mtDNA can be used to look at carryover and persistence of mtDNA with the spindle or pronucleus.
Studies in rhesus macaque monkeys14 suggest that MST is efficient, allowing replacement of almost all mitochondria and the birth of four healthy offspring that show no abnormities after two years15. Rates of development to blastocyst stages and pregnancy rates were essentially the same as for control unmanipulated embryos.
Studies using PNT with human zygotes are more difficult to gauge. The published study to date16 used only abnormally-fertilised human oocytes, for example those containing three pronuclei. Embryos were reconstituted so that they had a normal complement of one maternal and one paternal pronucleus. The efficiency of blastocyst formation was about half that of control blastocysts. However, as the control blastocysts were not always good quality embryos and the PNT embryos were from abnormally fertilised oocytes, it is difficult to assess the efficiency of blastocyst formation. Nevertheless, all evidence presented to the panel (both published and unpublished) suggested that the blastocysts obtained after PNT and MST were as normal as controls, including cell numbers in the trophoblast and inner cell mass, and markers of cell type and stage.
4.2.5 Overall MST appears less efficient than PNT, which may reflect problems in visualising the spindle and perhaps in transferring all the chromosomes. In mature metaphase II oocytes, the chromosomes are lined up attached to the spindle in preparation for the second meiotic division, rather than being enclosed in a pronuclear membrane, as is the case for PNT. Chromosomes may, therefore, be left behind, although this can be checked using fluorescent dyes that label DNA or chromatin. However, more recent studies have made use of new polarised light microscopy techniques, and these seem
14 Tachibana M., M. Sparman, et al. (2009) “Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 461, 367-372; Tachibana et al. (2010) Chromosome transfer in mature oocytes.” Nature P r o t o c o l s , 5 ( 6 ) : 1 1 3 8 - 1 1 4 7 . D r M i t a l i p o v , O r e g o n U n i v e r s i t y , o r a l p r e s e n t a t i o n t o p a n e l . 1 5 Although the experimental numbers are small, it would be both costly and ethically unacceptable to extend these studies to many macaques.
16 Craven L., H. A. Tuppen, et al. (2010) “Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease.” Nature 465(7294):82-5
Page 17 of 45