Mitochondrial Replacement Techniques: Ethical, Social, and Policy Considerations (2016)

• Enucleation in 3 percent sucrose
• Transfer of C57BL/6J spindle-chromosome complexes to Kunming enucleated oocytes
• 142 oocyte-karyoplast reconstructed pairs fused by 1-3 rounds of electrofusion
• 11 fused oocytes used in IVF
• Transfer of eight 1-4 cell stage embryos into two foster mothers
  
  

• 15 MST embryos transferred into 9 females: 6 with 1-2 blastocysts, 3 with 2 cleavage stage (4-8 cell) embryos

• mtDNA copy number in karyoplasts and cytoplasts
• 102 MST oocytes generated by MST
• Transfer of preselected female embryos; recovery of fetuses preterm (135 days post-embryo transfer)
  
  
  
  
  
  

• Transfer of fresh spindle to vitrified cytoplast and vice versa
• Implantation of four blastocysts derived by transfer of vitrified spindles into fresh oocyte cytoplasts

• Enucleation
• Fertilization (2 pronuclei and extrusion of sec ond polar body)
• Embryonic and developmental potential
• Nuclear-cytoplasmic relationship

• 100 percent enucleation
• 25 pairs (17.6 percent) successfully fused
• 9 fused oocytes (82 percent) successfully fertilized
• One foster mother (50 percent) delivered two “transfer” pups (C57BL/6J nucleus, Kunming cytoplasm/cellular or ganelles)
• Body weight of transfer offspring was in range for Kunming (oocyte donor) mice

• Visualization and isolation of intact MII spindle-chromosomal complexes
• Karyoplast fusion
• Developmental potential of embryos
• F1 health, mtDNA carryover

• Karyoplasts isolated by polarized microscopy contained approximately 1.5 percent of the volume of cytoplasts
• Fusion of karyoplast with SeV prevented premature activation of oocytes
• Live birth of four offspring (one set of twins, Mito and Tracker; two singletons, Spindler and Spindy)
• ND mtDNA carryover (using as says sensitive to detect >3 percent heteroplasmy)

• mtDNA carry over into karyoplasts
• Embryonic developmental potential
• Heteroplasmy in somatic tissues of preterm fetus and fetus oocytes, 135 days post-embryo transfer

• 0.6 percent carry over of mtDNA into karyoplast
• 62 percent of MST oocytes developed to blastocysts after fertilization
• Female MST embryos selected by TE biopsy; two female singlet on pregnancies generated from selected blastocysts
• mtDNA carry over (ND) or <0.5 percent (cerebrum, heart, and blood in fetus #2) in fetal somatic organs and tissues
• 11/12 oocytes in each fetus displayed low (< 5.5 percent) or ND levels of mtDNA heteroplasmy; one oocyte from each fetus contained substantial mtDNA carry over (16.2 percent and 14. 1 percent)

• Effect of cryo-thaw on fertilization, embryo development, and live birth following MST in oocytes

Cryo-thaw MST oocytes:

• Fresh spindle to vitrified cytoplast: impaired fertilization (50 percent) and blastocyst development (0 percent), compared with 91 percent and 57 percent in controls, respectively
• Vitrified spindle to fresh cytoplast: 88 percent fertilization and 68 percent blastocyst rate, similar to control (91 percent and 57 percent, respectively)

2009 rhesus offspring:

• Measurement of body weight, bloodwork, and mitochondrial function (birth-3 years)

Tachibana et al. (20 13)
(OHSU Group)


Human oocyte

• 106 donated oocytes: 65 underwent MST, 33 controls
• Reciprocal MST followed by ICSI

Paull et al. (2013)
(New York Stem Cell Foundation [NYSCF] Group)

Human oocyte

• 18 synchronized donated oocytes underwent reciprocal MST
• Fusion of spindle-chromosomal complex by SeV or electrical pulse
• Parthenogenetically activated
  
  
  
  

• MST and PNT: transfer of MII-SCC or pronuclei, respectively, from NZB/OlaHsd to B6D2/F1 oocytes and zygotes, respectively

