The Role of Cytoplasmic Transfer

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Assisted conception is evidently an inefficient technology and, overall, only ~3% of oocytes collected result in live-births. Embryos are often inviable or abnormal as a result of defects arising during oogenesis and/ or in culture. Advancing maternal age further reduces the fertile potential of oocytes. Cytoplasmic (ooplasmic) transfer aims to improve egg and embryo quality by the donation of ooplasm from supposedly fertile oocytes to patients whose cells are of poorer quality. Cytoplasm can be transferred by either 1) electrofusion or 2) direct injection (Cohen et al, 1998). In 1), a small ooplast recovered from a donor egg is injected under the zona pellucida of a recipient oocyte (after removing its polar body) before fusing the membranes. The cybrid is then fertilized by ICSI. In 2), a small amount of ooplasm (~10%) is aspirated into a pipette and injected into the recipient cell at the same time as the fertilizing sperm. Transfer of anucleate cytoplasm has been used on a small scale in clinical trials, and children have been born. An alternative strategy is to transfer the germinal vesicle nucleus (together with shreds of attached cytoplasm) to an enucleated recipient ooplast (Zhang et al, 1999). In either case, the children should be genetic offspring of the patients, apart from the variable degree of cytoplasmic inheritance from the donor egg. 

Cytoplasmic transfer is a technique of last resort after repeated IVF failure or at advanced maternal age: it is based on an assumption that a vital molecule, such as ATP or cell cycle-related kinase, or an organelle, such as mitochondria, is deficient at a critical stage in early development. Hormone replacement therapy is an obvious parallel but, until there is a better understanding of the molecular development of oocytes, a deficiency state remains hypothetical. Indeed, the opposite hypothesis is also plausible - that poor quality reflects exposure of oocytes to environmental toxins, including drugs, diet and smoking. These hypotheses are not mutually exclusive, but if either is true it will be important to test whether recent evidence that the mammalian egg is polarized carries implications for the site of cytoplasmic “biopsy” and injection.

Cytoplasmic transfer has had some impressive achievements in experimental science. For example, in certain strains of mice or culture conditions embryos halt in early cleavage, and cytoplasm from non-blocking strains helps to overcome the problem (Muggleton-Harris et al, 1982). Nevertheless, experiments also reveal potential pitfalls. In the DDK strain, “cytoplasmic incompatibility” due to a maternal RNA interaction with the paternal genome causes embryonic wastage (Renard et al, 1994), and there is evidence of a replicative race between mitochondria in somatic cells and after nuclear transfer in farm animal oocytes. This is less surprising in view of the natural phenomenon of paternal mitochondria exclusion, which creates homoplasmic embryos. It is therefore unsafe to assume that mitochondrial transplants will be proportionately represented in the tissues of the fetus and child.

In addition to uncertainties about development, there are concerns about the safety of cytoplasmic transfer for future children. There is an obvious danger of transferring stray chromosomes from the donor cell to produce a hyperploid embryo, especially in light of evidence showing that the polar body is not an infallible landmark for avoiding the spindle apparatus. Public health scares about prion disease in cattle and humans and concerns about the risks of xenotransplantation force us to take heed of the risks of disease transmission and complementation. Viral-like particles can be observed in mouse oocytes and endogenous retroviral proteins are detectable on the surface of human oocytes (Nilsson et al 1999).

Finally, it is questionable whether cytoplasmic transfer is justified on the basis of evidence-based medicine, which has been defined as “the process of systematically finding, appraising, and using contemporaneous research findings as the basis for clinical decisions” (Rosenberg & Donald, 1995). There is no firm evidence that it can improve oocyte quality either from animal studies or randomized clinical trials, and isolated reports of successful pregnancies cannot, by definition, provide assurance about reproducibility. Admittedly, some of the key breakthroughs in ART were made before studies of safety and reliability had been performed, and there are major practical and ethical problems designing and executing trials in ART to the same exacting standards as, say, clinical pharmacology. Yet, the onus is on practitioners of cytoplasmic transfer to offer a convincing justification, or to produce a persuasive and ethically defensible case for making reproductive medicine an exception to the principles of evidence-based medicine. I am not aware that a case has been made. Nevertheless, it is important to remember that the reproductive medicine community must tread a cautious path between unproven technology, on the one hand, and damping valuable innovation, on the other.

References:

References

COHEN J, SCOTT R, ALIKANI, M et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod 4:269-280,1998

MUGGLETON-HARRIS A, WHITTINGHAM DG, SCOTT L. Cytoplasmic control of preimplantation development in vitro in the mouse. Nature 299:460-462,1982

NILSSON BO, JIN MS, ANDERSSON AC et al. Expression of envelope proteins of endogenous C-type retrovirus on the surface of mouse and human oocytes at fertilization. Virus Genes 18:115-120,1999

RENARD J-P, BALDACCI P, RICHOUX-DURANTHON V et al. A maternal factor affecting mouse blastocyst formation. Development 120:797-802,1994

ROSENBERG W, DONALD A. Evidence-based medicine: an approach to clinical problem-solving. Brit Med J 310:1122-1126,1995

ZHANG J, WANG C-W, KREY L et al. In vitro maturation of human preovulatory oocytes reconstructed by germinal vesicle transfer. Fertil Steril 71:726-731,1999