# BRMAC Briefing Document for Day 1, May 9, 2002

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## Ooplasm transfer as method to treat female infertility

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# 

# Introduction

Several fertility clinics in the United States are experimenting with
ooplasm transfer. This technique is hypothesized to enhance fertility in
women who have failed conventional *in vitro* fertilization due to poor
embryo development. It involves injection of cellular material
(cytoplasm or ooplasm) from a donor egg into the mother's unfertilized
egg prior to fertilization *in vitro*. The transferred material includes
proteins, RNAs, small molecules, and organelles. Prominent among these
are mitochondria, the organelles responsible for cellular aerobic
respiration. Mitochondria contain their own genetic material, as they
are thought to have evolved from primitive bacteria. Thus, when ooplasm
is transferred, mitochondrial DNA from the donor is transferred to the
recipient egg. Several recent scientific reports document that some
children born following use of this technique do, indeed, carry genetic
material from three separate individuals: their biological parents and
the ooplasm donor. The technique is therefore *de facto* germ line gene
transfer and represents the first reported case of "human germline
genetic modification" \[1\]. This crosses a line drawn by many
scientists and bioethicists at altering the genetic profile of unborn
children \[2;3\] and, as a result, has occasioned attention in
scientific publications and the popular press.

# Ooplasm transfer in the United States

Over two dozen births attributed to ooplasm transfer have been reported
by three clinics since 1998. As of June, 2001, at least 8 U.S. clinics
were offering this procedure. Fresh oocytes, frozen oocytes, and
tripronucleate zygotes have all been used as sources of donor cytoplasm
\[4-6\]. Both electrofusion of cytoplasts and direct injection have been
investigated as methods of ooplasm transfer. It is not clear what defect
is being corrected by this technique. The active component or components
of the transferred ooplasm have not been identified, nor is there
consensus on this point. There are differences in energy content (ATP)
in human oocytes \[7\] and data that suggest that mitochondrial activity
is negatively correlated with advancing age \[8\]. These data support
the concept that mitochondria may confer at least a portion of the
proposed fertility-enhancing effect of ooplasm. In rodents, epigenetic
modifications of paternal or maternal genomes have been attributed to
differences in cytoplasm, indicating that additional activities of
ooplasm may also be relevant \[9\].

Of 30 fertilizations achieved after ooplasm transfer from fresh oocytes,
13 pregnancies were reported \[10\]. Two fetuses were karyotypically 45,
XO (Turner's syndrome). One of these fetuses aborted spontaneously and
the other pregnancy was terminated. It is unknown whether a connection
exists between these reported aneuploidies and the ooplasm transfer
procedure.

# Mitochondria and Heteroplasmy

The mitochondrial genome is small, approximately 15-17 kb. It encodes 13
proteins, and contains other non-protein coding genes \[11\]. Most of
the proteins used by the mitochondria are encoded by the nuclear (host)
genome and many of these must work in conjunction with proteins produced
by the mitochondrial genome. Regulation between the two compartments
must therefore be closely coordinated, but the details of this process
are still unclear \[12\].

Mitochondria play roles in programmed cell death (apoptosis) and in ion
fluxes, particularly calcium flux. However, the primary role of the
mitochondrion is aerobic respiration by oxidative phosphorylation. Thus
the mitochondrion is an environment rich in mutagenic oxygen radicals,
and mitochondrial DNA (mtDNA) has a mutation rate 5-10X that of the
nuclear genome. Mutations in human mtDNA continue to be identified. Many
of these are associated with serious disorders, including premature
aging, myopathies, neurodegenerative diseases, and diabetes\[13\].

In mammals, the inheritance of mtDNA is strictly maternal. In humans,
mitochondria derived from the spermatozoa are destroyed early in
embryogenesis. Therefore, all the mitochondria in the developing embryo
derive from the cytoplasm of the oocyte. The embryo normally inherits a
single sequence variant of mtDNA, a condition called homoplasmy.
Evolutionary biologists speculate that homoplasmy has arisen to avoid
cytoplasmic competition between mitochondrial genomes, and to avoid
difficulties in coordination between the nuclear and mitochondrial
genomes \[14;15\].

