P-226 Efficiency of different antioxidants in reducing the supra-physiological Oxidation Reduction Potential Levels in human embryo culture media
Authors: Maldonado Rosas I , Ramirez Dominguez L.B , Jimenez Medina I , Villar Munoz L.G , Solorzano Vazquez J.F , Simopoulou M , Agarwal A 
Published: June 2022

Abstract

Study question
Does every antioxidant compounds perform similarly in reducing the excessive levels of Oxidation Reduction Potential (ORP) in IVF culture media?

Summary answer
Not all antioxidant compounds perform similarly in maintaining physiological ORP levels in IVF culture media.

What is known already
During assisted reproduction technologies (ART), several external factors are responsible for disturbing the ORP equilibrium in gametes and embryos. Therefore, antioxidant supplementation of culture media has been evaluated and reported. However, there is lack of consensus on the efficiency of antioxidant action in embryo culture media. Antioxidants are known to lower the levels of free radical concentration to physiological levels, preventing cellular damage caused by oxidative stress. Therefore, it is important to determine which antioxidant compound are the most effective in reducing ORP levels in embryo culture media.

Study design, size, duration
In this prospective study was performed at CITMER Reproductive Medicine, Mexico city. ORP levels were measured by MiOXSYS System (UAB Caerus Biotechnologies, Vilnius, Lithuania) in embryo-free culture medium (Global® Total®, LifeGlobal®, Connecticut, US). Embryo-free culture medium was supplemented with cumene hydroperoxide and seven different antioxidants were tested each day for 5 continuous days (n = 40). Follicular fluid ORP levels from dominant follicles of 40 oocyte donors were used as control and considered as a physiological target.

Participants/materials, setting, methods
Cumene hydroperoxide (CH) 1mM, was utilized to increase ORP values in culture media from 224 mV to 415 mV. The following compounds considered as antioxidants according to literature were supplemented at 2 mM to the CH culture media: Ascorbic acid (ASC), L-Carnitine (CAR), L-Cysteine (CYS), Glutathione (GLT), Curcumin (CU), Resveratrol (RSV) and b-Mercaptoethanol (BME). ORP levels were quickly evaluated each time.

Main results and the role of chance
In this study, CH was used as a culture media oxidant to measure the antioxidants capacity in lowering the excessive ORP values of CH culture media. ORP in CH solution was 415 mV ± 4.3. All antioxidants showed a statistically different capacity (p &lt; 0.05) to lower the ORP values when added to CH solution: RSV decreased the ORP to 348.3 mV ± 3.6 p = 0.0449, CAR to 316.32 mV ± 4.7 (p &lt; 0.0001), CU to 315.7 mV ± 4.4 (p &lt; 0.0001) and GLT to 258.4 mV ± 15.1 (p = 0.00002). The compounds that showed higher antioxidant capacity were CYS 158.4 mV ± 4.7 (p &lt; 0.00001), ASC 154.1 mV ± 3.1 (p &lt; 0.00001) and BME 136.1 mV ± 9.6 (p &lt; 0.00001). CYS, ASC and BME ORP values reached the physiological ORP values found in follicular fluid from donors, which are 89 mV±23.6 mV.

Limitations, reasons for caution
Further studies should focus on the comparison between physiological dynamics of antioxidant supplementation on culture media and molecular and biochemical effect of antioxidants on embryos and gametes.

Wider implications of the findings
According to our results, CYS, ASC and BME were significantly more potent antioxidants in lowering the ORP in culture medium to physiological levels. The measurement of antioxidant capacity in lowering excess of ROS in culture media may assist in developing the most effective antioxidant supplements, thus improving ART outcomes.

Trial registration number
none