Effect of Systemic Ozone Therapy on Corneal Endothelium (OZON-CORNEA)

July 16, 2026 updated by: Sebahattin Celik MD, Yuzuncu Yil University

Effect of Systemic Ozone Therapy on Corneal Endothelium: A Prospective Controlled Cohort Study With Specular Microscopy

This study evaluates the safety of systemic ozone therapy on the corneal endothelium, a vital non-renewable tissue that maintains corneal transparency. Ozone therapy is increasingly used for chronic pain, osteoarthritis, fibromyalgia, and other conditions due to its immune-modulating and antioxidant effects. However, its impact on the corneal endothelium has not been thoroughly investigated.

This prospective controlled cohort study includes 96 participants: 48 patients receiving systemic ozone therapy (major autohemotherapy, 40 µg/mL, 10 sessions) and 48 age- and sex-matched healthy controls. Corneal endothelial parameters including endothelial cell density (ECD), hexagonality (Hex%), coefficient of variation (CV), central corneal thickness (CCT), and intraocular pressure (IOP) are measured by specular microscopy at baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3). Oxidative stress markers (MDA, SOD, GSH-Px) are also assessed in the ozone group.

The primary goal is to determine whether systemic ozone therapy causes any adverse effects on corneal endothelial cells over a 6-month period. Secondary goals include evaluating changes in oxidative stress markers and assessing the correlation between systemic antioxidant effects and corneal health. Results will help guide clinical practice regarding the safety of ozone therapy in patients with or without pre-existing corneal conditions.

Study Overview

Status

Status

Indicates the current recruitment status or the expanded access status.
Completed

Conditions

Conditions

The disease, disorder, syndrome, illness, or injury that is being studied. On ClinicalTrials.gov, conditions may also include other health-related issues, such as lifespan, quality of life, and health risks.

Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.

Detailed Description

STUDY DESIGN AND PARTICIPANTS

This is a prospective controlled cohort study conducted at [Institution Name]. The study protocol was approved by the Local Ethics Committee and adhered to the tenets of the Declaration of Helsinki. All participants provided written informed consent.

The ozone group comprised 48 consecutive patients scheduled to receive systemic ozone therapy for various indications including fibromyalgia (16.7%), chronic pain (22.9%), knee osteoarthritis (29.2%), disk herniation (10.4%), peripheral arterial disease (6.3%), and sports injuries (14.6%). The control group consisted of 48 healthy individuals matched for age (±2 years), sex, and body mass index (BMI, ±3 kg/m²) who did not receive ozone therapy.

Exclusion criteria for both groups included: (1) history of ocular surgery or trauma, (2) corneal dystrophy or degeneration, (3) glaucoma or ocular hypertension, (4) diabetes mellitus, (5) contact lens use within the preceding 3 months, (6) active ocular surface disease, and (7) systemic conditions known to affect corneal endothelium.

OZONE THERAPY PROTOCOL

Systemic ozone therapy was administered via major autohemotherapy (MAH) using a medical-grade ozone generator (Ozonosan Alpha Plus, Herrmann Apparatebau GmbH, Germany). Approximately 100 mL of the patient's blood was drawn into a sterile glass bottle containing 3.8% sodium citrate as an anticoagulant. The blood was then exposed to a precisely measured ozone-oxygen mixture at a concentration of 40 µg/mL. The ozonated blood was gently mixed and reinfused intravenously over 10-15 minutes. This procedure was performed once daily for 10 consecutive sessions.

OPHTHALMIC EXAMINATIONS

All participants underwent comprehensive ophthalmic examinations including: slit-lamp biomicroscopy, best-corrected visual acuity (BCVA) measurement using Snellen chart (converted to logMAR), intraocular pressure (IOP) measurement by non-contact tonometry (Topcon CT-80), and corneal endothelial evaluation by non-contact specular microscopy (Topcon SP-3000P, Japan).