The high mutation rate observed for mtDNA and the apparent lack of
variation in a given individual's mitochondrial genotype indicate that a
restriction event must occur to eliminate variation in a given
individual's mitochondrial genotype. This is referred to as the
'bottleneck' \[16;17\], and appears to occur during oogenesis. Whether
it occurs by selection or stochastically is unresolved \[18\], but
evidence suggests that when organisms are found to contain more than one
mitochondrial genotype, a condition known as heteroplasmy, this
variation disappears within in a few generations in the individual's
offspring, and one genotype prevails.

Homoplasmy therefore is the normal condition in mammals. There have been
observations of mitochondrial heteroplasmy at single loci in humans
\[19\]. Humans show mutations in a subset of their mitochondrial
populations during aging or in acquired mitochondrial disease, but a
marked lack of sequence variation in mitochondrial genotype is most
commonly observed in young, healthy individuals.

Segregation patterns of mitochondrial genotypes in artificially created
heteroplasmic animals have been analysed. Interestingly, segregation of
mitochondrial genotypes was not random. Instead, selection for
genetically distinct mitochondrial populations was tissue specific
\[20\]. This type of selection pressure does not seem to be based solely
on differences in respiration or in efficiency of replication \[21\].
Such examples of nonrandom tissue distribution of mtDNA genotypes have
also been seen in humans with mutant genotypes leading to disease
states. For example, a mutant mtDNA genotype associated with the
condition 'mitochondrial encephalomyopathy with lactic acidosis and
stroke-like episodes' (MELAS) has been found to have a nonrandom tissue
distribution \[22\].

**Localization of mitochondria in oocytes and embryos**

Oocytes and embryos have polarized cytoarchitecture. A prominent example
of such polarization is that mitochondria are not randomly located in
oocytes and early embryos. In several mammalian species, including
humans, relocation of mitochondria during oocyte maturation has been
reported \[8;23;24\]. Immediately after fertilization, mitochondria are
observed to move to a perinuclear position in rhesus monkey and human
embryos \[8;24;25\]. This relocalization is thought to be necessary to
support the increased energy requirements of the nucleus, particularly
the spindle apparatus.

The partitioning of mitochondria among blastomeres of the early cleaving
embryo also is not random \[23\]. Mitochondrial genotype segregation
among blastomeres was observed in artificially produced heteroplasmic
mouse embryos \[26\] and asymmetric distribution of mitochondria has
been reported for human embryos, with some blastomeres receiving a
greater proportion of the pool of mitochondria than others. Poor
developmental potential is observed in blastomeres with low
mitochondrial number \[23\]. In addition, mitochondria of oocytes and
early embryos do not appear to be an entirely homogeneous population, as
measured by their membrane potential, and may have specific subcellular
localizations that reflect differences in function \[27\]. It should be
noted that no mitochondrial replication occurs from fertilization until
perhaps as late as implantation, so the early embryo has a limited
supply of mitochondria \[16\].

All these observations support a hypothesis that cells, and particularly
oocytes and embryos, adjust mitochondrial density to support specific
metabolic demands, and that failure to maintain appropriate
mitochondrial distribution can have negative effects on embryonic
development.

**Mitochondria and ooplasm transfer**

In experimental procedures carried out in human embryos, the fate of
mitochondria transferred from fresh donor oocytes was monitored by
confocal microscopy using fluorescent dyes specific for active
mitochondria \[1\]. Active donor mitochondria were found in the
recipient oocyte and segregated to many of the blastomeres of the early
embryo. Active donor mitochondria were not evenly segregated throughout
the blastomeres after the ooplasm transfer procedure. The proportion of
the genotypes of mitochondria from donor and recipient was determined by
PCR, using primers from the hypervariable region of the mitochondrial
genome. This is the region used by population geneticists and forensic
biologists to track mitochondrial genotypes between individuals.
Mitochondrial genotypes were monitored in nonviable embryos, amniocytes,
placental tissue and fetal cord blood of pregnancies resulting from
ooplasm transfer. Donor mitochondria could be detected in multiple
numbers of these sites in more than one subject and the tissue
distribution of the mtDNA heteroplasmy was variable \[28\]. Further
follow-up indicated that this heteroplasmy persisted in two children at
9 months and 14 months of age, as determined by direct sequencing of DNA
from blood samples.

