Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease

Abstract

Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases1. Reproductive technologies designed to uncouple the inheritance of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof of concept studies involving abnormally fertilized human zygotes2 were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. Following optimisation, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.

Assisted reproductive technology in reproductive medicine — possibilities and limitations

“…12 www. journals.viamedica.pl/ginekologia_polska has been approved by the FDA in the US, it is the source of much controversy in many countries [28,29,36].…”

Assisted reproductive technologies to prevent human mitochondrial disease transmission

“…However, because mitochondria are thought to be quiescent during pre-implantation stages of development, the panel recommended carefully examining the behavior of carried-over mtDNA in a context where mtDNA replication occurs, namely in ES cells derived from the embryos and in differentiated cell types obtained from these (http://hfeaarchive.uksouth.cloudapp.azure.com/www.hfea.gov.uk/docs/Third_ Mitochondrial_replacement_scientific_review.pdf). All three studies summarized above [38][39][40] derived ES cells from blastocysts generated by MST or PNT and, in the case of Kang et al 38 and Yamada et al 42 , also by somatic cell nuclear transfer. In most cases, these lines were passaged extensively in order to determine whether the initial low levels of carried-over mtDNA were maintained after multiple rounds of cell division in vitro.…”

“…Some PNT protocols 40 yielded blastocysts in which OXPHOS transcript levels varied but were indistinguishable (by unsupervised hierarchical clustering) from controls, regardless of whether donor karyoplast and recipient cytoplasts contained the same or different mtDNA haplotypes 40 .…”

“…Similar observations were made when analyzing embryos generated using MST and artificial oocyte activation 39 . Hyslop et al 40 reported a new PNT protocol, involving the transplantation of pronuclei soon after the completion of meiosis, which was important to improve the survival rates of reconstituted embryos when using normally fertilized zygotes. Carryover in PNT blastocysts was <2% in 79% of blastocysts and none had >5% carryover.…”

Prévenir la transmission des mutations de l’ADN mitochondrial par don de cytoplasme : où en est-on ?

“…Le taux d'hétéroplasmie mesuré sur les embryons issus de cette reconstruction a montré une très faible contamination par l'ADNmt du zygote A (moins de 2 %), transféré de façon artéfactuelle avec le karyoplaste. Récemment, la même équipe a appliqué cette procédure à des zygotes humains normaux (diploïdes) et a montré i) que le taux d'ADNmt contaminant restait faible, ii) que les embryons dérivés ne présentaient pas un excès d'anomalies chromosomiques, et iii) qu'ils avaient un profil d'expression génique comparable aux témoins [9]. Cependant, la culture de cellules souches embryonnaires dérivées à partir de cinq embryons ainsi reconstitués, était associée dans un cas à une réversion géné-tique aboutissant à une colonisation de la lignée par l'ADNmt issu du karyoplaste, ADN qui devenait alors très rapidement majoritaire.…”