Applications for Ooplasmic Transplantation Jacques Cohen BRMAC, 2002

Oocyte Deficits AneuploidyAneuploidy Chromosome breakageChromosome breakage Gene dysfunctionGene dysfunction Genomic activation delayedGenomic activation delayed

Implantation 40% 10% 20% 30% Maternal age Aneuploidy Munne et al, 1995

Implantation Rate After Aneuploidy Testing Maternal Age %

Is there evidence of an ooplasmic deficit?

I II III I II III Alikani et al, 1995, 1999 CELLULAR FRAGMENTATION

IV V IVIV V Alikani et al, 1995, 1999

PED Gene

Ped Gene Phenotype in Human (n=1360) DevelopmentImplantation =< 7-cells 17.4% => 8-cells 30.3% P<0.001 Warner et al, 1998

9aa peptide Outside the cell Inside the cell Predicted protein characteristics of the Ped fast and Ped slow phenotype (mouse) No Qa-2 protein Qa-2 protein Ped slow Slow embryos morula stage-19 cells Absence of protein Ped fast Fast embryos blastocyst stage-32 cells Presence of protein Cell membrane Warner et al, 1998

Mad2 mRNA Concentration in Human Oocytes versus Maternal Age Maternal Age Concentration (100s) P<.001

Mitochondria Genes

I Q M ND1 MELAS Sporadic myopathy ND2 W A N C Y OLOL COXI S D COXII K A8/6 MERFF Sporadic myopathy Sporadic myopathy COXIII G ND3 R ND4L ND4 LSHLSH ND5 ND6 Cyt b E OHOH HSP LSP Sporadic myopathy

23 rearrangements P< N=702 Barritt et al, 1999

mtDNA Mutation T414G and Age Barritt et al., Reproductive Biomedicine Online, 2001

P<0.01 N=66 Barritt et al, Reprod. BioMed. Online, 2001 MtDNA Point Mutation (T414G) in Replication Control Region of Compromised Human Oocytes

Clinical Rationale Knowledge base for specific ooplasmic defect treatment does not exist Knowledge base for specific ooplasmic defect treatment does not exist Are all mRNA, proteins, mitochondria the same? Are all mRNA, proteins, mitochondria the same?

Animal Experimentation and Cytoplasmic Transplantation

Cyto-Transfer Using Cytochalasin in Outbred Mice (Muggleton-Harris et al, 1982,1988) Cyto-Transfer Using Cytochalasin in Outbred Mice (Muggleton-Harris et al, 1982,1988) Cytoplasmic Replacement Using Karyoplasts -Asynchrony (McGrath and Solter, 1983; Surani 80’s – 00’s; Willadsen, 1986; Zhang et al, 2000; Takumi et al, 2001) Cytoplasmic Replacement Using Karyoplasts -Asynchrony (McGrath and Solter, 1983; Surani 80’s – 00’s; Willadsen, 1986; Zhang et al, 2000; Takumi et al, 2001) Cytoplasmic Transfer in Mouse, Monkey, Human (Flood et al, 1990; Smith et al, 1991: Levron et al, 1996; Jenuth et al, 1996; Meirelles and Smith, 1997, 1998; Van Blerkom et al, 1998; Cohen et al, 1997, 1998; Takeda et al, 1998) Cytoplasmic Transfer in Mouse, Monkey, Human (Flood et al, 1990; Smith et al, 1991: Levron et al, 1996; Jenuth et al, 1996; Meirelles and Smith, 1997, 1998; Van Blerkom et al, 1998; Cohen et al, 1997, 1998; Takeda et al, 1998) Cybrids in mouse and primates (Kenyon and Moraes, 1997; Yamaoka et al, 2000) Cybrids in mouse and primates (Kenyon and Moraes, 1997; Yamaoka et al, 2000) Cytoplasmic Transfer Experimentation

LC Smith Lab: Heteroplasmic Mammals after Manipulation of Cytoplasm in Early Embryos Healthy, normal mice produced following karyoplast and cytoplast transfer between inbred mouse strains w/ differing mitochondrial backgrounds Hundreds of such heteroplasmic animals have been produced and bred over 15 generations with no developmental or health problems

Levron et al (1996) : Cytoplast transfer across early developmental stages in mouse eggs/embryos (F1 Hybrid) The transfer of cytoplasm between mature mouse oocytes and zygotes led to development that was identical with controls. Following embryo transfers – implantation and viability of such cyto-transfer embryos was in one scenario significantly improved over controls.