# 

# FDA's actions and concerns

In June, 2001 FDA sent a letter to fertility clinics and practitioners
in the U.S. identified either by publications on ooplasm transfer or
advertisements offering the procedure. We advised practitioners that FDA
has jurisdiction over the use of human cells that have received
transferred genetic material by means other than the union of gamete
nuclei. We indicated that any further ooplasm transfer protocols should
be done under Investigational New Drug (IND) exemptions and that an IND
submission to the agency would be required to treat additional patients.

Ooplasm transfer is an experimental technique that has not been
evaluated extensively in animals. There has been little oversight of
this clinical intervention beyond that of local Institutional Review
Boards. There is a paucity of published preclinical data supporting
either the safety or the effectiveness of this procedure. As outlined
above, the regulation of mitochondria is complex and incompletely
understood. However, it is clear that stringent mechanisms have evolved
to insure homogeneity of mitochondrial genotypes at the initiation of
human development. FDA has concerns about the safety of perturbing this
process. In addition, this technique changes the genetic makeup of the
resulting offspring. Appropriate follow-up of children born after
ooplasm transfer and their progeny must therefore be considered
carefully. FDA feels further public discussion is necessary to: 1)
evaluate the potential risks of this procedure, 2) recommend how safety
should be monitored, 3) assess how efficacy might best be determined,
and 4) determine what further nonclinical data will be needed to support
additional human clinical trials.

References

1\. **Barritt,J.A., Brenner,C.A., Malter,H.E., and Cohen,J.,**
Mitochondria in human offspring derived from ooplasmic transplantation.
*Human Reproduction* 2001. **16**: 513-516.

2\. **Recombinant DNA Advisory Committee**. Prenatal Gene Transfer:
Scientific, Medical and Ethical Issues. 2000.

3\. **Frankel, M. S. and Chapman A.R**. Human Inheritable Genetic
Modifications. 2000. American Association for the Advancement of
Science.

4\. **Cohen,J., Scott,R., Alikani,M., Schimmel,T., Munne,S., Levron,J.,
Wu,L., Brenner,C., Warner,C., and Willadsen,S.,** Ooplasmic transfer in
mature human oocytes. *Molecular Human Reproduction* 1998. **4**:
269-280.

5\. **Lanzendorf,S.E., Mayer,J.F., Toner,J., Oehninger,S., Saffan,D.S.,
and Muasher,S.,** Pregnancy following transfer of ooplasm from
cryopreserved- thawed donor oocytes into recipient oocytes. *Fertility
and Sterility* 1999. **71**: 575-577.

6\. **Huang,C.C., Cheng,T.C., Chang,H.H., Chang,C.C., Chen,C.I.,
Liu,J.E., and Lee,M.S.,** Birth after the injection of sperm and the
cytoplasm of tripronucleate zygotes into metaphase II oocytes in
patients with repeated implantation failure after assisted fertilization
procedures. *Fertility and Sterility* 1999. **72**: 702-706.

7\. **Van Blerkom J, Davis PW, and , Lee J**. ATP content of human
oocytes and developmental potential and outcome after in-vitro
fertilization and embryo trans. *Human Reproduction* **10**, 415-424.
1995.

8\. **Wilding,M., Dale,B., Marino,M., di Matteo,L., Alviggi,C.,
Pisaturo,M.L., Lombardi,L., and De Placido,G.,** Mitochondrial
aggregation patterns and activity in human oocytes and preimplantation
**embryos.** *Human Reproduction* **2001. 16: 909-917.**

**9. Hawes SM, Sapienza C, and Latham KE.** Ooplasmic donation in
humans: The potential for epigenic modifications. *Human Reproduction*
**14**, 850-852. 2002.

10\. **Barritt,J.A., Willadsen,S., Brenner,C., and Cohen,J.,**
Epigenetic and experimental modifications in early mammalian
development: Part II - Cytoplasmic transfer in assisted reproduction.
*Human Reproduction Update* 2001. **7**: 428-435.

11\. **Lightowlers,R.N., Chinnery,P.F., Turnbull,D.M., and Howell,N.,**
Mammalian mitochondrial genetics: heredity, heteroplasmy and disease.
*Trends in Genetics* 1997. **13**: 450-455.

