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 human recombinant hyaluronidase (Cumulase®) improve fertilization and blastocyst formation rates in sibling human oocytes compared to bovine-derived hyaluronidase? Summary answer The use of Cumulase® for oocyte denudation does not appear to affect fertilization or usable blastocyst formation rate in patients undergoing intracytoplasmic sperm injection (ICSI). What is known already Bovine-derived hyaluronidase has been widely employed to denude the cumulus-oocyte complexes (COCs) prior to ICSI or oocyte vitrification. Nonetheless, the recent concern for its safety, due to its animal origin, has led to the development of alternatives ascertaining the enzyme’s purity. It has been reported that human recombinant hyaluronidase is not equally effective as its bovine-derived analogue, leading to a longer time required for complete denudation coupled by the subsequent toxicity risk for oocytes. This may result to lower fertilization and blastocyst formation rates. Although there are some studies on IVF outcomes with recombinant hyaluronidase, its use remains rather limited. Study design, size, duration This randomized double-blinded clinical trial was conducted at Citmer Reproductive Medicine, in Puebla and Monterrey, Mexico from February to December 2021. The impact of denudation on sibling oocytes from 103 patients employing bovine-derived hyaluronidase (InVitroCare®) or human recombinant hyaluronidase (ICSI Cumulase® (rHuPH20), CooperSurgical®) was evaluated. A total of 1237 oocytes were assigned as follows: The Hyaluronidase Group included 626 oocytes, while the Cumulase® Group included 611 oocytes. Preimplantation Genetic Testing was performed in 18 cycles. Participants/materials, setting, methods After retrieval, COCs were incubated for 2-4 h and placed in microdropets with 10 IU/mL bovine-derived hyaluronidase or Cumulase® in culture media GlobalTotal® (LifeGlobal®) for granulosa cells removal. COCs were gently pipetted up and down employing 20 μL and 150 μm tips. Metaphase II oocytes were injected and incubated for 17-20 hours in 8% CO2, 20% O2, 37°C. Normally fertilized zygotes were cultured to blastocyst stage and embryo development was evaluated. Seventy-nine blastocysts were biopsied. Main results and the role of chance The mean age of the patients was 32.12±1.11 years old. A total of 506 and 493 metaphase II oocytes from Groups 1 and 2 respectively were subjected to ICSI. Normal fertilization rates for oocytes treated with Hyaluronidase and Cumulase® were 71.34% (n = 361) and 72.41% (n = 357) respectively, with no statistically significant differences between the two groups (p = 0.35). Usable blastocysts rate (selected for embryo transfer or blastocyst cryopreservation) did not differ with any statistical difference between the bovine-derived hyaluronidase (n = 170, 47.09%) and the Cumulase® treated oocytes (n = 158, 44.25%), (p = 0.44). Preimplantation genetic testing for aneuploidies (PGT-A) was performed in 18 cycles. Seventy-nine blastocysts were biopsied and tested. Fourty-seven embryos from the bovine-derived hyaluronidase group were biopsied, presenting with a euploidy rate of 51.06% (n = 24), compared to 32 embryos from the recombinant hyaluronidase group with a euploidy rate of 50% (n = 16). Results indicated no statistically significant difference (p = 0.92). These data support that human recombinant hyaluronidase presents with similar efficacy to bovine-derived hyaluronidase. in terms of normal fertilization, embryo development as well as euploidy rates. Limitations, reasons for caution The small sample size regarding the euploidy outcome stands as a limitation of the study. Further studies should focus on the effect of recombinant hyaluronidase on genetic anomalies on blastocysts. Wider implications of the findings Fertilization, usable blastocyst and euploidy rates did not differ when employing bovine-derived or human recombinant hyaluronidase, rendering the latter a safe and efficient alternative. However, it is important to assess long-term effects, as only a limited number of studies report on perinatal and neonatal outcomes Trial registration number 25-12-21MTYPUECIT
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