Effects of Telomerase Reactivation With Danazol in Ovarian Function.

Study Overview
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Brief Summary
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This project explores the implication of the telomere pathway in ovarian premature and regular aging. Telomere length and maintenance underlie several biological processes such cancer, aging, human diseases and the biology of stem cells. The reactivation of telomerase should lead to a rejuvenation of the ovarian tissue and the improvement of fertility. The correlation of telomeric factors in blood and granulosa cells will be studied with the aim of finding telomeric biomarkers of ovarian aging.

Detailed Description
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This is a pilot study, randomized, controled, blind, parallelo arm clinical trial with inactive substance and medicine. A pilot study will be developed with a total of 45 individuals from 30 to 45 years old, who represent the most frequent population of women seeking for ART. A control group composed of women with normal ovarian reserve (30 to 45 years old) is needed to compare telomeric and fertility parameters with the group of women with compromised ovarian reserve. Women in the control group irrespective of their age, will have a greater number of follicles compared to women belonging to the group with compromised ovarian reserve. A group of women with diminished ovarian reserve that will take an inactive substance has been incluided to avoid biases and to set the fertility base line for women with compromised ovarian reserve. The use of an inactive substance or placebo will help obtain better quality results. For instance, if Danazol happened to improve the fertility outcome, then, there could be a possibility that the IVF improvement might be due to other components of the pill. Using placebo, this possibility would be eliminated, since the placebo will contain all components of the pill, except Danazol. The development of a pilot study will help us understand the statisticl behavior or the telomeric factors as well as to determine the appropriate number of individuals that shoyuld be recruited in a clinical trial to have results with statistical significance. In addition, a pilot study will help us learn errors or undesired events that may happen during the clinical trial. For instance, if patients cannot follow the indications of their doctors and why, or what proportion of patients will drop the study and the reasons for it. Furthermore, it will also help us understand if there exist a tendency, an indication or even a clear beneficial effect for patients without harming them. The number of participants selected for the study is considered adequate for each group, since each group will be measured in an independent manner.

Official Title
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Effects of Telomerase Reactivation With Danazol in Ovarian Function.

Conditions
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Ovarian Reserve

Intervention / Treatment
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* Drug: Danazol/Placebo
* Other: Without intervention


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Provide signed and dated informed consent form. Willing to comply with all study procedures and be available for the duration of the study. This include the decisión to use contraception methods different to sexual hormones, such as the use of condoms, during the treatment with Danazol. In good general health as evidenced by medical history or diagnosed with body mass index between 18 and 30 kg/m2. Women with normal (AMH valued must be equal or higher tan 2ng/ml) or compromised ovarian reserve (defined as AMH < 2 ng/ml) Not having had any steroid hormones for one month. Exclusion Criteria: Pregnancy o lactation. Taking other sexual hormones. Women with diseases in heart, liver or kidney or tumors which depend on male sexual hormones or hormone-dependent tumour. Women taking anticonvulsants, medicaments for diabetes, anticoagulants and anti-hypertension: ciclosporin and tacrolimus and other steroids and statins. Women suffering irregular genital bleeding or with thrombus or thromboembolicdiseases. Known allergic reactions to components of the study product (cornstarch and lactose). Having received ovulation induction drugs within one month before the inclusión in the study. Anything that would place the individual at increased risk or preclude the individual´s full compliance with or completion of the study. Simultaneous participation in another clinical trial or previous participation in this study. Participation in another clinical study 2 months before inclusión in the present study that could affect its objectives.

Ages Eligible for Study
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Minimum Age: 30 Years
Maximum Age: 45 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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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: A control group composed of women with normal ovarian reserve to compare telomeric and fertility parameters with the group with compromised ovarian reserve.
Masking: Quadruple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Sham Comparator: CONTROL<br>A control group composed of women with normal ovarian reserve (30 to 45 yerras old) is needed to compare telomeric and fertility parameters with the group of women with compromised ovarian reserve. Note that the term normal ovarian reserve referred to older women indicates women that still have follicles in their ovaries -and thus, normal AMH values-, even though the number may be lower tan at a younger age or the quality of oocytes may be lower tan in younger women. In other words, women in the control group irrespective of their age, will have a greater number of follicles compared to women belonging to the group with compromised ovarian reserve. | Other: Without intervention<br>* Women with normal ovarian reserve will not be treated in anyway, thus, randomization will not be necessary.<br>|
| Experimental: EXPERIMENTAL<br>A group of women with diminished ovarian reserve that will take an inactive substance has been included to avoik biases and to set the fertility base line for women with compromised ovarian reserve. | Drug: Danazol/Placebo<br>* This group will be randomized 1:1 for either inactive substance or Danazol, according to a computer-generated randomization list prepared. Women will be treated with Danazol/Placebo oral way for 3 months.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Telomere length in granulosa cells | Measured in arbitrary units of fluorescence (a.u.) from 0 to 255 gray intensity (8bit) or Kb (continuous variable). Below 3 kb are considered critically short telomeres in humans. | The evaluation of the main assessment criterion will be done both after eighth visit (36 hours post induction) for women with low ovarian reserve and fifth visit for woman with normal ovarian reserve | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Accumulation of short telomeres | Measured in arbitrary units of fluorescence (a.u.) from 0 to 255 gray intensity (8bit) or Kb (continuous variable). Below 3 kb are considered critically short telomeres in humans. | Through study completion, an average of 1 year | 
| Telomerase activity | Product of PCR amplification, run in acrylamide gels and quantified as band intensity (continuous variable) | Through study completion, an average of 1 year | 
| DNA damage measurement | If damage is positive, then different foci should be apparent in the nucleus of the cell. The number of foci present in the nuclei of at least 100 cells will be counted. If there is DNA damage at telomeres, then yH2AX or 53BPI foci will colocalize with telomeres (labelled with anti TRF1 antibody). Spots will be counted (continuous variable). | Through study completion, an average of 1 year | 
| Other telomeric factors. | The mRNA expression levels of the telomerase gene and the shelterins, which are involved in telomere lengthening and protection, will be measured by qPCR. (continuous variable). | Through study completion, an average of 1 year | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Blood, Ovarian reserve., Telomeric factor, Granulosa cells