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Brave New Embryology

Credit: Mopic/Adobe

After 30 years of IVF, only around 25% of the embryos created have the capacity to develop to term. Credit: Mopic/Adobe

By Chris O’Neill

New technologies are being developed to improve fertility, but the effects on the embryo are uncertain.

The development of in vitro fertilisation (IVF) for the treatment of infertility has opened up new and unexpected methods of alleviating disease. Creating embryos in a test tube allows the diagnosis of most known genetic conditions within days of fertilisation, and new gene editing techniques offer the prospect of correcting these conditions.

While this brings great hope, it also prompts fears of genetic manipulation for non-medical reasons, unintended biological consequences for the child, and even consequences across successive generations. For example, evidence suggests that the manipulation of gametes and early embryos may cause maladaptive errors in the programming of normal gene expression, leading to an increased burden of life-long chronic diseases.

The pace of innovation is breathtaking, yet our knowledge of the underlying biology may be too immature to allow the confident prediction of all outcomes. Future research must focus on these perceived risks, as well as the technical innovations.

Genetic Manipulation of the Embryo

Genetic diagnosis in the pre-implanted embryo is a widely used technology. The microsurgical biopsy of a small number of cells from the embryo in the test tube allows most genetic information to be retrieved. The diagnosis of common genetic conditions, as well as determining the sex of the embryo, allows couples to choose whether an embryo carrying a genetic disease will be transferred back to the uterus. Sex selection is used to eliminate genetic conditions that only affect males, such as Duchenne muscular dystrophy and haemophilia. In these cases, the couple may choose to transfer an affected female embryo but not its male sibling embryos. In Australia, it is currently not permitted to choose the sex of the embryo purely for social reasons, although this is under review by the National Health and Medical Research Council.

An alternative to the destruction of embryos that carry genetic diseases is now approaching feasibility. This involves the use of gene-editing technology to replace or repair the defective gene in the oocyte or early embryo. New tools for gene editing exploit the capacity of bacteria to recognise and remove invading foreign DNA. The bacterial molecules that perform this task (e.g. CRISPR/CAS9 and TALENs) have been isolated and can be used to perform sophisticated editing of the human genome. The technique is highly efficient and has a low error rate compared with earlier developments in genetic modification. It has been very widely used in animal models and is now under active discussion in many international jurisdictions for use in human embryos.

A key issue of concern is that genetic changes to the cells of the early embryo will inevitably be passed on to every cell in the body, including the sperm or oocyte (egg). As a result, these changes will be passed down through future generations.

There are many valid questions about how quality assurance can be performed to ensure that only the desired genetic changes are induced and that no off-target effects are generated. There are also inevitable questions about how the technology can be regulated to ensure that it’s not used for non-medical purposes that would bring a number of science fiction scenarios into the real world.

Under current legislation, gene editing of a human embryo cannot be performed in Australia but its future use in other jurisdictions will inevitably lead to calls for this to be reviewed.

Treatment of Mitochondrial Diseases

Every cell contains small membrane-bound structures called mitochondria. These structures convert nutrients into the energy used to perform cellular functions. Curiously, the proteins that carry out the most critical functions of the mitochondria are encoded by a small genome contained within the mitochondrion itself. Mutations in this genome can result in a serious loss of normal mitochondrial function and severe disease, typically affecting the neurological and neuromuscular systems. While these mitochondrial mutations can occur spontaneously within individuals, they are often inherited with the oocyte.

A remarkable feature of sexual reproduction in almost all organisms is that mitochondria are only inherited from one parent. In mammals, this is the female. This means that if the mother’s mitochondrial DNA has defects, this cannot be compensated for by mitochondria from the sperm.

One potential therapy for inherited mitochondrial defects is to remove the nucleus from a genetically affected oocyte (or a fertilised one-cell embryo) and transfer it to a donor oocyte that has normal mitochondria but from which the host nucleus has been removed. The resulting embryo is commonly referred to as a three-parent embryo. There is some controversy surrounding the use of this technique.

Apart from any ethical qualms about having “three parents”, scientific questions arise about the safety of this approach. It is argued that the pervasive matrilineal inheritance of mitochondria throughout the evolution of complex organisms has resulted in the genetic co-selection of the nuclear and mitochondrial genomes to ensure their close functional co­ordination. Modelling studies suggest that the use of donor mitochondria may uncouple this tight coordination and potentially pre­dispose individuals to disease. It is difficult to decide how serious such concerns would be for the resulting individual and what, if any, mitigating strategies could be put in place to alleviate potentially adverse outcomes.

