Applications for Ooplasmic Transplantation

Jacques CohenBRMAC, 2002

Oocyte DeficitsOocyte Deficits• Aneuploidy Aneuploidy

• Chromosome breakageChromosome breakage

• Gene dysfunctionGene dysfunction

• Genomic activation delayedGenomic activation delayed

Implantation

40%

10%

20%

30%

20-34 35-39 40-45

Maternal age

Aneuploidy

Munne et al, 1995

Implantation Rate After Aneuploidy Testing

0

10

20

30

40

50

60

28-31 31-34 34-37 37-40 40-44

Maternal Age

%

Is there evidence of an ooplasmic deficit?

I II III

I II III

Alikani et al, 1995, 1999

CELLULAR FRAGMENTATION

IV V

IV

IV V

Alikani et al, 1995, 1999

PED Gene

Ped Gene Phenotype in HumanPed Gene Phenotype in Human(n=1360)

Development Implantation=< 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 proteinQa-2 protein

Ped slowSlow embryosmorula stage-19 cellsAbsence of protein

Ped fastFast embryosblastocyst stage-32 cellsPresence of protein

Cell membrane

Warner et al, 1998

Mad2 mRNA Concentration in Human Oocytes versus Maternal Age

Maternal Age

Co

nce

ntr

atio

n (

100

s)

P<.001P<.001P<.001

Mitochondria Genes

IQM

ND1

MELAS

Sporadic myopathy

ND2

W A N C Y

OL

COXI S D

COXII K

A8/6

MERFF

Sporadic myopathy

Sporadic myopathy

COXIII

G

ND3

R

ND4L

ND4

LSH

ND5

ND6

Cyt b

E

OH

HSP

LSP

Sporadic myopathy

0

10

20

30

40

50

60

Any mtDNArearrangement

Multiplerearrangements

OocyteEmbryo

23 rearrangements

P<0.0001N=702 Barritt et al, 1999

mtDNA Mutation T414G and Age

Barritt et al., Reproductive Biomedicine Online, 2001

0

10

20

30

40

24-36 years 37-42P<0.01N=66 Barritt et al, Reprod. BioMed. Online, 2001

MtDNA Point Mutation (T414G) in Replication Control Region of Compromised Human Oocytes

Clinical RationaleClinical Rationale

• Knowledge base for specific Knowledge base for specific

ooplasmic defect treatment ooplasmic defect treatment

does not existdoes not exist• Are all mRNA, proteins, Are all mRNA, proteins,

mitochondria the same?mitochondria the same?

Animal Experimentation and Cytoplasmic Transplantation

• Cyto-Transfer Using Cytochalasin in Outbred MiceCyto-Transfer Using Cytochalasin in Outbred Mice (Muggleton-Harris et al, 1982,1988)(Muggleton-Harris et al, 1982,1988)

• Cytoplasmic Replacement Using Karyoplasts -AsynchronyCytoplasmic Replacement Using Karyoplasts -Asynchrony(McGrath and Solter, 1983; Surani 80’s – 00’s; Willadsen, 1986; Zhang et (McGrath and Solter, 1983; Surani 80’s – 00’s; Willadsen, 1986; Zhang et

al, 2000; Takumi et al, 2001)al, 2000; Takumi et al, 2001)

• Cytoplasmic Transfer in Mouse, Monkey, HumanCytoplasmic Transfer in Mouse, Monkey, Human(Flood et al, 1990; Smith et al, 1991: Levron et al, 1996; Jenuth et (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, al, 1996; Meirelles and Smith, 1997, 1998; Van Blerkom et al, 1998; Cohen et al, 1997, 1998; Takeda et al, 1998)1998; Cohen et al, 1997, 1998; Takeda et al, 1998)

• Cybrids in mouse and primates Cybrids in mouse and primates (Kenyon and Moraes, 1997; Yamaoka et al, 2000)(Kenyon and Moraes, 1997; Yamaoka et al, 2000)

Cytoplasmic Transfer ExperimentationCytoplasmic Transfer Experimentation

LC Smith Lab: Heteroplasmic Mammals after Manipulation of Cytoplasm in Early Embryos

Healthy, normal mice produced following Healthy, normal mice produced following karyoplast and cytoplast transfer between inbred karyoplast and cytoplast transfer between inbred mouse strains w/ differing mitochondrial mouse strains w/ differing mitochondrial backgroundsbackgrounds

Hundreds of such heteroplasmic animals have been Hundreds of such heteroplasmic animals have been produced and bred over produced and bred over 15 generations15 generations with no with no developmental or health problemsdevelopmental 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 The transfer of cytoplasm between mature mouse oocytes and zygotes led to development mouse oocytes and zygotes led to development that was identical with controls.that was identical with controls.

