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Taken from www.rbmonline.com - August 2003

Toward scientific discussion of human reproductive cloning

Joe Leigh Simpson
Baylor College of Medicine, Houston, Texas, USA
Correspondence: e-mail: jsimpson@bcm.tmc.edu

Science advances in time honoured ways. First, an investigator generates a hypothesis and then proposes experiments. Concurrently, ethics of the proposed research must also be considered, based on principles of beneficence. Do benefits outweigh risks, for society as well as for individuals? Will the research be conducted under the aegis of the appropriate oversight, Institutional Review Boards in the United States? Next follows the actual study, its publication and eventual validation through replication. Ideally, scientists, individuals, and society synchronize these time-honoured sequences. However, in reproductive medicine we are often accused of deviating. Promising clinical advances are said to be incorporated into practice without prior ethical deliberation. Conversely, others in society would, strictly on ethical grounds, proscribe many advances beneficial to patients. A considerable minority still disagrees with women exercising any reproductive choices, undergoing prenatal genetic diagnosis or availing themselves of assisted reproduction treatment. Many would seem quite happy to turn the reproductive clock back half a century.

The predictable effect of attempting to ban or criminalize reproductive cloning is to drive investigators underground, and cause patients to become complicit. Paradoxically, the result is to hide the science, rather than have it conducted under a spotlight that would generate public confidence. Zavos’ (2003) note could have the salutary effect of helping lead us out of this scientific imbroglio. True, I would have preferred more data in it, and sceptics are justified in holding opinions in abeyance until molecular studies confirm that the 8–10 cell embryo is truly a clone. However, the experiments proceeded logically. First, human granulosa cells were placed in enucleated bovine oocytes, electrostimulated, and shown to develop at least as often as occurs in parthenogenesis. Next, human granulosa cells were placed in enucleated human oocytes, one in nine yielding the embryo whose photograph is published. Whether the cloned embryo is normal is not yet known.

Setting aside for the moment concerns about either scientific veracity or ethical acceptability, how successful might human reproductive cloning be? Is success a priori so unlikely that reproductive cloning should not be pursued on scientific grounds? In non-human primates this was indeed the conclusion of Simerly et al. (2003), who believe disturbances of the mitotic spindle assembly precluded development in any of 33 rhesus embryos created by non-embryonic somatic cell nuclear transfer. Many believe this will be the fate of human clones, justifying the de-facto ban on human reproductive cloning in most of the world. Yet, how definitive for humans could be evidence derived from rhesus monkeys, a species differing from humans with respect to oocyte size, form of implantation, and perhaps also response to micromanipulation procedures such as intracytoplasmic sperm injection (ICSI)?

Could the work of Zavos (2003) and others ever lead us to alter beliefs that clinical success in reproductive cloning is unlikely if not inconceivable? In fact, perusal of animal studies tabulated by the US National Academy of Sciences (2002) reveals promising results in several animal studies, using adult cells. A few random examples: Polejaeva et al. (2000) in pigs (using adult granulosa); Ogura et al. (2000) in mice (immature adult Sertoli cells), and Wells et al. (1999) in cattle (adult mural granulosa cells). True, many reports have documented perinatal complications. Consider, however, the experience of Chesné et al. (2002), which suggests that the conclusion may be less categorical and definitive than originally believed. These workers initially observed that pregnancies with cloned rabbits resulted in death and anomalies; however, delaying transfer by a day or so (through use of an asynchronous recipient) resulted in normal full-term rabbits. That is, a problem existed but the solution proved relatively simple. In fact, discrepant results, some highly successful, others abject failures, are not unprecedented in reproductive medicine. Following initial IVF successes in the early 1980s, several centres experienced embarrassingly long stretches without a single baby; only after individual technical prowess evolved did the ‘take home baby rate’ improve to the current US rates. (In the year 2000 US cycles, 32.8% produced a livebirth in women <35 years; 26.7% in women aged 35–37; 18.5% in women aged 38–40; and 10.1% in women aged 41–42; Centres For Disease Control, 2002). Our IVF pioneers did not assume that the few successful early series were aberrations, and clinical IVF thus not worthy of pursuit. Rather, these workers learned from and built on the initial successes. All this occurred quite openly – peer reviewed publications, presentations at established scientific meetings, ubiquitous conferences. Given that attempts at cloning are clearly proceeding somewhere, the open scientific forum exemplified by early IVF workers should be the paradigm.

A pivotal reason for open discussion is the lack of full explanation for exactly why reproductive cloning might be scientifically unwise, notwithstanding many statements of certitude. The distinguished US National Academy of Sciences (2002) panel systematically considered potential explanations, but concluded that failure of an adult somatic cell to transmutate properly into a zygote (reproductive cloning) must be the result of imprinted genes unable to reset their genetic ‘switch’ from adult to gamete or embryo. Alberio and Campbell (2003) succinctly summarized reasons for epigenetic factors underlying all offspring derived from nuclear transfer, somatic or embryonic in origin. Yet other evidence shows that exogenous factors (e.g. culture conditions) actually determine the precise outcome, and that these are not immutable. Even though imprinting disturbances occur in cloned animals, perhaps always (Humpherys et al., 2002), less is known about such molecular aspects than some would have us believe. It follows that consequences in human embryos are even less well understood, and extrapolation from animal studies could be hazardous. Animal studies are always desirable, but not necessarily obligatory and even potentially misleading. Recall that in the 1970s, many insisted that non-human primate success was necessary before embarking on human IVF. Those ignoring this admonition made history, and non-human primate IVF rates are still not high. In humans, the clinical consequence of imprinting perturbations in reproductive cloning might merely be failure to implant, or failure to survive early embryogenesis. Those embryos, however few, that successfully make the ‘switch’ might just prove as normal as in-vivo biparental conceptions. Naturally, it is not possible at this time to predict the likelihood of this sanguine sequence, but I personally consider it more than mere speculation. Reproductive clones are a fact in many animals, and for every well publicized premature demise (Dolly) there are many quiet successes.

