mitochondrial manipulation technology, MMT, mitochondrial replacement therapy, pronuclear transfer, maternal spindle transfer, ooplasmic transfer, cytoplasmic transfer, three-parent baby
Mitochondrial donation techniques create children born with nuclear DNA from their parents’ sperm and egg plus healthy mitochondria from an egg donor, unrelated to the mother. Mitochondrial donation techniques figure as an optional therapy to eliminate mitochondrial mutations, thereby allowing the birth of mitochondrial mutation-free healthy children, because mitochondria have their own set of DNA (mtDNA) separate from the rest of our body that is inherited through the female line.
Mitochondrial donation is possible through three main ways:
The key distinction is that pronuclear transfer (PNT) occurs after fertilization. It involves the creation and use of embryos, or more accurately 1 day old zygotes (one of which is discarded). The mother’s egg is fertilized by the father’s sperm and a zygote with two pronuclei is formed. The zygote contains faulty maternal mitochondrial genes. The parental DNA has the form of two pronuclei. Furthermore, we need a donor egg which contains defect free mitochondria. The donor egg is fertilized by the father’s sperm and second zygote is formed. Second zygote has healthy mitochondria and also two pronuclei. These pronuclei (donor plus father) are removed and replaced by pronuclei from first zygote (mother plus father). The resulting zygote contains nuclear DNA from mother and father and healthy mitochondria from the donor.
Maternal spindle transfer
This process includes a genetic manipulation technique where the donor's nuclear DNA but not her mitochondrial DNA is transferred to the receiving egg. The technique involves taking nuclear DNA from an egg cell and transferring that DNA into another egg cell, leaving the defective mitochondrial DNA behind. The cell is then implanted using in-vitro fertilization techniques. The final mitochondrial DNA, however, is from the receiving egg cell, causing the original donor's mitochondrial DNA to not be passed on to the eventual offspring.
In this type of mitochondrial donation, a part of the ooplasm (egg cytoplasm) with healthy mitochondria is placed from donor egg into recipient egg with faulty mitochondria (see Pic. 1). An ooplasm, including mitochondria from fresh, mature or immature, or cryopreserved-thawed donor eggs (preferably from a younger woman) is directly injected into recipient oocytes via a modified ICSI technique to enhance the viability of the oocytes. The resulting healthy egg is fertilized and then implanted into the uterus (usually that of the recipient egg). This method is used with confidence as cytoplasmic manipulation of oocytes and early embryos was shown to be compatible with normal development.
Mitochondrial donation involves the transfer of genetic but not nuclear material, and this has led to uncertainty as to whether it should be regulated as egg donation or as tissue donation. Mitochondria play an important role in many bodily processes, and therefore the genetic contribution of the donor might be significant: there are complex interactions between nuclear DNA and mitochondrial DNA and organelles contained in the cytoplasm might introduce epigenetic alterations in nuclear DNA.
The legislation specifies who are eligible to use the techniques: women at risk of transmitting mitochondrial disease to their offspring. This would mean that those wishing to use the techniques to enhance fertility and lesbian couples who wish to use the techniques so that the child has a genetic contribution from both (one would be mitochondria donor) would not be permitted.
The United Kingdom became the first country to legalize the procedure after the Parliament and House of Lords approved The Human Fertilisation and Embryology (Mitochondrial Donation) Regulations in February 2015, and which came into force on 29 October 2015. The current regulations in the UK state that mitochondrial IVF techniques should be limited to selected cases where “there is a particular risk that the patient's egg or an embryo created using the patient's egg will carry a mtDNA abnormality and that there is a significant risk that a child born from the use of that egg will have or develop a serious mitochondrial disease”. The use is closely overseen by that country’s Human Fertilisation and Embryology Authority (HFEA), which regulates assisted reproduction and embryo research.
In the US, the legality of mitochondrial manipulation techniques is still under discussion.
Another area of controversy is the impact of the techniques on the human genome. The UK Department of Health identified that the techniques involved germ-line modification, “in that the result of mitochondrial donation – the avoidance of the transmission of a serious mitochondrial disease – will be passed down to future generations”. It concluded that the techniques did not involve genetic modification; there is no universally agreed definition of “genetic modification” in humans – people who have organ transplants, blood donations or even gene therapy are not generally regarded as being “genetically modified”. While there is no universally agreed definition, the Government has decided to adopt a working definition for the purpose of taking forward these regulations. The working definition that we have adopted is that genetic modification involving the germ-line modification of nuclear DNA (in the chromosomes) that can be passed on to future generations. Opponents of the regulation appear to be concerned that it will lead to the creation of “designer babies”. Another ethical problem is how to prevent other possible uses of the technique.
Genetically, the child will, indeed, have DNA from three individuals but all available scientific evidence indicates that the genes contributing to personal characteristics and traits come solely from the nuclear DNA, which will only come from the proposed child's mother and father. The donated mitochondrial DNA will not affect those characteristics. If our character and physical appearance is considered to be solely determined by our nuclear genes then as the Department of Health suggested, altering mitochondrial genes might not have a significant impact on the child. But many reject this kind of genetic essentialism. Identity is difficult to define, but it is more than our character and physical traits. Reproductive medicine further complicates questions of identity. Being born without mitochondrial disease would, of course, have a significant impact on the child.
