Involvement of Reticulum Endoplasmic Stress in the Physiopathology of Polycystic Ovary Syndrome

Study Overview
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Brief Summary
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The main objective of the present project is to evaluate the relevance of reticulum stress in the pathogenesis of polycystic ovary syndrome (PCOS), focusing particularly on the underlying mechanisms of insulin resistance, which is the origin of metabolic comorbidities. Furthermore, the investigators will assess the potential of insulin sensitizers as a treatment to control endoplasmic reticulum stress markers in PCOS patients.

Detailed Description
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To do this, the investigators will evaluate anthropometric, biochemical and hormone parameters, lipid profile and cardiovascular risk markers (using enzymatic and biochemical techniques, nephelometry, enzyme-linked immunosorbent assay, radioimmunoassay), and markers of endoplasmic reticulum stress and the insulin pathway and inflammatory and apoptotic parameters (by means of Western blot, Real Time- Polymerase Chain Reaction (RT-PCR), Luminex® xMAP® Technology ) in patients with and without PCOS. The investigators' second objective is to evaluate (using the abovementioned methodology) the efficacy of different insulin sensitizers (myoinositol and metformin) administered to PCOS patients during a 3-month period after which the investigators will analyze different parameters of oxidative stress and mitochondrial function (using Clark electrode and fluorometric techniques).

Official Title
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Involvement of Reticulum Endoplasmic Stress in the Physiopathology of Polycystic Ovary Syndrome: Possible Therapeutic Implications of Insulin Sensitizers.

Conditions
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Polycystic Ovary Syndrome

Intervention / Treatment
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* Drug: Metformin
* Dietary Supplement: Myo-inositol + folic acid


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Women diagnosed with PCOS using the Rotterdam criteria Women of reproductive age Exclusion Criteria: Organic, malignant, haematological, infectious or inflammatory disease History of ischaemic heart disease (stroke or thromboembolism) Diabetes mellitus, Secondary causes of obesity (hypothyroidism, Cushing's syndrome) Severe hypertension. Smoking or alcohol habit

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 45 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: Non-Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Metformin<br>PCOS women began treatment with ER 500 mg metformin per day, and the dose was increased to 1000 mg after 2 weeks, and to 1700 mg/d after a further 2 weeks, and was maintained at this dose for a total of 12 weeks. | Drug: Metformin<br>* Dose: metformin (1700 mg / day) for 12 weeks<br>|
| Experimental: Myo-inositol + folic acid<br>PCOS women received a dietary supplement (Ovusitol® : 4 g myo-inositol plus 400 micrograms of folic acid) for 12 weeks | Dietary Supplement: Myo-inositol + folic acid<br>* Dose: Ovusitol® (4 g myo-inositol plus 400 micrograms of folic acid/day) for 12 weeks<br>|
| No Intervention: Healthy women<br>Healthy untreated women adjusted for age and body mass index | |

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in markers of endoplasmic reticulum stress in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Markers of endoplasmic reticulum stress (78-kDa glucose-regulated protein (GRP78), ubiquitous translation initiation factor 2α (eIF2α), double-stranded RNA-activated protein kinase (PERK), inositol requiring enzyme 1 (IRE1α), X-box binding protein 1 (XBP-1)) were assessed by Western Blot and Real Time- Polymerase Chain Reaction (RT-PCR) in polymorphonuclear cells | 3 months | 
| Changes in markers of the insulin pathway in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Markers of the insulin pathway (c-Jun N-terminal kinase (JNK), insulin receptor substrate (IRS)) were assessed by Western Blot and Real Time- Polymerase Chain Reaction (RT-PCR) in polymorphonuclear cells | 3 months | 
| Changes in inflammatory parameters in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Inflammatory parameters (nuclear factor κB (NF-κB), interleukin-6 (IL6), tumor necrosis factor α (TNFα)) were assessed by Western Blot, Real Time- Polymerase Chain Reaction (RT-PCR), or Luminex® xMAP® Technology in polymorphonuclear cells and serum | 3 months | 
| Changes in apoptotic parameters in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Apoptotic parameters (transcription factor C/EBP homologous protein (CHOP) and caspase 12) were assessed by Western Blot and Real Time- Polymerase Chain Reaction (RT-PCR) in polymorphonuclear cells | 3 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in anthropometric parameters in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Anthropometric (weight, height, body mass index and waist) and blood pressure parameters were evaluated | 3 months | 
| Changes in biochemical parameters in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Glucose levels were measured using enzymatic techniques. Insulin was measured by an enzymatic luminescence technique. IR was calculated by homeostasis model assessment (HOMA). Total cholesterol and triglycerides were measured by employing enzymatic assays, and high density lipoprotein cholesterol (HDLc) concentrations were recorded with an autoanalyser using a direct method. Low-density lipoprotein cholesterol (LDLc) concentration was calculated using the Friedewald method. Luteinizing hormone (LH), follicle stimulating hormone (FSH) and testosterone were measured by specific radioimmunoassays. Dehydroepiandrosterone-sulfate (DHEAS), sex hormone-binding globulin (SHBG), androstenedione and testosterone were measured by specific chemiluminescence techniques. High-sensitive C-reactive protein (hsCRP) was quantified by a latex-enhanced immunonephelometric assay | 3 months | 
| Changes in mitochondrial function parameters in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Oxidative stress markers ( mitochondrial oxygen (O2) consumption, membrane potential, glutathione, reactive oxygen species (ROS) and hydrogen peroxide levels, mitochondrial mass) were assessed by Clark electrode and fluorometric techniques | 3 months | 
| Changes in endothelial function parameters in controls and pcos women before and after metformin/Myo-inositol + folic acid administration | Interactions between leukocytes and human umbilical vein endothelial cells were evaluated by flow chamber microscopy (leukocyte rolling velocity, leukocyte rolling flux and leukocyte adhesion). The vascular cell adhesion molecule 1 (VCAM-1), Intercellular adhesion molecule 1 (ICAM-1) and E-selectin were evaluated in serum by Luminex® 200 flow analyzer system | 3 months | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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reticulum endoplasmic stress, Polycystic Ovary Syndrome, mitochondrial function, insulin resistance, inflammation, myoinositol, metformin, oxidative stress