The beauty is that all this is testable scientifically. Molecular studies should tell us if imprinting perturbations exist, and hence if Zavos’ embryo could develop normally. Will Oct-4 or other genes fail to be expressed, as may occur in mice (Boiani et al., 2002)? If imprinting perturbations exist, however, this need not provide scientific justification for proscription in perpetuity. This follows from ongoing work in humans, on the various clinical disorders caused by disturbances in imprinting (e.g. Rett syndrome, caused by perturbation of the transcriptional repressor MeCP2). Jaenisch and Bird (2003) recently wondered whether one might correct errors of methylation or demethylation, i.e. to treat disorders like Rett syndrome. If successful, the temptation to apply the same technology to reproductive cloning would be irresistible. If such a technical tour de force were possible, ethical issues once dismissed as moot would once again become topical.

To take into account potentially sinuous scientific pathways, I wonder if the ethical discussion should not be stratified to include different outcome scenarios. This would not only respect the fact that not all members of the public are rigid in their opposition to reproductive cloning, but would also address their belief that the concern is not so much ethics per se as safety and efficacy (Harris, 2002; Simpson and Edwards, 2003). We might start the dialogue with the usual assumption that liveborn clinical anomaly rates in human reproductive cloning will be prohibitively high (like or unlike animals?) and the motives nefarious, i.e. creation of a clone identical in appearance and temperament to the egotistical, and doubtless boastful donor. Probably we would all agree that reproductive cloning should not be pursued. But what if anomaly rates prove no greater than the 2–3% background in human liveborns? What if liveborn success rates prove unexpectedly high? Higher than traditional IVF? Cheaper to achieve? What if biparental genetic contributions prove facile, either through generation of chimeras or insertion of mitochondrial or nuclear genes? Or, if somatic cell hybridization, literally a branch of cloning, could offer sterile couples an alternative way to have their own biparental child (Tesarik et al., 2001)? What if perturbations of imprinting can be corrected? Then, there is the paradoxical possibility that imprinting could be the silver lining. Suppose it is stochastically impossible to reset all imprinting genes to the same state that exists in the parental somatic progenitor. Suppose some genes randomly reset in a way distinct from the adult progenitor (i.e. maternal versus paternal allele), yielding not necessarily pathological but still distinctive phenotypic differences. The cloned embryo would now differ from its somatic progenitor, in fact more so than monozygotic twins. Clone and parent would neither look, nor be, exactly the same. Their DNA sequences would be identical, but gene expression and thus phenotype would differ. This might disappoint an egotistical donor, but assuage critics. Irrespective, reproductive cloning would scientifically become merely another tool in the pantheon of infertility treatments.

In conclusion, reproductive cloning raises enough questions to keep us all busy. Zavos (2003) and others will continue their pursuit in one venue or another. Given this, I find it unconscionable to stifle scientific openness and ethical deliberation. Closing either avenue would be at the scientific community’s peril, inviting untoward and unpredictable outcomes below the horizon of scrutiny.

References

Alberio R, Campbell KH 2003 Epigenetics and nuclear transfer. Lancet 361, 1239–1240.

Boiani M, Eckardt S, Scholer HR et al. 2002 Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes and Development 16, 1209–1219.

Centers For Disease Control 2002 Assisted Reproductive Technology Success Rates; National Summary and Fertility Clinic Reports.

Chesné P, Adenot PG, Viglietta C et al. 2002 Cloned rabbits produced by nuclear transfer from adult somatic cells. Nature Biotechnology 20, 366–369.

Harris M 2002 Americans Deeply Divided About Use of Genetic Technologies in Reproduction Genetics. Genetics and Public Policy Center, Office of Communications and Public Affairs, John Hopkins Medical Institutions, Baltimore, USA.

Humpherys D, Eggan K, Akutsu H et al. 2002 Abnormal gene expression in cloned mice derived from embryonic stem cell and cumulus cell nuclei. Proceedings of the National Academy of Sciences of the USA 99, 12889–12894.

Jaenisch R, Bird A 2003 Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nature Genetics 33 (suppl.), 245–254.

National Academy of Sciences 2002 Scientific and Medical Aspects of Human Reproductive Cloning. National Academy Press, Washington D.C., pp. 1–272.

Ogura A, Inoue K, Ogonuki N et al. 2000 Production of male cloned mice from fresh, cultured, and cryopreserved immature Sertoli cells. Biology of Reproduction 62, 1579–1584.

Polejaeva IA, Chen SH, Vaught TD et al. 2000 Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407, 86–90.

Simerly G, Dominko T, Navara C et al. 2003 Molecular correlates of primate nuclear transfer failures. Science 300, 297.

Simpson JL, Edwards RG 2003 Public objections to designer babies and cloning in USA: not quite what was expected. Reproductive BioMedicine Online 6, 147–148.

Tesarik J, Nagy ZP, Sousa M et al. 2001 Fertilizable oocytes reconstructed from patient’s somatic cell nuclei and donor ooplasts. Reproductive Biomedicine Online 2, 160–164.

Wells DN, Misica PM, Tervit HR 1999 Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. Biology of Reproduction 60, 996–1005.

Zavos PM 2003 Human reproductive cloning: the time is near. Reproductive BioMedicine Online 6, 397–398.


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