The successful treatment of infertility through in vitro manipu by bcs24005

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									Does Nuclear Transplantation Have a Place in Clinically Assisted

Reproductive Technology?

Henry Malter and Jacques Cohen
Tyho-Galileo Research Laboratories and Reprogenetics Inc., West Orange, NJ 07052,
USA


Introduction

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 techniques

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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