Specular microscopy was performed by a single experienced technician blinded to group allocation. Three images were captured from the central cornea of each eye, and the image with the highest cell count and clarity was selected for analysis. The following parameters were recorded: endothelial cell density (ECD, cells/mm²), percentage of hexagonal cells (Hex%), coefficient of variation in cell area (CV, %), and central corneal thickness (CCT, µm). Measurements were obtained at four time points: baseline (T0, before ozone therapy), 1 month (T1, immediately after completing 10 sessions), 3 months (T2), and 6 months (T3) after baseline.

BIOCHEMICAL ANALYSIS

In the ozone group, venous blood samples were collected at each time point. Serum levels of malondialdehyde (MDA, µmol/L) were measured by the thiobarbituric acid reactive substances (TBARS) method. Superoxide dismutase (SOD, U/mL) and glutathione peroxidase (GSH-Px, U/mL) activities were determined by spectrophotometric methods.

STATISTICAL ANALYSIS

Statistical analysis was performed using SPSS version 26.0 and Python 3.11 with SciPy and StatsModels libraries. Normality was assessed using the Shapiro-Wilk test. Between-group comparisons at baseline were performed using independent t-test or Mann-Whitney U test. Within-group changes over time were analyzed using repeated measures ANOVA or Friedman test, followed by Wilcoxon signed-rank test. Between-group comparisons of changes from baseline (delta values) were performed using independent t-test or Mann-Whitney U test. Effect sizes were calculated as Cohen's d and r (Z/√N). Statistical significance was set at p < 0.05.

PRIMARY OUTCOME MEASURES

  • Change in endothelial cell density (ECD) from baseline to 6 months
  • Change in hexagonality (Hex%) from baseline to 6 months
  • Change in coefficient of variation (CV) from baseline to 6 months
  • Change in central corneal thickness (CCT) from baseline to 6 months
  • Change in intraocular pressure (IOP) from baseline to 6 months

SECONDARY OUTCOME MEASURES

  • Change in malondialdehyde (MDA) levels from baseline to 6 months (ozone group only)
  • Change in superoxide dismutase (SOD) levels from baseline to 6 months (ozone group only)
  • Change in glutathione peroxidase (GSH-Px) levels from baseline to 6 months (ozone group only)
  • Correlation between changes in oxidative stress markers and corneal endothelial parameters
  • Subgroup analyses by age, sex, BMI, and ozone therapy indications

FOLLOW-UP SCHEDULE

  • T0: Baseline assessment (before ozone therapy)
  • T1: 1 month after baseline (immediately after completing 10 ozone sessions)
  • T2: 3 months after baseline
  • T3: 6 months after baseline (primary endpoint)

SAFETY MONITORING

All adverse events were documented throughout the study. Slit-lamp biomicroscopy was performed at each visit to detect any ocular abnormalities. Best-corrected visual acuity was monitored to ensure no visual deterioration occurred.

Study Type

Study Type

Describes the nature of a clinical study. Study types include interventional studies (also called clinical trials), observational studies (including patient registries), and expanded access.
Interventional

Enrollment (Actual)

Enrollment

The number of participants in a clinical study. The "anticipated" enrollment is the target number of participants that the researchers need for the study.
96

Phase

Phase

The stage of a clinical trial studying a drug or biological product, based on definitions developed by the U.S. Food and Drug Administration (FDA). The phase is based on the study's objective, the number of participants, and other characteristics. There are five phases: Early Phase 1 (formerly listed as Phase 0), Phase 1, Phase 2, Phase 3, and Phase 4. Not Applicable is used to describe trials without FDA-defined phases, including trials of devices or behavioral interventions.
  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Eligibility Criteria

The key requirements that people who want to participate in a clinical study must meet or the characteristics they must have. Eligibility criteria consist of both inclusion criteria (which are required for a person to participate in the study) and exclusion criteria (which prevent a person from participating). Types of eligibility criteria include whether a study accepts healthy volunteers, has age or age group requirements, or is limited by sex.

