P-197 Nuclear transfer with almost nonexistent mitochondrial carryover -a mouse model-
Authors: Okamoto R , Xiao W , Fukasawa H , Hirata S , Masuyama H , Otsuki J 
Published: June 2023

Abstract

Study question
Can the transfer of aggregated chromosomes (AC) as compared to spindle-chromosome transfer (SCT) reduce mitochondrial DNA (mtDNA) carryover and improve development rates?

Summary answer
The rate of mtDNA carryover after AC transfer was only 0.1%, which was much less than that of 2.0% after SCT.

What is known already
Nuclear transfer techniques such as SCT and pronuclear transfer have been applied to prevent mitochondrial diseases and to treat early embryonic arrest, however, the problem of mtDNA carryover has not yet been resolved. We previously reported on an AC transfer (ACT) method in humans which could overcome the above problem, however, consequent embryonic development rates have not been investigated in Japan due to legal restrictions and no animal study has been conducted, as the formation of ACs has not been found in any other species.

Study design, size, duration
Following our success in the creation of ACs in mouse oocytes using IBMX (3-Isobutyl 1-methylxanthine), a phosphodiesterase inhibitor, a mouse model of ACT was established. This was followed by a comparison of the rates of embryo development and mtDNA carryover in both ACT and SCT in over 100 oocytes. The distribution of mitochondria around SC and AC were also examined by confocal laser microscope (FV3000, Olympus, Japan).

Participants/materials, setting, methods
GV stage oocytes were collected from 8-12 week B6D2F1 mice. After GVBD, oocytes were incubated in KSOM medium, containing 3 mM IBMX, for 10-14 hours until AC is formed. ACs and SCs were injected into enucleated MII stage donor oocytes and then fertilized at the MII stage. Embryos were cultured for 5 days to compare the rates of embryo development. Enucleated ACs and SCs were subjected to real-time PCR, to analyze the carryover of mtDNA.

Main results and the role of chance
The rates of fertilization, embryo cleavage and blastocyst formation in AC oocytes without AC transfer (control), in ACT and in SCT were 60.7%, 55.9%, 13.2% (control), 82.5%, 80.8% and 30.8% (ACT), and 81.0%, 74.5% and 31.4% (SCT) respectively. The ACT group showed a significant increase in fertilization (P = 0.007), embryo cleavage (p = 0.008) and blastocyst formation (p = 0.034) rates, as compared to those in the control group. There were no significant differences between ACT and SCT regarding the rates of fertilization, embryo cleavage and blastocyst formation. The relative, real-time PCR showed that mtDNA carryover in the ACT group was 0.1% ± 0.07%, which was significantly lower than that in the SCT group of 2.0% ± 0.6% (p = 0.027). 3D confocal laser microscopy revealed that mitochondria were not localized around either SCs or ACs, as they were around pronuclei.

Limitations, reasons for caution
Although we succeeded in establishing a mouse model of ACT, validation in human oocytes is required to confirm the benefit of this method. Further experiments are required to confirm if healthy offspring can be obtained after ACT.

Wider implications of the findings
ACT could reduce the amount of mtDNA carryover and overcome heteroplasmy. As ACT improved the low embryo development rate caused by IBMX and had similar embryo development rates to SCT, the findings of this study may give rise to ACT being employed as a novel method for human nuclear transfer.

Trial registration number
not applicatable