Does Nuclear Transplantation Have a Place in Clinically Assisted
Henry Malter and Jacques Cohen
Tyho-Galileo Research Laboratories and Reprogenetics Inc., West Orange, NJ 07052,
Human clinical assisted reproductive technology (ART) current exhibits considerable
success in addressing a variety of issues in infertility and genetic disease. However, many
hurdles still remain in attempting to facilitate the birth of a single healthy child to all
couples seeking treatment. Successfully addressing this may require the application of
more invasive techniques to diagnose and potentially correct genetic and functional
deficits in oocytes and early embryos. Also, advanced assisted reproductive techniques
are being proposed for new applications involving the creation of embryos with selected
genomes for use in medical scenarios that fall outside of standard infertility treatment.
These techniques are sometimes unfortunately lumped together under a heading of
“cloning”, since historically a common use of nuclear transfer involves the creation of
identical clonal offspring. All of these techniques involve both the physical and
constitutional manipulation of cellular and/or nuclear components in the oocyte or early
embryo. Unfortunately, such manipulation during the critical early stages of
development is a complicated matter with potential negative consequences for the
subsequent developmental process. The limited basic and applied scientific knowledge
available would suggest that application in the human may be at least problematic from
an efficiency point of view and possibly dangerous affecting the health of offspring.
Furthermore, such manipulation is seen by many as inherently unethical and taboo,
though cloning can at least logically be considered as a form of identical twinning, a
completely natural process in the human affecting many births
There currently exists a wide range of opinion and action within the pursuit and
application of such techniques. Some human ART workers suggest that even the most
extreme scenarios involving human nuclear transfer are well within the grasp of current
or near future technology (1,2). Others conclude that any manipulative intervention for
the establishment of pregnancy will forever be out of the question due to safety issues,
while the use of such techniques for so-called “therapeutic” intervention (stem cell
development) should be pursued. This position seems to be held by the majority of the
basic science community. Finally, some would suggest a complete restriction on all
interventional strategies including the majority of the current standard of medical
treatment for human infertility. This is obviously a very complex issue. It is also clearly a
defining issue for the future of reproductive medicine and ART and one that our
community has neglected to discuss publicly often out of fear of negative publicity. A
critical and honest understanding of these techniques and their potential problems and
benefits is therefore very important. The techniques under consideration fall into four
main categories: techniques for diagnostic purposes, techniques for ART-related
purposes, techniques for therapeutic stem-cell-related purposes and techniques to study
processes such as imprinting and reprogramming for basic science purposes. The latter
studies form in our opinion one of the most essential questions in medicine to date and
should be addressed separately.
Diagnostic scenarios have already successfully used nuclear transfer-based techniques for
facilitating the genetic assessment of gametes and embryos. In general, this involves
obtaining a metaphase chromosome complement from gametic or embryonic nuclei (3-5).
These techniques involve the combination (by injection or electrofusion) of the cell to be
analyzed with the oocytes or early embryos of experimental animals. In such a scenario,
the introduced nuclei reliably enter the cell cycle leading to the production of metaphase
chromosomes. Such techniques, using readily available bovine or mouse material allow
for a complete chromosomal analysis as necessary in the case of certain complex
translocations. These techniques have been applied both in the assessment of human
spermatozoa and in the assessment of individual blastomeres from human embryos
during ART. Recently a similar technique, termed “gamete duplication” has been
suggested by Steen Willadsen (Tyho-Galileo Research Laboratories) for creating two
haploid chromosome complements from a single spermatozoon. This would theoretically
allow one complement to be used for genetic analysis while the other could be used in a
nuclear transfer-based clinical scenario as discussed below (6). The metaphase diagnostic
techniques perhaps fall outside of what is currently considered invasive clinical nuclear
manipulation, since apart from a standard biopsy procedure, the actual clinical embryos
are not disturbed. However, they are important to consider since they constitute
experimental protocols involving nuclear manipulation with human material. Also, as in
the case of “gamete duplication,” these techniques could be extended in the future to
become part of more invasive interventional strategies.