Although this therapy has been approved for use in the UK, caution is needed in this area until a deeper understanding of the biological and safety issues is available. This controversy reflects the understandable conflict between the wishes of a couple to conceive a child and our responsibility to the unconceived child.

An alternative genetic editing approach has been proposed and tested in animals. This involves the use of gene-editing technology to significantly reduce the number of defective mitochondria in the embryo. It uses the same form of technology described above for nuclear gene editing, but it is instead directed at the mitochondrial genome. Because affected oocytes typically contain a mix of normal and genetically abnormal mitochondria, the approach is to edit out many of the abnormal copies, allowing the normal copies to gain numerical ascendancy. It is unclear whether current legislation in Australia would prohibit this approach.

Embryonic Stem Cells and Regenerative Medicine

When embryos grow to around 80 cells, they form a structure resembling a soccer ball called the blastocyst. The cells in the outer layer of this ball (~50 cells) will eventually form the placenta. A small group of cells (~30) form a clump inside the ball (the inner cell mass), and these eventually form the embryo proper after the blastocyst invades the lining of the uterus.

The microsurgical isolation of the inner cells and their propagation under special culture conditions creates a population of cells that will proliferate indefinitely. These embryonic stem cells are pluripotent – they have the potential to form every different cell type within the organism.

As these pluripotent stem cells can be directed to differentiate into any of the specialised cells of the body, they are a potential source of “spare parts” for our bodies. Currently, regenerative medicine trials for the treatment of type 1 diabetes, spinal injury and macular degeneration are underway using the transplantation of specialised cells derived from embryonic stem cells.

A limitation of the use of embryonic stem cells is that they are immunologically foreign to the person receiving them, and are therefore potential targets for immune destruction. In the case of diabetes, treatment is achieved by encasing the cells in a membrane that is impermeable to the host’s immune cells.

However, this strategy is not applicable when transferred cells must be integrated into the structure of an organ. A potential solution is the conversion of adult cells into embryonic stem cells.

The changing of adult cells to those resembling the embryo is called “induced pluripotency”. The discovery that a small number of key molecules determine the pluripotent state of the inner cell mass has allowed scientists to force the expression of these factors in adult cells. Remarkably, this has resulted in some adult cells resuming the characteristics of embryonic stem cells.

The potential of these cells for regenerative medicine is being fully investigated, but perhaps the most striking discovery is their ability to convert into either sperm or oocytes. In mouse models, gametes produced from induced pluripotent stem cells are fertile and can produce viable offspring. Obvious questions regarding the safety of using gametes created from converted adult tissue must be addressed before any thought should be given to it as a treatment for infertility.

Induced pluripotent stem cells allow the development of banks of cells from people with known genetic conditions. These can be induced to differentiate into tissues of interest , such as nerve cells in cases of neurodegenerative disease, and serve as powerful tools for the investigation of disease mechanisms and for drug screening. Induced pluripotent cells can also be used to test the toxicity of new chemicals.

Unexpected Long-term Consequences of Working on Embryos

All of these exciting advances in embryo-based therapies may be associated with unexpected biological risks. A new field called epigenetics points to the important role of the early embryo in setting the program that determines the timing and level of expression of key regulators of homeostasis throughout life. A host of stresses experienced by gametes and early embryos can adversely change these settings.

The technologies used to create and manipulate the embryo in vitro generate stress responses in the embryo that alter epigenetic settings. When this is sufficiently severe, it leads to early embryo death.

It is chastening to realise that after 30 years of developing these techniques, only around 25% of the embryos created have the capacity to develop to term. The vast majority die within the first week or so after IVF. Those that survive have an increased risk of epigenetic changes in gene expression patterns throughout life, which causes an increased incidence of hypertension, poor glucose control and a range of other chronic diseases. Animal modelling shows that the more extreme the manipulations of the embryo, the greater the stress response and epigenetic disturbance.

These studies point to a need for a greater understanding of how environmental conditions disturb the process of epigenetic programming in the gametes and early embryo, as well as the development of strategies to mitigate their maladaptive effects.

Conclusion

The confluence of our new molecular understanding of embryology and an increased capacity for the microsurgical and genetic manipulation of the embryo provides a panorama of new opportunities for alleviating human disease. Each of these new opportunities suggests questions about their safety and the possible unintended consequences for future generations.

The potential benefits are such that many are certain to be fully explored, but it is equally important that the long-term safety of any new treatments receives equal attention. It is unlikely that the market will attend to these needs, and this market failure will mandate institutional intervention.

Chris O’Neill is Professor of Reproductive and Developmental Medicine at the Kolling Institute for Medical Research and Sydney Medical School, President of the Society for Reproductive Biology, and a past member of the National Health and Medical Research Council’s Embryo Licensing Committee.