In the process of ART treatment, patients encounter many cycles of treatment failure due to poor embryo quality. There are many factors affecting embryo development, such as culture medium [16], CO2 concentration [17], temperature [17], in vitro operations [18, 19], as well as sperm and oocyte quality [20, 21]. It can be considered that during ART treatment, the quality of oocytes is one of the most critical factors that determine the embryo status and clinical outcome. Any factor affecting the quality of the oocyte will affect its fertilization and subsequent embryo development [22], and ultimately the cycle outcome of ART treatment. In this study, an appropriate concentration of MT was added to the embryo culture medium and the embryo development and clinical treatment outcomes of the patients were observed, to understand whether the application of MT has a positive impact on embryo development.
According to research reports, embryos are in a relatively high oxygen environment during in vitro culture, which will cause oxidative stress due to the imbalance of the antioxidant defense system, resulting in a large number of ROS produced by oocytes and embryos during development, thus affecting the quality of oocytes and outcome of the embryo development in vitro [23]. MT is a highly effective antioxidant with strong antioxidant activity. Animal study has shown that the addition of MT to the embryo culture medium can improve embryo development in vitro, and it has also been confirmed that the improvement is concentration-dependent [4]. In 2020, our team found that adding MT to the human IVM medium can improve the IVM outcome of human immature oocytes by promoting mitochondrial function and inhibiting damage due to oxidative stress, and 10− 5 M was found to be the optimal concentration [10]. Based on the conclusions of the previous studies, two main questions were posed: (1) can adding MT to the culture medium of the human embryos improve the in vitro development and clinical outcome of the embryos? And (2) is its effect also correlated to concentration? Therefore, in this study, human immature oocytes in the COH cycle were collected for IVM to obtain IVM-MII oocytes and ICSI insemination was conducted. Next, the fertilized oocytes were cultured in vitro in culture medium containing 0, 10− 11, 10− 9, 10− 7 or 10− 5 M MT, and a systematic comparative analysis was performed on the fertilization of oocytes and early embryo development in each group. The fertilization, cleavage, blastocyst and high-quality blastocyst rates of the 10− 9 M group were all significantly higher than those of the 0 M group. The blastocyst and high-quality blastocyst rates of the 10− 9 M group were significantly different from those of the 10− 5 M group, and the blastocyst rate was also significantly different compared to the 10− 11 M group. In addition, the fertilization, cleavage, blastocyst and high-quality blastocyst rates of the 10− 9 M group were higher than those of the 10− 7 M group, but these differences were not significant. It has been reported that the fertilization rate, cleavage rate and total blastocyst cell number of porcine IVF embryos cultured in medium containing 10− 9 M MT were significantly increased [24]. Wang et al. [4] added different concentrations of MT to the culture medium of bovine embryos in vitro and found that 10− 9 M MT could significantly promote embryo development. These results are consistent with the results of the present study. These results showed that the addition of 10− 9 M MT to human embryo culture medium could significantly improve the fertilization of human oocytes and embryo development in vitro obtained after fertilization, and 10− 9 M was the optimal concentration.
Next, 10− 9 M was selected as the MT concentration to be added to the embryo culture medium of patients with failed IVF/ICSI cycles for embryo culture in vitro. The study found that in terms of fertilization, cleavage, high-quality embryo, blastocyst and high-quality blastocyst rates, the values of the 10− 9 M group were significantly higher than those of the 0 M group. In this study, further group analysis was also carried out according to the number of failed IVF/ICSI cycles and it was discovered that the application of 10− 9 M MT to embryo culture in vitro could significantly improve embryonic development in the repeated cycle of each group. A large number of studies have found that adding MT to the culture medium can reduce the levels of ROS in oocytes and promote embryonic development of cattle, mice, sheep, and pigs [7, 25–27]. Nakano et al. [28] confirmed that the addition of MT during embryo culture in vitro can reduce the level of ROS in parthenogenetic embryos and promote embryo development. These findings are consistent with the results of the current study, suggesting that the improvement of embryonic development in patients with failed IVF/ICSI cycles by MT may be related to the highly effective antioxidant properties of MT. Oxidative stress inhibits oocyte maturation and embryo development, and MT has a strong antioxidant effect, which can resist oxidative stress, maintain the balance of antioxidant system, reduce ROS content in the embryo, and promote gamete maturation and embryo development. In this study, 50 cycles of vitrified-warmed embryo transfers with MT intervention have been performed, and a biochemical pregnancy rate of 64% was achieved, as well as a clinical pregnancy rate of 40%. This result shows that the application of 10− 9 M MT to embryo culture medium in vitro can significantly improve the clinical treatment effect of patients with failed IVF/ICSI cycles, which greatly enhance our confidence in treating such patients, and thereby increases the overall success rate of ART treatment.
In conclusion, the application of MT to embryo culture medium in vitro can improve embryonic development for patients with repeated cycles after failed IVF/ICSI cycles and lead to good clinical outcomes. The optimal concentration of MT was 10− 9 M.