12\. **Poyton,R.O. and McEwen,J.E.,** Crosstalk between nuclear and
mitochondrial genomes. *Annual Review of Biochemistry* 1996. **65**:
563-607.

13\. **Wallace,D.C.,** Mitochondrial diseases in man and mouse.
*Science* 1999. **283**: 1482-1488.

14\. **Cummins,J.,** Mitochondrial DNA in mammalian reproduction.
*Reviews of Reproduction* 1998. **3**: 172-182.

15\. **Cummins,J.M.,** Mitochondria: potential roles in embryogenesis
and nucleocytoplasmic transfer. *Human Reproduction Update* 2001. **7**:
217-228.

16\. **Jansen,R.P.S. and de Boer,K.,** The bottleneck: mitochondrial
imperatives in oogenesis and ovarian follicular fate. *Molecular and
Cellular Endocrinology* 1998. **145**: 81-88.

17\. **Chinnery,P.F., Thorburn,D.R., Samuels,D.C., White,S.L.,
Dahl,H.H.M., Turnbull,D.M., Lightowlers,R.N., and Howell,N.,** The
inheritance of mitochondrial DNA heteroplasmy: random drift, selection
or both? *Trends in Genetics* 2000. **16**: 500-505.

18\. **Jenuth,J.P., Peterson,A.C., Fu,K., and Shoubridge,E.A.,** Random
genetic drift in the female germline explains the rapid segregation of
mammalian mitochondrial DNA. *Nature Genetics* 1996. **14**: 146-151.

19\. **Wilson,M.R., Polanskey,D., Replogle,J., DiZinno,J.A., and
Budowle,B.,** A family exhibiting heteroplasmy in the human
mitochondrial DNA control region reveals both somatic mosaicism and
pronounced segregation of mitotypes. *Human Genetics* 1997. **100**:
167-171.

20\. **Jenuth,J.P., Peterson,A.C., and Shoubridge,E.A.,**
Tissue-specific selection for different mtDNA genotypes in heteroplasmic
mice. *Nature Genetics* 1997. **16**: 93-95.

21\. **Battersby,B.J. and Shoubridge,E.A.,** Selection of a mtDNA
sequence variant in hepatocytes of heteroplasmic mice is not due to
differences in respiratory chain function or efficiency of replication.
*Human Molecular Genetics* 2001. **10**: 2469-2479.

22\. **Chinnery,P.F., Zwijnenburg,P.J.G., Walker,M., Howell,N.,
Taylor,R.W., Lightowlers,R.N., Bindoff,L., and Turnbull,D.M.,**
Nonrandom tissue distribution of mutant mtDNA. *American Journal of
Medical Genetics* 1999. **85**: 498-501.

23\. **Van Blerkom,J., Davis,P., and Alexander,S.,** Differential
mitochondrial distribution in human pronuclear embryos leads to
disproportionate inheritance between blastomeres: relationship to
microtubular organization, ATP content and competence. *Human
Reproduction* 2000. **15**: 2621-2633.

24\. **Bavister BD and Squirrell JM**. Mitochondrial distribution and
function in oocytes and early embryos. *Human Reproduction*. **15**
Suppl 2, 189-198. 2000.

25\. **Van Blerkom,J., Sinclair,J., and Davis,P.,** Mitochondrial
transfer between oocytes: potential applications of mitochondrial
donation and the issue of heteroplasmy. *Human Reproduction* 1998.
**13**: 2857-2868.

26\. **Meirelles,F.V. and Smith,L.C.,** Mitochondrial genotype
segregation during preimplantation development in mouse heteroplasmic
embryos. *Genetics* 1998. **148**: 877-883.

27\. **Van Blerkom,J., Davis,P., Mathwig,V., and Alexander,S.,** Domains
of high-polarized and low-polarized mitochondria may occur in mouse and
human oocytes and early embryos. *Human Reproduction* 2002. **17**:
393-406.

28\. **Brenner,C.A., Barritt,J.A., Willadsen,S., and Cohen,J.,**
Mitochondrial DNA heteroplasmy after human ooplasmic transplantation.
*Fertility and Sterility* 2000. **74**: 573-578.