart rate at 12 weeks' gestation or beyond, after transfer of all embryos from the started treatment cycle. | At 12 weeks' gestation at 6 months after randomization. After 6 months, most patients doing IVM have finished all their frozen embryos; therefore, we consider this time point for analyzing the cumulative ongoing pregnancy rate. | 
| Adverse events (OHSS rate, miscarriage rate, ectopic rate) | OHSS rate: Routine assessments for OHSS were performed on day 3 post oocyte retrieval in both groups. At other times, OHSS was evaluated if symptoms were reported by the patient. OHSS was classified using the flow diagram developed by Humaidan and colleagues for use in clinical trial settings Miscarriage defined as spontaneous loss of a clinical pregnancy before week 22 of gestational age, in which the embryo(s) or fetus(es) is/are nonviable and is/are not spontaneously absorbed or expelled from the uterus. Ectopic pregnancy defined as a pregnancy in which implantation takes place outside the uterine cavity | OHSS rate: at 03 days after oocytes pick-up and 14 days after embryo transfer, Miscarriage rate: Before 22 weeks of gestational age, ectopic rate: At 5-7 weeks of gestation | 
| The relative expression ratio ( R ) of human cumulus cell genes | Cumulus cells (CCc) will be collected, cDNA synthesis after mRNA purification, relative quantification PCR for detecting gene expression between CCs of GM-CSF group and CCs of control group. | cumulus cells will be collected after at least 30 hours of maturation culture, storaged at -80oC until RNA purification | 
| Infant DNA methylation expression level | Newborn's material (cord blood, buccal smears, placental tissue) will be collected for epigenetics analysis. Computational pipeline for genome-wide bisulfite sequencing data analysis. Reads from bisulfite sequencing are first aligned to the reference genome. The alignment data may be visualized in different tracks for comparison (control group vs GM-CSF group). After methylation calling, the bulk methylation level and genome-wide methylation level are calculated and plotted, and DMRs (differentially methylated regions) are determined. To perform an integrative analysis, DNA methylation data are coupled with gene expression, e.g., differentially genes (DEGs), to delineate the regulatory role of DNA methylation. | At the time of delivery | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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GM-CSF, CAPA-IVM