Clinical ART-related techniques
ART interventional techniques could potentially address a variety of clinically relevant
deficits in the gametes or pre-implantation embryos of infertile patients or of those with
heritable genetic lesions. Conceptually, these techniques range from mildly invasive
strategies for “improving” oocytes to full-fledged somatic cell nuclear transfer for the
creation of gametes or embryos. In addressing cases of poor development and
implantation failure associated with dysfunctional or genetically abnormal oocytes, such
compromised oocytes can simply be replaced by donor substitutes but at the price of
losing the patient’s genetic contribution. A variety of controversial strategies have been
proposed to get around this problem including oocyte to donor ooplast nuclear transfer
(7) and the creation of functional “oocytes” from the patient’s somatic cells (8). Another
concept involves attempts to “improve” the patient’s own eggs by an infusion of
theoretically healthy ooplasm or ooplasmic components from a donor egg (9,10).
Potentially addressable deficits in the oocyte fall into two categories that may be inter-
related, those directly related to the oocyte aneuploidy and those related to oocyte
function and developmental problems. The incidence of chromosomal abnormalities such
as aneuploidy is one of the biggest challenges facing the treatment of infertility in
patients of advanced reproductive age (11,12). Cytoplasmic deficits related to poor
oxidative conditions or the lack of critical meiotic components have been proposed as
causative factors in the generation of such meiotic abnormalities (13-16). Theoretically, a
correction of these cytoplasmic deficits could result in promoting correct spindle
behavior and chromosome segregation. Since the majority of aneuploidy in the human
apparently arises in the meiotic divisions involved in the generation of the mature
metaphase II oocyte, manipulative intervention would need to occur prior to the
completion of meiosis (17,18). The main manipulative strategy that has been proposed is
the transfer of the oocyte nucleus at the germinal vescicle (GV) stage from a theoretically
compromised patient egg to an enucleated donor oocyte (7,19-21) In this way, the entire
cytoplasmic component is exchanged between the patient and donor. Experimental
protocols in the mouse and human have demonstrated that such GV stage nuclear transfer
can be performed and that reconstituted oocytes can exhibit successful maturation and
subsequent fertilization by intracytoplasmic sperm injection (7, 20). However, to date, the
extent of such studies is still very limited and a complete and satisfactory evaluation of
even the physical aspects of this methodology in the human is lacking. Furthermore, the
overall concept that such cytoplasmic exchange can correct spindle function and lead to a
reduced rate of meiosis-associated aneuploidy remains to be established.
The second area in which manipulative techniques could be used is to address ooplasmic
deficits. Ooplasmic-mediated activities are critical in setting up much of the subsequent
developmental program with stable and heritable downstream effects (22,23). There can
be little question that dysfunction in ooplasmic components is a causative factor in
human infertility. Preliminary work in the mouse demonstrated the lack of a detrimental
effect for cytoplasmic transfer and suggested the presence of a positive effect under some
scenarios. (24). A variety of other animal research also supports such a positive effect
(25-27). Based on these experiments and others, the direct transfer of synchronous
cytoplasm from donor to patient eggs was attempted in patients with recurring
developmental deficits and implantation failure (4,28). This technique is based on the
concept that healthy donor cytoplasm contains components that are lacking or
compromised in oocytes of certain patients and that an infusion of such components can
have a positive effect on development. The initial results from applying the technique in a
defined group of patients seemed to be positive, but potential problems have been
discussed extensively. Implantation was improved with a 43% clinical pregnancy rate
(and several births) in 27 couples with a consistent combined history of almost 100 failed
assisted reproduction cycles (29). To date, several variations on the oocyte infusion
concept have been reported including the use of cryopreserved oocytes or polyspermic
zygotes for donor cytoplasm and the injection of purified mitochondria from the patient’s
own cumulus cells (30,31,10). Following its initial clinical trail, the United States Food
and Drug Administration issued an order stating that ooplasmic transfer and similar
protocols are subject to approval under an Investigative New Drug application (32). This
intervention was based solely on the issue of genomic transfer, since mitochondrial
genomes were shown to be transferred from the donor to patient oocyte during the
procedure. This matter is currently under analysis and review and will no doubt have an
impact on the future applications of similar manipulative human clinical protocols.
