Oxidative Stress-related Gene Expression in Cumulus Cells Among Low Responders

Study Overview
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Brief Summary
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In Vitro Fertilization is considered one of effective treatment for infertile couple. However, some women do not have adequate response to controlled ovarian hyperstimulation, resulting in low response which defined as less than 5 retrieved oocytes. There are studies claiming that live birth rates in these patient lower than normal responder (10-15 retrieved oocytes). Among factors contributing low response, oxidative stress, resulting from increased pro-oxidant and/or reduced antioxidant, is considered to effect the functions of follicles and quality of oocytes. However, there are few studies demonstrating this relation. Objective of our study is to find the difference of oxidative stress-related gene expression in cumulus cell and oocyte competence biomarkers from follicular fluid between low responders and normal responder to controlled ovarian hyperstimulation.

Detailed Description
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Low response to controlled ovarian hyperstimulation (less than 5 retrieved oocytes) is a problem in some of couple receiving treatment for infertility. Studies showed that success rate in treatment for these patients is lower than normal response group. Among many factors contributing low response, oxidative stress is suspected to be involved in malfunction of follicular environment and oocyte maturation. However, direct relationship between oxidative and oocyte function cannot be studied due to ethical concern, so many studies had studied oocyte's surrounding environment such as cumulus cell and follicular fluid instead. In our study , we aim to study the difference in level of oxidative stress-related gene expression and oocyte competence biomarkers among low responders and normal responders. For oxidative stress-related gene expression of cumulus cell , we will measure mRNA expression by quantitative PCR for Superoxide dismutase 1 (SOD1) , SOD2 , Glutathione peroxidase (GPX) and Inducible nitric oxide synthase (iNOS) . For oocyte competent biomarkers, we will use ELISA technique to measure the level of GDF(, BMP15, Amphiregulin and CoQ10 in follicular fluid. Sample size calculation was done with data from study performed by Donnabela et al , which found that Mean SOD gene expression in control group was 2.06 2-Δ ΔCT (SD = 0.53) . The formula for comparing two different mean was used with expectation that low responders group would have lower SOD gene expression by 20%.: n/group = 2(Z α/2 + Z β)2 σ2 / (X̅ 1- X̅ 2)2 α = 0.05 , β = 0.2 Z α/2 = Z0.05/2 = 1.96 (two tail) Z β = Z0.8 = 0.84 Mean in group1 (X̅ 1) = 2.06, SD. in group1 (σ₁) = 0.53 Mean in group2 (X̅ 2) = 1.64, SD. in group2 (σ₂)= 0.53 Sample size: Group1 (n₁) = 25, Group2 (n₂) = 25 Total number is 56 patients (added 10% for possible data loss) Statistical analysis Mean and Standard deviation will be used for descriptive data. Mann-Whitney U test , unpaired t-test and chi square will be used as appropriate p value less than 0.05 is considered statistical significant.

Official Title
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Oxidative Stress-related Gene Expression in Cumulus Cells Among Low Responders to Controlled Ovarian Hyperstimulation During in Vitro Fertilization

Conditions
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Low Responder

Intervention / Treatment
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* Diagnostic Test: Oxidative stress-related gene expression (RNA expression)
* Diagnostic Test: oocyte competence biomarkers


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Low responder : women aged 25-49 years old with less than 5 oocytes (1-4 oocytes) retrieved during controlled ovarian hyperstimulation Normal responder women aged 25-49 years old with 5-15 oocytes retrieved during controlled ovarian hyperstimulation Exclusion Criteria: Active ovarian pathology ( ovarian cyst or tumor) Basal FSH more than 20 IU/L or Antimullerian hormone less than 0.5 ng/ml Ovarian hyperstimulation syndrome

Ages Eligible for Study
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Minimum Age: 25 Years
Maximum Age: 49 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 |
| --- | --- |
| Low responder<br>Low responder : women aged 25-49 years old with less than 5 oocytes (1-4 oocytes) retrieved during controlled ovarian hyperstimulation Oxidative stress-related gene expression and oocyte competence biomarkers will be performed to access the difference between low and normal responders. | Diagnostic Test: Oxidative stress-related gene expression (RNA expression)<br>* Oxidative stress-related gene expression (RNA expression),Superoxide dismutase 1 (SOD1) , SOD2 , Glutathione peroxidase (GPX) and Inducible nitric oxide synthase (iNOS) , measured by Real time PCR technique.<br>Diagnostic Test: oocyte competence biomarkers<br>* Oocyte competence biomarkers ,GDF9, BMP15, Amphiregulin and CoQ10 measured by ELISA<br>|
| Normal responder<br>Normal responder women aged 25-49 years old with 5-15 oocytes retrieved during controlled ovarian hyperstimulation Oxidative stress-related gene expression and oocyte competence biomarkers will be performed to access the difference between low and normal responders. | Diagnostic Test: Oxidative stress-related gene expression (RNA expression)<br>* Oxidative stress-related gene expression (RNA expression),Superoxide dismutase 1 (SOD1) , SOD2 , Glutathione peroxidase (GPX) and Inducible nitric oxide synthase (iNOS) , measured by Real time PCR technique.<br>Diagnostic Test: oocyte competence biomarkers<br>* Oocyte competence biomarkers ,GDF9, BMP15, Amphiregulin and CoQ10 measured by ELISA<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Oxidative stress-related gene expression | mRNA expression by quantitative PCR for Superoxide dismutase 1 (SOD1) SOD2 , Glutathione peroxidase (GPX) and Inducible nitric oxide synthase (iNOS). | June 2017-May 2018 | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| oocyte competent biomarkers | ELISA technique to measure the level of GDF9, BMP15, Amphiregulin and CoQ10 in follicular fluid. | June 2017-May 2018 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Low responder, Oxidative stress, Cumulus cell, Controlled ovarian hyperstimulation, In vitro fertilization