Effect of COVID-19 on Platelet Mitochondrial Bioenergetic, Antioxidants and Oxidative Stress in Infertile Men.

Study Overview
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Brief Summary
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To verify the hypothesis that infertility and the effect of SARS-CoV-2 on infertility may damage platelet mitochondrial bioenergetics and endogenous coenzyme Q10 levels in infertile men.

Detailed Description
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Infertility is defined as the failure of the reproductive system to achieve pregnancy after 12 months of unprotected sex life. The pathobiochemical mechanisms of male fertility disorders include reduced sperm motility and quality, oxidative stress, reduced antioxidant capacity, mtDNA fragmentation, and sperm mitochondrial dysfunction. Sperm contain a number of mitochondria that are spirally arranged around the middle part of the axomen. The main role of mitochondria in spermatozoa is to generate the energy needed for their motility (1, 2). Endogenous sources - coenzyme Q10 and carnitine - are key for energy production (ATP) in sperm mitochondria. Physiological functions of sperm require a minimal amount of reactive oxygen species (ROS), but uncontrolled ROS production contributes to reduced motility and sperm count, fragmentation of mtDNA (3). In recent years, blood cells (platelets, lymphocytes and monocytes) have been used to diagnose mitochondrial disorders. Isolated peripheral blood platelets are an available source of mitochondria to assess mitochondrial health. Platelets receive energy mainly through glycolysis and oxidative phosphorylation. Platelet mitochondrial dysfunction has been demonstrated in patients with chronic kidney disease (4, 5), in patients with rheumatoid arthritis (6), in patients with acute COVID-19 (7). An O2k-respirometer (Oroboros, Austria) (8, 9) is used for respirometric analysis of platelet mitochondrial bioenergetics. None information is available on the effect of infertility on platelet mitochondrial function, none on the effect of SARS-CoV-2 on platelet mitochondrial function in infertile patients, or the effect of vaccination on sperm function. Testicular damage and subsequent infertility due to SARS-CoV infection is expected. -2, directly via SARS-CoV-2 binding to ACE2 receptors or secondarily, in relation to the immunological and inflammatory response (10). SARS-CoV-2 virus induces excessive production of pro-inflammatory cytokines, mainly interleukin 6 (IL6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Cytokines can impair sperm movement and reduce sperm count. High levels of pro-inflammatory cytokines have been found in infertile men (with oligozoospermia, asthenozoospermia, teratozoospermia) (11). SARS-CoV-2 virus can manipulate mitochondrial function in patients with post-COVID-19 syndrome, which may persist for a long time (12). In previous our study the investigators found modulation of platelet mitochondrial respiration, reduction ATP production via oxidative phosphorylation, reduces endogenous coenzyme Q10 production, reprogramming of cellular metabolism patients after 4-7 weeks overcoming acute COVID-19, SARS-CoV-2 (7). In another studies the investigators confirmed platelet mitochondrial bioenergetic deficiency, reduced endogenous coenzyme Q10 production in patients with post-COVID-19 syndrome, 3-6 months after overcoming COVID-19 (13, 14, 15). Results of this study contribute to the understanding of the pathobiochemical mechanisms of infertility on subcellular level and to verify the hypothesis that infertility and the effect of SARS-CoV-2 on infertility may affect platelet mitochondrial bioenergetics and endogenous coenzyme Q10 levels.

Official Title
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Effect of COVID-19 on Spermiogram, Platelet Mitochondrial Bioenergetic, Antioxidants and Oxidative Stress in Infertile Men

Conditions
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Infertility, Male, COVID-19

Intervention / Treatment
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* Other: diagnostic test and sperm analysis


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Infertile patients without COVID-19 Infertile patients after COVID-19 Control group: healthy volunteers Exclusion Criteria: disagreement with informed consent

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 40 Years

Sexes Eligible for Study
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Male

