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 < 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 < 0.0001), CU to 315.7 mV ± 4.4 (p < 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 < 0.00001), ASC 154.1 mV ± 3.1 (p < 0.00001) and BME 136.1 mV ± 9.6 (p < 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
Study question Does the evaluation of oxidation-reduction potential (ORP) levels in blood plasma from subfertile patients and oocyte donors represents the ORP status in follicular fluid? Summary answer ORP levels in blood plasma from oocyte donors and sub fertile patients could be an indicator of oxidative stress in follicular fluid What is known already Oxidation-reduction potential (ORP) measurement of seminal plasma is one method to diagnose oxidative stress in male patients, which can help physician to recommend antioxidants administration when it is elevated. Seminal plasma is easily accessible by masturbation. An empirical analogue for semen could be follicular fluid (FF). Unfortunately, it is very difficult to access to FF, unless a surgical procedure is performed. Hence, it is necessary to find indirect parameters to evaluate the ORP status in FF. Since blood plasma (BP) circulation provides antioxidants to the FF, we studied the BP ORP measurement to indirectly determine ORP levels in FF. Study design, size, duration Prospective study conducted at CITMER, Mexico from December 2022 to January 2023. We included under informed consent 15 oocyte donors (age 26.8 ± 4.06 years old) and 55 patients (age 35.2 ± 4.8 years old) undergoing IVF/ICSI. Participants/materials, setting, methods ORP levels in BP and FF from dominant follicles were measured at the same time of oocyte collection with MiOXSYS system. Oocytes were inseminated and zygotes were cultured until blastocysts stage. We calculated correlation of a) ORP levels in FF vs BP, b) ORP of FF and BP vs number of retrieved oocytes, c) ORP of FF and BP vs number of fertilized oocytes, d) ORP of FF and BP vs number of blastocysts Main results and the role of chance A total of 172 oocytes from oocyte donors were collected. After fertilization check, 135 zygotes (78%) were observed. A total number of 55 blastocysts (31%) were observed at day 5 + day 6. For patients, a total of 550 oocytes were collected, 360 zygotes were observed (65%) and 164 blastocysts at day 5 + day 6 were obtained (45%). The correlation between ORP levels from FF and BP for oocyte donors was 0.90 and 0.51 for patients. The overall ORP levels in FF and BP in patients were 136.2 ± 14.9 mV and 148.3 ± 15.97 mV, respectively. For oocyte donors, ORP levels in FF and BP were 160.3 ± 13.8 mV and 170.28 ± 17.67mV, respectively. There was a mild correlation of ORP levels in BP with numbers of oocytes retrieved (r = 0.25). The correlation of BP ORP with oocyte fertilization was 0.24 in patients with autologous oocyte utilization. The mean ORP levels of FF were 136.2 ± 14.9 mV, versus a mean ORP of 148.3 ± 15.97 mV in BP. The FF ORP levels between 100-150 mV [LRD1] were moderately correlated and predictive of blastocyst development [LRD2]. For donor oocytes there was a weak negative correlation [LRD3] of FF ORP with oocyte fertilization (-0.06) and a weak negative correlation for blastocyst formation was found (-0.18). Limitations, reasons for caution The MiOXSYS System ( Careous Biotechnology, Lituania) is a device calibrated to measure ORP levels in seminal plasma but not to measure ORP levels in FF nor BP. The number of analyzed data should be increased. Wider implications of the findings According to this study, the use of blood plasma to surrogate the measurements of ORP levels in follicular fluid could represent a further strategy to include female patients to be treated by antioxidants before an IVF treatment. Trial registration number None
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