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. 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