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: Other
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: infertile men with post-COVID-19 (vaccinated or without vaccination)<br>15 infertile men with post-COVID-19 (vaccinated or without vaccination) | Other: diagnostic test and sperm analysis<br>* Diagnostic Test: 2x14 ml of peripheral blood collected in the tube with anticoagulant, for platelet isolation, respirometry mitochondrial analysis, antioxidants (coenzyme Q10, vitamin E, gamma-tocopherol, beta-carotene) and TBARS estimation. Sperm analysis: standard spermiogram examination (volume, pH, number, motility and pathology) in the broker chamber, as well as extended examinations: mioxsys for redox potential, Vitalsperm (eosin-nigrosine staining) for sperm vitality and anti-sperm antibody (IgG) test.<br>|
| Active Comparator: infertile men without post-COVID-19 (vaccinated or without vaccination)<br>15 infertile men without post-COVID-19 (vaccinated or without vaccination) | Other: diagnostic test and sperm analysis<br>* Diagnostic Test: 2x14 ml of peripheral blood collected in the tube with anticoagulant, for platelet isolation, respirometry mitochondrial analysis, antioxidants (coenzyme Q10, vitamin E, gamma-tocopherol, beta-carotene) and TBARS estimation. Sperm analysis: standard spermiogram examination (volume, pH, number, motility and pathology) in the broker chamber, as well as extended examinations: mioxsys for redox potential, Vitalsperm (eosin-nigrosine staining) for sperm vitality and anti-sperm antibody (IgG) test.<br>|
| Active Comparator: Control: 15 healthy men volunteers (no COVID-19, no other pathologies)<br>15 healthy men volunteers (no COVID-19, no other pathologies) as control group | Other: diagnostic test and sperm analysis<br>* Diagnostic Test: 2x14 ml of peripheral blood collected in the tube with anticoagulant, for platelet isolation, respirometry mitochondrial analysis, antioxidants (coenzyme Q10, vitamin E, gamma-tocopherol, beta-carotene) and TBARS estimation. Sperm analysis: standard spermiogram examination (volume, pH, number, motility and pathology) in the broker chamber, as well as extended examinations: mioxsys for redox potential, Vitalsperm (eosin-nigrosine staining) for sperm vitality and anti-sperm antibody (IgG) test.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Clinical symptoms | Clinical symptoms: infertile patients without COVID-19 (vaccinated, or none vaccinated) Clinical symptoms patients after COVID-19 | 15 minutes | 
| Damaged platelet mitochondrial bioenergetics 1 | Basal oxygen consumption rate in intact platelets (ce) | 1 day | 
| Damaged platelet mitochondrial bioenergetics 2 | rate of mitochondria LEAK respiration with CI-linked substrates (1PM - state 4) | 1 day | 
| Damaged platelet mitochondrial bioenergetics 3 | CI-linked respiration coupled with ATPproduction (2D-CI-linked oxidative phosphorylation capacity), respiration after addition of cytochrome c (2C). | 1 day | 
| Damaged platelet mitochondrial bioenergetics 4 | Maximal oxidative capacity (the electron transfer capacity -ET), after uncoupler titration (3U). | 1 day | 
| Damaged platelet mitochondrial bioenergetics 5 | After addition of exogenous substrate glutamate (4G) non-coupled mitochondrial oxygen consumption. | 1 day | 
| Damaged platelet mitochondrial bioenergetics 6 | Non-coupled oxygen consumption with CI&CII-linked substrate (5S) improvement of mitochondrial parameters representing OXPHOS- and electron tranport capacity (ET-capacity). | 1 day | 
| Endogenous coenzyme Q10-TOTAL 1 | CoQ10-TOTAL in: Platelets (pmol.10-9 cells) | 1 day | 
| Endogenous coenzyme Q10-TOTAL 2 | CoQ10-TOTAL in: Blood (µmol.L-1) | 1 day | 
| Endogenous coenzyme Q10-TOTAL 3 | CoQ10-TOTAL in: Plasma (µmol.L-1) | 1 day | 
| Endogenous coenzyme TBARS | Endogenous concentration of CoQ10-TOTAL (ubiquinone + ubiquinol) in platelets, blood and plasma CoQ10-TOTAL in: TBARS in plasma (µmol.L-1). | 1 day | 
| Sperm analysis 1 | standard spermiogram examination (volume, pH, number, motility and pathology) in the broker chamber | 1 day | 
| Sperm analysis 2 | mioxsys for redox potential | 1 day | 
| Sperm analysis 3 | Vitalsperm (eosin-nigrosine staining) for sperm vitality | 1 day | 
| Sperm analysis 4 | anti-sperm antibody (IgG) test | 1 day | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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men infertility, after-COVID-19, platelets, mitochondrial bioenergetics, coenzyme Q10, oxidative stress