• Overall health and postnatal development of rhesus macaque offspring from 2009 study

• Live birth of a female offspring (Crysta)

 2009 rhesus offspring:

• Normal de velopment
• No change in het eroplasmy in blood and skin samples

• Developmental potential
• Establishment and pluripotency of embryonic stem cell (ESC) lines
• mtDNA carry over in oocytes and ESCs
• Cytogenetic analyses
• Efficacy of MST in cryopreserved oocytes

• Significant proportion of MST oocytes (52 percent) showed abnormal fertilization compared with controls (13 percent)
• Normally fertilized MST oocytes had statistically significant level of blastocyst development (62 percent) similar to that of controls (76 percent)
• Mean mtDNA carry over in MST embryos 0.5 percent
• Mean carry over in derived ESC lines 0.6 percent
• No structural or numerical chromosomal abnormalities in ESC lines

• Developmental potential of MST embryos
• mtDNA copy number and volume in karyoplasts
• mtDNA carry over in preimplantation embryos

• Efficient development to blastocyst stage (39 percent), statistically similar to controls (33 percent)
• mtDNA copy number in karyoplasts was 0.36 percent of total mtDNA in MII oocytes; corresponded to volume of karyoplasts (0.89 percent of intact MII oocytes)
• Mean mtDNA carry over 0.31 percent in preimplantation embryos
• Depolimerization via cooling or cryo-thaw prevents premature oocyte activation following fusion by electrical pulse

• mtDNA carry over
• Embryonic developmental competence

• Parthenogenesis: NS difference in fusion, reconstruction, two-cell and blastocyst formation rate between activated MII and control groups; blastocyst quality similar to that of controls
• ICSI: NS difference in ICSI survival, 8-16 cell embryo formation between fertilized MII and control groups; no blastocyst formation in either group

Wang et al. (2014)


Mouse

• Developmental potential (in vitro and in vivo)
• mtDNA carryover (F1 and F2 generations)

Newcastle group (unpublished)^a


Human oocyte

MST versus PNT

• NS difference in successful fusion, cleavage rate, and blastocyst formation rate between MST (parthenogenetic) and PNT
• NS difference in mean mtDNA carryover:
  • MST oocytes: 0.29 percent (ND in 17/21, <2.15 percent in 4/21)
  • PNT zygotes: 0.29 percent (ND in 21/25, <2.6 percent in 4/25)

• 27 metaphase II spindle-chromosomal complexes (MII-SCCs) transferred
• 18 MST embryos transferred to pseudopregnant females
• mtDNA carryover: tail tip/brain tissue and internal organs (F1) and toe tips (F2)

• 85.7 percent developed to blastocyst
• 44.4 percent live, healthy births
• 5.5 percent mtDNA carryover (F1 tail tip/brain)
• ND-6.88 percent mtDNA carryover (F1 internal organs)
• ND-7.1 percent mtDNA carryover (F2 tail tip)

McGrath and Solter (1983)

Mouse

• C3H/HeJ
• C57BL/6J
• (ICR)

• Transfer of male and female pronuclei in karyoplast from zygote to enucleated zygote of genetically distinct substrain
• Development of reconstructed zygote in vitro to day 5 morula of blastocyst
• Transfer of reconstructed (64) and control (34) zygotes to pseudopregnant females
  
  
  
  
  

Sato et al. (2005)



Mito-mouse (ΔmtDNA): C57BL/6J (B6) with 4696-bp deletion

Normal control: C57BL/6J (B6)

• Transplantation of both pronuclei in karyoplast from mito-mouse zygote to enucleated normal zygotes
• Avg. ΔmtDNA levels in zygotes estimated by second polar body biopsy
• 39 PNT zygotes, ΔmtDNA/total mtDNA estimated to be 17-53 percent (average 25 percent), transferred into two pseudo-pregnant females
• 34 non-PNT zygotes (est. 11-47 percent ΔmtDNA; avg. 32 percent) implanted in two pseudo pregnant females
  
  
  
  
  

Craven et al. (2010)
(Newcastle Group)



Human zygotes (abnormally fertilized [unipronuclear/tripronuclear])

