The press-assisted fusion scheme greatly reduces the amount of HVJ-E required in mitochondrial replacement techniques

Abstract

Abstract Background Mitochondrial replacement techniques (MRTs) afford pathogenic mitochondria carried women an opportunity to have related disease-free offspring with a genetic link. Among the fusion methods, HVJ-E-induced fusion has been considered the most promising method for MRTs clinical translation. Although HVJ-E has been confirmed to have no RNA activity, a decrease in blastocyst quality was observed in several MRTs studies with HVJ-E-induced fusion scheme. Nevertheless, HVJ-E has not been proven to be a single factor affecting embryonic development in MRTs. Safety has been the biggest obstacle for its clinical application. Methods Pronuclear transfer (PNT) was performed on mouse zygotes and human abnormal zygotes (3PN,1PN) with the traditional HVJ-E-induced fusion (original HVJ-E) and press-assisted HVJ-E-induced fusion (1%HVJ-E). Fusion rates and residual amount of HVJ-E (the relative HVJ-E fluorescence intensity) in reconstructed mouse and human zygotes were assessed. Cleavage rate, blastocyst formation rate, intracellular ROS levels and double-stranded DNA breaks (γH2A.X) of reconstructed mouse zygotes in traditional fusion and press-assisted fusion groups were assessed. Results No significant differences were observed in the fusion rates of the press-assisted fusion and traditional fusion group in mouse zygotes and human 3PN/1PN zygote. The relative HVJ-E fluorescence intensity of the press-assisted fusion group was greatly lower than traditional fusion group in mouse and human. The relative ROS fluorescence intensity and the γH2A.X loci of the press-assisted fusion group were lower than that in the traditional group. The blastocyst formation rates in the press-assisted fusion were higher than hat in the traditional fusion group. Conclusions In this study, we proved high concentration of HVJ-E used in traditional HVJ-E fusion scheme is an independent factor affecting embryonic development in MRTs, which might be caused by enhanced DNA damage due to increased ROS levels in reconstructed embryos. In order to minimize the amount of HVJ-E attached to the reconstructed zygotes without reducing the fusion efficiency in MRTs, we designed a new scheme for HVJ-E-induced fusion: the press-assisted fusion, which is beneficial to decrease the adverse factors affecting embryo development in MRTs.