Ages Eligible for Study

  • Adult
  • Older Adult

Accepts Healthy Volunteers

Yes

Description

Inclusion Criteria:

  • Age 18 years or older
  • For ozone group: Patients scheduled to receive systemic ozone therapy for fibromyalgia, chronic pain, knee osteoarthritis, disk herniation, peripheral arterial disease, or sports injuries
  • For control group: Healthy individuals matched for age (±2 years), sex, and body mass index (BMI, ±3 kg/m²)
  • Willing and able to provide written informed consent
  • Willing and able to comply with all study procedures and follow-up visits

Exclusion Criteria:

  • History of ocular surgery or trauma
  • Corneal dystrophy or degeneration
  • Glaucoma or ocular hypertension
  • Diabetes mellitus
  • Contact lens use within the preceding 3 months
  • Active ocular surface disease
  • Systemic conditions known to affect corneal endothelium
  • Any condition that, in the opinion of the investigator, would interfere with study participation or evaluation of outcomes

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

  • Primary Purpose: Other
  • Allocation: Non-Randomized
  • Interventional Model: Parallel Assignment
  • Masking: None (Open Label)

Arms and Interventions

Participant Group / Arm

Participant Group / Arm

A group or subgroup of participants in a clinical trial that receives a specific intervention/treatment, or no intervention, according to the trial's protocol.
Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.
Experimental: Ozone Therapy Group
48 participants receiving systemic ozone therapy via major autohemotherapy (MAH) at a concentration of 40 µg/mL, administered once daily for 10 consecutive sessions.
Other: Systemic Ozone Therapy
Major autohemotherapy (MAH) using a medical-grade ozone generator (Ozonosan Alpha Plus, Herrmann Apparatebau GmbH, Germany). Approximately 100 mL of the patient's blood is drawn into a sterile glass bottle containing 3.8% sodium citrate as an anticoagulant. The blood is then exposed to an ozone-oxygen mixture at a concentration of 40 µg/mL. The ozonated blood is gently mixed and reinfused intravenously over 10-15 minutes. The procedure is performed once daily for 10 consecutive sessions.
No Intervention: Control Group
48 age- and sex-matched healthy individuals who did not receive ozone therapy.

What is the study measuring?

Primary Outcome Measures

Primary Outcome Measures

In a clinical study's protocol, the planned outcome measure that is the most important for evaluating the effect of an intervention/treatment. Most clinical studies have one primary outcome measure, but some have more than one.
Outcome Measure
Measure Description
Time Frame
Change in Coefficient of Variation (CV) of cell area from baseline to 6 months
Time Frame: Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Coefficient of variation in endothelial cell area (%), measured by non-contact specular microscopy (Topcon SP-3000P).
Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Change in Endothelial Cell Density (ECD) from baseline to 6 months
Time Frame: Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Endothelial cell density (cells/mm²) measured by non-contact specular microscopy (Topcon SP-3000P) from the central cornea.
Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Change in Hexagonality (Hex%) from baseline to 6 months
Time Frame: Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Percentage of hexagonal corneal endothelial cells measured by non-contact specular microscopy (Topcon SP-3000P).
Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)

Secondary Outcome Measures

Secondary Outcome Measures

In a clinical study's protocol, a planned outcome measure that is not as important as the primary outcome measure for evaluating the effect of an intervention but is still of interest. Most clinical studies have more than one secondary outcome measure.
Outcome Measure
Measure Description
Time Frame
Change in Malondialdehyde (MDA) levels from baseline to 6 months
Time Frame: Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Serum malondialdehyde (MDA) levels (µmol/L) measured by thiobarbituric acid reactive substances (TBARS) method. Assessed only in the ozone group.
Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Change in Superoxide Dismutase (SOD) activity from baseline to 6 months
Time Frame: Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)
Serum superoxide dismutase (SOD) activity (U/mL) measured by spectrophotometric method. Assessed only in the ozone group.
Baseline (T0), 1 month (T1), 3 months (T2), and 6 months (T3)

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Sponsor

The organization or person who initiates the study and who has authority and control over the study.
Yuzuncu Yil University