• 1 or 2 pronuclei transferred from abnormally fertilized zygote to enucleated recipient zygote
• Monitored 6-8 days in vitro for embryonic developmental potential
• Optimized procedure to minimize cytoplasm carried in karyoplast
• mtDNA carryover measured in blastomeres
• Total mtDNA copy number in oocytes

• Embryonic development
• Live birth of offspring

• Overall efficacy of enucleation and fusion of the pronuclei to enucleated zygote: 91 percent
• 96 percent of PNT zygotes developed to morula or blastocyst at day 5 (versus 100 percent in nonmanipulated controls)
• Live birth of 10 PNT offspring (16 percent) compared with 5 control offspring (15 percent); 7/10 PNT offspring survived to adulthood compared with 3/5 control offspring
• Coat color phenotype of PNT offspring was that of the nuclear donor
• 5/7 PNT offspring surviving to adulthood were fertile (no control value given)

• Rescue from disease phenotype
• mtDNA carryover
• Change in ΔmtDNA levels during embryogenesis, postnatal development and aging

• 11 mice born following transfer of PNT embryos (compared to 9 in controls)
• Avg. carryover of ΔmtDNA in PNT mice: 11 percent at weaning (range 6-21 percent), increased to 33 percent +300 days from weaning (range 5-44 percent); estimated to be 43 percent at 800 days
• Avg. ΔmtDNA levels in non-PNT mice: 66 percent at weaning (range 51-73 percent), 80 percent at +170 days
• PNT offspring rescued from disease phenotypes: all (11) PNT mice survived >300 days after birth; comparable weight gain, no observed renal abnormalities, steady blood lactate and urea levels compared with normal controls
• Non-PNT mice died at 218-277 days; exhibited renal abnormalities, elevated blood lactate and urea levels, decreased weight gain compared with normal control mice

• mtDNA carryover
• Embryonic developmental potential

• 22.7 percent and 22.2 percent of 1- and 2-pronuclei transfer PNT zygotes, respectively, developed past 8-cell stage; 8.3 percent to blastocyst stage (50 percent of nonmanipulated, abnormally fertilized controls)
• Optimized PNT to reduce cytoplasm volume: mtDNA carryover ND in 4 of 9 embryos; remaining five embryos: average <2 percent mtDNA carryover (range in blastomeres: ND-11.4 percent)
• Range of mtDNA copy number in oocytes: approx. 100,000-850,000

Neupane et al. (2014)


Mouse (NZB/OlaHsd & B6D2/F1)

• MST and PNT: transfer of MII-SCC and pronuclei, respectively, from NZB/OlaHsd to B6D2/F1 oocytes and zygotes, respectively

• 38 PNT zygotes reconstructed
• 13 PNT embryos transferred to pseudopregnant females
• mtDNA carryover: examined in tail tip/brain tissue and internal organs (F1) and toe tips (F2)
  
  
  

Newcastle group, (unpublished)^a

• mtDNA carry over
• Embryonic developmental competence

• Blastocyst quality similar to that of controls
• 1/8 blastomeres from cleavage stage embryos presented with 4.9 percent mtDNA carryover (7/8 ND); sensitivity of assay not disclosed

MST versus PNT

• NS difference in successful fusion, cleavage rate, and blastocyst formation rate between MST (parthenogenetic) and PNT
• NS difference in mean mtDNA carryover:
  • MST oocytes: 0.29 percent (ND in 17/21, <2.15 percent in 4/21)
  • PNT zygotes: 0.29 percent (ND in 21/25, <2.6 percent in 4/25)

• Developmental potential
• mtDNA carryover (F1 and F2 generations)

• 81.3 percent developed to blastocyst
• 53.8 percent live, healthy births
• 23.7 percent mtDNA carryover (F1 tail tip/brain)
• 5.5-39.8 percent mtDNA carryover (F1 internal organs)
• 22.1 percent mtDNA carryover (F2 toe tip)

• mtDNA carryover
• Chromosomal makeup

• High rates of development to blastocyst stage
• Subtle differences in embryo development