Recently, a more invasive form of cytoplasmic “correction” has been attempted involving
the transfer of pronuclei between patient zygotes and donor zygote-stage cytoplasts (2).
This procedure was used in a patient exhibiting consistent developmental failure during
ART and led to the establishment of a complicated triplet pregnancy which failed prior to
30 weeks gestation. The fetuses derived from this procedure were shown to carry the
patient’s genome, overtly normal karyotypes, and mitochondrial genotype derived solely
from the donor zygote cytoplasts.
The most invasive manipulative techniques proposed for ART-related treatment concern
the use of genomic manipulation to create gametes or embryos for individuals who lack
functional germ cells. A variety of strategies might be possible based on nuclear transfer
scenarios using premature or non-functional germ cells or somatic cells as the genome
source. One strategy would involve the creation of patient-derived haploid cells that
could be combined with an ooplast to create a functional “oocyte”. A second strategy
would be to simply create an embryo through nuclear transfer from a patient-derived
somatic cell. This would constitute true somatic cell nuclear transfer (SCNT) “cloning”
and the resulting offspring would harbor the identical genome of their parent. Such
proposals are highly controversial from both scientific and ethical standpoints although
for some individuals they might represent the only reproductive option available.
Creating artificial gametes from diploid germline or somatic cells via nuclear transfer
would require the additional step of haploidizing the source genome to allow for
compatible combination with a germ cell genome or second haploidized genome.
Experiments in the mouse and human have demonstrated limited development following
protocols with diploid somatic cells (33,34). In the human, somatic cells injected into
enucleated oocytes apparently underwent haploidization by the emission of a polar body.
The resulting artificial “oocytes” could be fertilized and fluorescent in situ hybridization
analysis of the second polar body indicated a haploid complement (34). Haploid
pronuclei have also been observed following diploid cell injection into enucleated human
oocytes (35). Theoretically, a variety of techniques are possible based on the basic
premise of allowing diploid chromosomes to segregate in an oocyte. The resulting
pronuclei or polar bodies can theoretically be re-transferred to a second enucleated oocyte
or to a zygote from which the appropriate pronucleus has been removed. To date,
experimental protocols inducing haploidization have been very limited and a definitive
demonstration that such chromosome segregation produces a normal functional haploid
genome is lacking.
A secondary application for some of the invasive ART techniques would be
circumventing inheritance of the deleterious mitochondrial genotypes associated with
several rare but serious human diseases. Individuals harboring such genotypes transmit
them to their offspring through the ooplasm. Theoretically, by manipulation or
exchanging compromised ooplasm with healthy donor ooplasm such inheritance could be
prevented. This might be possible either via ooplasmic injection or through nuclear
transfer. In this regard, it is interesting to note that donor mitochondrial genomes could be
detected in a limited subset of offspring produced from ooplasmic transfer (36-38).
Conversely, the patient’s mitochondrial genome could not be detected in pronuclear
transfer-derived fetuses which harbored at least a majority donor mitochondrial genotype
(2). Mitochondrial inheritance is still not fully understood and further work will be
needed to establish the basis for such interventional strategies.
Therapeutic “cloning” techniques
A final category for invasive manipulation involves the use of nuclear transfer for the
creation of embryonic stem cell lines. This has been termed “therapeutic cloning.”
