Comparative analysis of different nuclear transfer techniques to prevent the transmission of mitochondrial DNA variants

Abstract

Prevention of mitochondrial DNA (mtDNA) diseases may currently be possible using germline nuclear transfer (NT). However, scientific evidence to compare efficiency of different NT techniques to overcome mtDNA diseases is lacking. Here, we performed four types of NT, including first or second polar body transfer (PB1/2T), maternal spindle transfer (ST) and pronuclear transfer (PNT), using NZB/OlaHsd and B6D2F1 mouse models. Embryo development was assessed following NT and mtDNA carry-over levels were measured by next generation sequencing (NGS). Moreover, we explored two novel protocols (PB2T-a and PB2T-b) to optimize PB2T using mouse and human oocytes. Chromosomal profiles of NT-generated blastocysts were evaluated using NGS. In mouse, our findings reveal that only PB2T-b successfully leads to blastocysts. There were comparable blastocyst rates amongst PB1T, PB2T-b, ST and PNT embryos. Furthermore, PB1T and PB2T-b had lower mtDNA carry-over levels than ST and PNT. After extrapolation of novel PB2T-b to human in vitro matured (IVM) oocytes and in vivo matured oocytes with smooth endoplasmic reticulum aggregates (SERa) oocytes, the reconstituted embryos successfully developed to blastocysts at a comparable rate to ICSI controls. PB2T-b embryos generated from IVM oocytes showed a similar euploidy rate to ICSI controls. Nevertheless, our mouse model with non-mutated mtDNAs is different from a mixture of pathogenic and non-pathogenic mtDNAs in a human scenario. Novel PB2T-b requires further optimization to improve blastocyst rates in human. Although more work is required to elucidate efficiency and safety of NT, our study suggests that PBT may have the potential to prevent mtDNA disease transmission.

Improvement of early developmental competence of postovulatory‐aged oocytes using metaphase II spindle injection in mice

“…However, the blastocyst rate of the FF group was lower than the rate (87.3%) reported by a previous study using ST, 45 probably because in MESI, the oocyte membrane was broken, decrease the durableness. In addition, MESI requires higher technical expertise than that required by ST.…”

New avenues for therapy in mitochondrial optic neuropathies

“…54 There are two main methods being used in the UK, namely maternal spindle transfer (ST), which involves transferring spindle-chromosome complexes from donors, and pronuclear transfer (PNT), which involves transferring pronuclei from donors. 55 MtDNA carryover in PNT blastocytes is less than 2% 56 while the carryover in ST is less than 1%. 57 Aside from ethical issues, inheriting disease-associated mutated DNA does not mean that the individual will develop vision loss in LHON or ADOA, due to incomplete penetrance.…”

State of the art of nuclear transfer technologies for assisting mammalian reproduction

“…Interestingly, 25 years have passed, and no reports of births in other mammal species have been published. The advantages of PB2‐NT are like those of PB2‐NT concerning the genetic potential, the number of mitochondria introduced during the procedure, and the possibility of easy micromanipulation without inducing chromosome damage (Tang et al, 2019). Murine blastocyst PB2‐NT rates are like those observed for the other NT technologies (Tang et al, 2019; T. Wang et al, 2014).…”

“…The advantages of PB2‐NT are like those of PB2‐NT concerning the genetic potential, the number of mitochondria introduced during the procedure, and the possibility of easy micromanipulation without inducing chromosome damage (Tang et al, 2019). Murine blastocyst PB2‐NT rates are like those observed for the other NT technologies (Tang et al, 2019; T. Wang et al, 2014). In this species, pronuclei can be easily visualized under a bright light microscope, and the PN size and closeness to the PB2 can reveal its origin.…”