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

  • 1. PubMed: (Makalenin PMID'si varsa girin, yoksa boş bırakın) 2. Other: Bocci V. Ozone: A New Medical Drug. 2nd ed. Dordrecht: Springer; 2011. 3. Other: Franzini M, Valdenassi L, Pandolfi S, Tirelli U, Ricevuti G, Chirumbolo S. The role of ozone as an Nrf2-Keap1-ARE activator in the anti-microbial activity and immunity modulation of infected wounds. Antioxidants. 2023;12(11):1985. doi:10.3390/antiox12111985 4. Other: Malatesta M, Tabaracci G, Pellicciari C. Low-dose ozone as a eustress inducer: Experimental evidence of the molecular mechanisms accounting for its therapeutic action. Int J Mol Sci. 2024;25(23):12657. doi:10.3390/ijms252312657 5. Other: Cheng Y, Lu Y, Du Y, Zhao Y, Zhuang X. A clinical study on ozone autohemotherapy for the treatment of acute ischemic stroke. Front Neurol. 2025;16:1583726. doi:10.3389/fneur.2025.1583726

Helpful Links

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

Study Start

The actual date on which the first participant was enrolled in a clinical study. The "anticipated" study start date is the date that the researchers think will be the study start date.
January 14, 2026

Primary Completion (Actual)

Primary Completion

The date on which the last participant in a clinical study was examined or received an intervention to collect final data for the primary outcome measure. Whether the clinical study ended according to the protocol or was terminated does not affect this date. For clinical studies with more than one primary outcome measure with different completion dates, this term refers to the date on which data collection is completed for all the primary outcome measures. The "anticipated" primary completion date is the date that the researchers think will be the primary completion date for the study.
July 14, 2026

Study Completion (Actual)

Study Completion

The date on which the last participant in a clinical study was examined or received an intervention/treatment to collect final data for the primary outcome measures, secondary outcome measures, and adverse events (that is, the last participant's last visit). The "anticipated" study completion date is the date that the researchers think will be the study completion date.
July 14, 2026

Study Registration Dates

First Submitted

First Submitted

The date on which the study sponsor or investigator first submitted a study record to ClinicalTrials.gov. There is typically a delay of a few days between the first submitted date and the record's availability on ClinicalTrials.gov (the first posted date).
July 16, 2026

First Submitted That Met QC Criteria

First Submitted That Met QC Criteria

The date on which the study sponsor or investigator first submits a study record that is consistent with National Library of Medicine (NLM) quality control (QC) review criteria. The sponsor or investigator may need to revise and submit a study record one or more times before NLM's QC review criteria are met. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
July 16, 2026

First Posted (Actual)

First Posted

The date on which the study record was first available on ClinicalTrials.gov after National Library of Medicine (NLM) quality control (QC) review has concluded. There is typically a delay of a few days between the date the study sponsor or investigator submitted the study record and the first posted date.
July 21, 2026

Study Record Updates

Last Update Posted (Actual)

Last Update Posted

The most recent date on which changes to a study record were made available on ClinicalTrials.gov. There may be a delay between when the changes were submitted to ClinicalTrials.gov by the study's sponsor or investigator (the last update submitted date) and the last update posted date.
July 21, 2026

Last Update Submitted That Met QC Criteria

Last Update Submitted That Met QC Criteria

The most recent date on which the study sponsor or investigator submitted changes to a study record that are consistent with National Library of Medicine (NLM) quality control (QC) review criteria. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
July 16, 2026

Last Verified

Last Verified

The most recent date on which the study sponsor or investigator confirmed the information about a clinical study on ClinicalTrials.gov as accurate and current. If a study with a recruitment status of recruiting; not yet recruiting; or active, not recruiting has not been confirmed within the past 2 years, the study's recruitment status is shown as unknown.
July 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

Other Study ID Numbers

Identifiers or ID numbers other than the NCT number that are assigned to a clinical study by the study's sponsor, funders, or others. These numbers may include unique identifiers from other trial registries and National Institutes of Health grant numbers.
  • GOKAEK/2026-01-10 (Other Identifier: No other identifiers)

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

YES

IPD Plan Description

ndividual participant data (IPD) will be shared with researchers who provide a methodologically sound proposal for meta-analysis or secondary analyses.

IPD Sharing Time Frame

Data will be available starting 6 months after publication and ending 3 years after publication.

IPD Sharing Access Criteria

Data access will be granted upon reasonable request to the corresponding author after approval of a research proposal.

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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