Cybrid Experiments Creation of cell hybrids w/ disparate nuclear and Mitochondrial makeup – cross species/genus Normal mitochondrial function in many scenarios

Nuclear transplantation of F1 hybrid mouse zygotes Malter and Schimmel, unpublished

Cytoplasmic Transfer in Mouse Zygote Using Karyoplast Fusion 12 mice – (F1 hybrids) 12 mice – (F1 hybrids) No apparent problems No apparent problems First generation 30 months old First generation 30 months old One more generation (n=13) One more generation (n=13) No apparent problems No apparent problems

NZBoocyte CB6F1 cytoplasm Ooplasmic Transplantation By Piezo Facilitated Injection Thinned tool Multiple donor aspiration Similar ratio to human procedure

Clinical Experience

Informed Consent First IRB 1995 First IRB 1995 Second IRB 1999 Second IRB 1999 Third IRB pending Third IRB pending

Normal donor stage Compromised recipient stage

Recipient Donor Ooplasmic donation by injection

Ooplasmic Transfer by injection 28 patients (33 cycles): egg donation candidates28 patients (33 cycles): egg donation candidates Recurrent implantation failure (RIF)Recurrent implantation failure (RIF) Recurrent poor embryo morphologyRecurrent poor embryo morphology 9 male factors9 male factors 5 with repeated miscarriages5 with repeated miscarriages June 2001

33 attempts 28 couples 31 transfers 13 clinical pregnancies 2 biochemical 1 first tri miscarriage (XO) 1 twin with XO (IRB ) Barritt et al, 2000 Hum Reprod 15,2:207

one twin born one quadruplet born 17 babies pediatric follow-up one PDD-NOS (18 month)

Cycle Attempts Ooplasmic transfer cases n=2477 n=8487

10µm 30µm a 10µm Barritt et al, 2001

DNA Sequencing mtDNA Sequence Analysis of Hyper-variable Region

‘spare’ embryos 17/35 amniocentesis 3/10 placenta 3/13 fetal blood 3/13 Babies tested 2/13 Incidence of “Heteroplasmy”

Molecular Beacon for Recipient mt DNA

“Germ-line Genetic Modification” Barritt et al, 2001

Tully et al, 2000 Wilson et al, 1997 Mitochondrial diversity in the hyper-variable area occurs in % of ‘normal’ humans

Hyper-variable area is in a non-coding region

No known cases of mitochondrial disease after…….egg donation

Risk To Offspring: 1.Mechanical Damage? ICSI-derived procedure CTr Survival >90% CTr Fertilization >65% > 100,000 babies with ICSI Preimplantation like IVF Malformation rate like IVF

Risk To Offspring: 2. Inadvertent Transfer? Unique organelles Avoid spindle Cytogenetic analysis Detailed study of zygote Centriole is sperm derived

Risk To Offspring: 3. Enhanced survival? Aneuploidy is common Enhanced in ICSI? XO most common

Risk To Offspring: 4. Heteroplasmy? Three polymorphisms confirmed Heteroplasmic polymorphisms common in population Very common in experimental embryology No evidence of risk between outbred individuals in same species

Risk To Mother: Elevated Incidence of Chromosomal Anomalies? No statistical evidence for this Aneuploidy most common form of miscarriage (>60%) XO most common form of aneuploidy

What Cellular Issues? Donor Screening? Donor Screening? Abnormal Zygotes? Abnormal Zygotes? Source of Mitochondria Source of Mitochondria Video For Evidence Video For Evidence Frozen oocytes? Frozen oocytes? Chromosome screen Chromosome screen Screen embryos? Screen embryos?

Further Non-clinical Experimentation Mouse model: profound genetic and functional differences Mouse model: profound genetic and functional differences Primate model? Primate model? Costs? Costs?

Possible Clinical Applications for Cytoplasmic Transfer: Treating other groups with RIF Treating other groups with RIF Avoiding Aneuploidy Avoiding Aneuploidy Egg Freezing Egg Freezing Diagnosing Sperm Dysfunction Diagnosing Sperm Dysfunction Treating Mitochondrial Disease Treating Mitochondrial Disease Haploidization Haploidization

Mitochondria infusion (Tzeng et al, 2001) (Tzeng et al, 2001) Argues there is a single cause Argues there is a single cause Somatic mitochondria Somatic mitochondria Isolation process? Isolation process? Age-related mutations Age-related mutations Replication Replication Multiple mtDNA genomes Multiple mtDNA genomes