Therapeutic cloning would be technically similar to the previously discussed techniques
for use in GV or somatic cell nuclear transfer. However, in this case such “cloning’
methodology would be applied in achieving the creation of patient-specific embryonic
stem cell lines for use in cellular-replacement therapies. (39,40) Blastocysts created by
patient SCNT to an enucleated donated oocyte would be used as the source for the
derivation of such cell lines (41). In this way, the stem cells or derived tissues such as
neurons, muscle or blood cells would be immunologically identical with the patient and
would not be subject to rejection. (42-44). Recently the first successful demonstration of
the derivation of human ES cells from cloned embryos has been reported (45).
Safety and clinical application of invasive manipulative techniques
While some might suggest otherwise, manipulation of gametes and early embryos via
nuclear transfer is far from perfected. Nuclear transfer and related techniques has been
applied in mammalian embryology for over 20 years (46,47). Although much has been
learned, the process and developmental consequences are still poorly understood. One
clear aspect that has emerged from this work is that a considerable amount of critical
genomic “remodeling” and processing occurs during early development and this process
can be perturbed by manipulative intervention (22,23). For the most part, development
following nuclear transfer experiments has been compromised. Problems with the
efficiency of nuclear transfer cloning technology have been associated with aberrant
epigenetic processing and altered genomic imprinting(48-50). However, this connection
is complicated by the fact that simple in vitro culture is known to result in certain
epigenetic effects in animal models (51-53). The connection between altered imprinting,
gene expression and developmental consequences is far from defined although
developmental abnormalities, pathological conditions, and other phenotypic
consequences of imprinting defects are clearly present in mammals including the human
(54,55). However, it is also recognized that differences in epigenetic processing exist
between model species and the human (56). These differences suggest that
epigenetically-derived abnormalities observed following in vitro manipulation in
experimental animals may not be fully relevant to the human. In primates, a secondary
issue may arise from depletion of critical maternal ooplasmic proteins during enucleation
(57). This phenomenon has been suggested to underlie a failure to obtain experimental
primate offspring from SCNT. However, the recent success of SCNT in producing
apparently normal human blastocysts and ES cells (which used a unique enucleation
technique) indicates that this problem may be avoidable (45).
The identified and potential developmental problems that could arise from imperfect
genetic re-programming, imprinting deficits, and other developmental perturbations make
the immediate clinical application of invasive manipulative techniques questionable.
However, as stated before, there are clear differences in genetic, developmental and
physical aspects between the experimental animal systems in which techniques such as
SCNT have been developed and the human. Obviously, much basic research remains to
be done in the human. For instance, the simple expression analysis of imprinted genes in
experimental nuclear transfer human embryos (as has already been accomplished in
normal ooctyes and embryos) would be a critical step in assessing potential aberrant
epigenetic aspects (58). There is reason to hope that the problematic aspects of nuclear
transfer can be successfully addressed in the human. A consistent lesson from the past 12
years of human oocyte micromanipulation, including the development of enucleation
techniques and intracytoplasmic sperm injection, is that human oocytes and early
embryos exhibit much greater tolerance of physical manipulation compared with rodent
and possibly even non-human primate material (59-63) Furthermore, other critical
differences may exist that will facilitate nuclear transfer in the human. This has recently
been supported by the identification of clear differences in the imprinting process
between human and rodent/large animal species and in the successful production of
blastocysts and stem cells following human SCNT(56,45).
It is at least premature to suggest that success with these or related techniques in clinical
applications will be impossible. As in any medical practice issue, examining this issue
should involve an honest recognition of potential benefits along with potential risks. The
tremendous potential of such invasive manipulative techniques needs to be recognized
while accepting that safety and efficacy remains to be determined. Individuals,
organizations and governments are currently involved in considerable debate as to the
proper course to take in regulating human research and medical practice in this area. It is
clear that considerable further research, including human experiments and clinical trials,
will be necessary to begin to answer the many questions that will help to define this
assessment. Hopefully, the coming years will see an increase in support for such basic
research while the ethical debate continues.
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