Following embryo transfers – implantation Following embryo transfers – implantation and viability of such cyto-transfer embryos and viability of such cyto-transfer embryos was in one scenario was in one scenario significantly improvedsignificantly improved over controls. over controls.

Cybrid ExperimentsCybrid ExperimentsCreation of cell hybrids w/ disparate nuclear Creation of cell hybrids w/ disparate nuclear and and Mitochondrial makeup – cross species/genusMitochondrial makeup – cross species/genus

Normal mitochondrial function in many Normal mitochondrial function in many

scenariosscenarios

Nuclear transplantation of F1 hybrid mouse zygotes

Malter and Schimmel, unpublished

Cytoplasmic Transfer in Mouse Cytoplasmic Transfer in Mouse Zygote Using Karyoplast FusionZygote Using Karyoplast Fusion

• 12 mice – (F1 hybrids)12 mice – (F1 hybrids)• No apparent problemsNo apparent problems• First generation 30 months oldFirst generation 30 months old• One more generation (n=13) One more generation (n=13) • No apparent problemsNo apparent problems

NZB oocyte

CB6F1 cytoplasm

Ooplasmic Transplantation By Piezo Facilitated Injection

• Thinned tool• Multiple donor aspiration• Similar ratio to human procedure

Survival – up to 90+ %

Good preimplantation development

Clinical ExperienceClinical Experience

Informed ConsentInformed Consent

• First IRB 1995First IRB 1995• Second IRB 1999Second IRB 1999• Third IRB pendingThird IRB pending

Normal donor stage

Compromised recipient stage

RecipientDonor

Ooplasmic donation by injection

Ooplasmic Transfer Ooplasmic Transfer by injectionby injection

• 28 patients (33 cycles): egg donation 28 patients (33 cycles): egg donation candidatescandidates

• 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 2001June 2001

• 33 attempts• 28 couples• 31 transfers• 13 clinical pregnancies• 2 biochemical• 1 first tri miscarriage (XO)• 1 twin with XO (IRB - 1999)

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)

0

10

20

30

40

50

60

70

Clinical Pregnancy FHB / embryo

123454.8

Cycle Attempts

Ooplasmic transfer cases

n=2477n=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 16224 mt

DNA

““Germ-line Genetic Modification”Germ-line Genetic Modification”

Barritt et al, 2001Barritt et al, 2001

Tully et al, 2000Wilson et al, 1997

Mitochondrial diversity in the hyper-variable area occurs in 10-

15% of ‘normal’ humans

Hyper-variable area is in a non-coding region

No known cases of mitochondrial disease

after…….egg donation

Risk To Offspring:Risk To Offspring:1.1.Mechanical Damage?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:Risk To Offspring:

2. Inadvertent Transfer?2. Inadvertent Transfer?

• Unique organelles• Avoid spindle• Cytogenetic analysis• Detailed study of zygote• Centriole is sperm derived

Risk To Offspring:Risk To Offspring:

3. Enhanced survival?3. Enhanced survival?

• Aneuploidy is common• Enhanced in ICSI?• XO most common

Risk To Offspring:Risk To Offspring:

4. Heteroplasmy?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:Risk To Mother:

Elevated Incidence of Elevated Incidence of Chromosomal Anomalies?Chromosomal Anomalies?

• No statistical evidence for this• Aneuploidy most common form of miscarriage (>60%)• XO most common form of aneuploidy

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

Further Non-clinical Further Non-clinical ExperimentationExperimentation

• Mouse model: profound genetic Mouse model: profound genetic

and functional differencesand functional differences• Primate model?Primate model?• Costs?Costs?

Possible Clinical Possible Clinical Applications for Applications for Cytoplasmic Transfer:Cytoplasmic Transfer:

• Treating other groups with RIFTreating other groups with RIF• Avoiding AneuploidyAvoiding Aneuploidy• Egg FreezingEgg Freezing• Diagnosing Sperm DysfunctionDiagnosing Sperm Dysfunction• Treating Mitochondrial DiseaseTreating Mitochondrial Disease• HaploidizationHaploidization

Mitochondria infusionMitochondria infusion (Tzeng et al, 2001)(Tzeng et al, 2001)

• Argues there is a single causeArgues there is a single cause• Somatic mitochondriaSomatic mitochondria• Isolation process?Isolation process?• Age-related mutationsAge-related mutations• ReplicationReplication• Multiple mtDNA genomesMultiple mtDNA genomes