# Presentation: 205901

## Applications for Ooplasmic Transplantation 

- Applications for Ooplasmic Transplantation

- Jacques Cohen
*BRMAC, 2002*

## Oocyte Deficits

- Aneuploidy
- Chromosome breakage
- Gene dysfunction
- Genomic activation delayed

## Maternal age

- Implantation

- 40%

- 10%

- 20%

- 30%

- 20-34

- 35-39

- 40-45

- Aneuploidy

- Munne et al, 1995

## Implantation Rate After Aneuploidy Testing 

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

- Maternal Age

- %

## Is there evidence of an ooplasmic deficit?

## CELLULAR FRAGMENTATION

*Alikani et al, 1995, 1999*

## Alikani et al, 1995, 1999

*Alikani et al, 1995, 1999*

##  PED Gene

-  PED Gene

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

- Development		Implantation
- =< 7-cells 			17.4%
- => 8-cells      		30.3%

- P<0.001

- Warner et al, 1998

## Ped slow

- 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 

- Mad2 mRNA Concentration in
- Human Oocytes versus Maternal Age

- Maternal Age

- Concentration (100s)

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

## Mitochondria Genes

## Slide 13

**Notes:**
Contemporary mitochondria contain at least one ring of DNA seen here in an artist rendering that codes for less than 40 genes. Some yeasts have larger DNA chromosomes that code for 97 genes.  Most of the ancient mitochondrion's genome has moved to the nucleus, probably to limit its independence, although this is all speculation. The mitochondrion's role in respiration and oxidative phosphorylation is therefore incomplete. The majority of gene products of this metabolic pathway are coded by the nucleus. Even so, current thinking usually puts the mitochondrion on a biological pedestal  calling it the powerhouse, firefly or battery of the cell.
If you, like me, happen to have developed a more than common interest in mitochondria you may have developed a gentle condition. Eric Schon from Columbia University, who is one of the leading mitochondrial experts and has a special interest in the role of mitochondria dring reproduction calls such people mitochondriacs. I have had the good fortune during the past few years to be associated with some of these mitochondriacs, depicted here.

## 23 rearrangements

- P<0.0001
- N=702

- Barritt et al, 1999

## mtDNA Mutation T414G and Age

- Barritt et al., Reproductive Biomedicine Online, 2001

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

- P<0.01
- N=66

- Barritt et al, Reprod. BioMed. Online, 2001

## Clinical Rationale

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

## Animal Experimentation and Cytoplasmic Transplantation

## Cytoplasmic Transfer Experimentation

-  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 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)

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

- _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)
-  No apparent problems
-  First generation 30 months old
-  One more generation (n=13)
-  No apparent problems

##  Thinned tool

- NZB

- oocyte

- CB6F1

- cytoplasm

- Ooplasmic Transplantation By Piezo Facilitated Injection

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

## Slide 26

## Clinical Experience

## Informed Consent

-  First IRB 1995
-  Second IRB 1999
-  Third IRB pending

## Slide 29

## Ooplasmic donation 

**Ooplasmic donation **
**by injection**

## Slide 31

## Ooplasmic Transfer by injection

- 28 patients (33 cycles): egg donation candidates
- Recurrent implantation failure (RIF)
- Recurrent poor embryo morphology
- 9 male factors
- 5 with repeated miscarriages

- June 2001

##  33 attempts

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

## Cycle Attempts

- n=2477
- n=8487

## Slide 36

**Notes:**
The mitochondrion is an ancient organelle, developed before the separation of multi-cellular organisms, billions of years ago. It is postulated that a proto-bacterium fused with a organism and formed a symbiotic bond that still remains essential for cellular function in all organisms today. This symbiotic pre-mitochondrion was capable of metabolizing complex carbohydrates and formed hydrogen and CO2 as by-products, which the host cell needed to survive. Later in the more oxygen rich world, pyruvate became the mitochondrion's main source of food, and it started generating ATP for its host cell. All but red blood cells have mitochondria. 

## a

**10μm**

**30μm**

**a**

- Barritt et al, 2001

## Slide 38

## mtDNA Sequence Analysis 

- DNA Sequencing

- mtDNA Sequence Analysis
- of Hyper-variable Region

##  ‘spare’ embryos	 17/35 

-  ‘spare’ embryos	 17/35
-   amniocentesis 	   3/10
-   placenta			   3/13
-   fetal blood		   	   3/13
-   Babies tested		   2/13

- Incidence of “Heteroplasmy”

## Molecular Beacon for 

**Molecular Beacon for **
**Recipient 16224 mt DNA**

## “Germ-line Genetic Modification”

*“**Germ-line Genetic Modification”*

- Barritt et al, 2001

## Mitochondrial diversity in the hyper-variable area occurs in 10-15% of ‘normal’ humans

- Wilson et al, 1997

## Hyper-variable area is in a non-coding region

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

## Risk To Offspring:

- 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?
-  Abnormal Zygotes?
-  Source of Mitochondria
-  Video For Evidence
-  Frozen oocytes?
-  Chromosome screen
-  Screen embryos?

## Further Non-clinical Experimentation

-  Mouse model: profound genetic and functional differences
-  Primate model?
-  Costs?

## Possible Clinical Applications for Cytoplasmic Transfer:

-  Treating other groups with RIF
-  Avoiding Aneuploidy
-  Egg Freezing
-  Diagnosing Sperm Dysfunction
-  Treating Mitochondrial Disease
-  Haploidization

## Mitochondria infusion

-  (Tzeng et al, 2001)

-  Argues there is a single cause
-  Somatic mitochondria
-  Isolation process?
-  Age-related mutations
-  Replication
-  Multiple mtDNA genomes