Any children born via the technique should be monitored over the long term. That requires parents' permission; on the other hand, you can’t give consent for people who have not yet been conceived.
This technique is used in cases when mothers carry genetic mitochondrial diseases, and conventional IVF techniques do not work. Currently, women who wish to have children and avoid passing on mtDNA conditions have few options. They can seek to use donated eggs, provided by a woman without a history of mitochondrial condition(s), but it does not offer the opportunity to have a genetically related child; apply for adoption, or seek an arrangement with a surrogate mother willing to use her own eggs or donated eggs.
In order to reduce the risk of transmission, women can elect to have preimplantation genetic diagnosis (PGD) to estimate the levels of mutated mitochondria in a given embryo. However, the PGD route is not suitable for all conditions, especially those which – when severe – have fatal outcomes, as the levels of mutated mitochondria in early embryos are a poor predictor of the severity of the disorder. Nor can PGD predict if the embryo will develop into an individual with a high level of mutated mitochondria in all their tissue types, or only in some or one. Hence, PGD is not suitable for all women, including those with high levels of mutated mitochondria; leaving aside any issues pertaining to accessing these technologies. Finally, prenatal diagnosis (PND) can be utilised, but in addition to the limitations outlined above regarding PGD, women (and partners) may be faced with deciding whether or not to terminate their pregnancies, a (potentially) difficult decision exacerbated by the uncertainty in predicting mitochondrial conditions and their severity. Hence, the use of mitochondrial donation techniques would enable these women to have a genetically related child (through their nuclear DNA), if they so wished, whilst simultaneously avoiding the possibility of transmission of “faulty” mtDNA to future generations.
Ooplasmic transfer is recommended for woman with deficient or damaged mitochondria and patients with poor embryo quality. Mitochondrial dysfunctions associated with infertility have clearly been shown in women affected by diseases or metabolic disorders such as diabetes and obesity as well as changes in metabolism resulting from oocyte aging. Advanced maternal age (near to age of 35) can affect mitochondrial functions too. Other mitochondrial dysfunctions are still unexplained but have resulted in an increased number of women requiring IVF or other ARTs.
With every ooplasmic transfer, it is possible that there would be a small amount of the donor’s mitochondrial DNA present. This means that some children born through ooplasmic transfer test positive for having the DNA of three parents instead of two. This creation of genetic hybrids is possibly the first example of human germ-line genetic modification (manipulation of genes that can affect future generations).
Also, because this procedure hasn’t been around for long, there is no data on the effects of ooplasmic transfer on maturing childrens’ health. The cytoplasm containing the mitochondria that is injected into the recipient egg could have an unknown reaction to the egg’s DNA. It is not known for certain whether the injection of the cytoplasm does not alter the egg’s primary function, and the creator of ooplasmic transfers Jacques Cohen himself has helped publish a paper which claims that if this happens there would be probable deficiencies.
It is also not known for certain if the early embryo goes unaffected by the foreign material injected into it and grows without deficiency. The possible mitochondrial DNA transfer brings the risk of the embryo being exposed to diseases connected with mitochondrial DNA, such as diabetes, Lou Gehrig’s disease and general developmental disorders. Disorders in ooplasmic transfer children could be because of the DNA “mishmash“. If the embryo is indeed affected, then the mitochondrial DNA can be passed down from a mother to child (it is only transmitted through eggs).
The regulations being proposed will maintain the ban on altering this nuclear DNA. This means there is no way the technique can be used to choose hair colour, personality traits, intelligence or any other characteristic associated with creating “designer babies”.
Genetically, the child will, indeed, have DNA from three individuals but all available scientific evidence indicates that the genes contributing to personal characteristics and traits come solely from the nuclear DNA, which will only come from the proposed child's mother and father. The donated mtDNA will not affect those characteristics - genetic contribution from the second woman is so small, just 0.01%, that she could not.
Another important thing is that the genes in the mitochondria are entirely separate from the genes in the nucleus and they cannot combine with each other. The only genes that would be affected by the technique would be the damaged mitochondrial genes. A child born using this technique would still receive all its nuclear genetic material - controlling its physical and psychological traits - from its mother and father. Children born after mitochondrial transfer would be free of mitochondrial disease - and would eventually pass this healthy mitochondrial DNA on to their own children.
Importantly, severity of disease in offspring cannot be predicted on the basis of the severity of the disease of the mother. One reason put forward for this is the “bottleneck theory”. Put simply, replication of mitochondrial DNA between cells and redistribution during egg maturation can lead to extreme differences in levels of mutation. This means that an asymptomatic mother can have a very severely or fatally affected child. Indeed many of the parents whose stories have been the focus of the mitochondrial debate did not know that they were at risk of having a child with mitochondrial disease, until their child was diagnosed.
In general, most somatic cells that would be obtained from the reproductively mature oocyte donor would be expected to contain mitochondrial DNA deletions and mutations. Such mitochondrial DNA would not be considered safe for the offspring, as the transmission of these mutations and deletions to all embryonic tissue would be a potential risk. Somatic cell mitochondria appeared to adversely affect embryonic development.
Autologous mitochondria obtained from the patient’s own granulosa cells have been used to successfully improve fertility outcomes. However, concerns were raised about the use of somatic cell mitochondria, as they would be expected to contain point mutations and deletions, similar to all other somatic cells. In addition, death (apoptotic) signals from mitochondria within granulosa cells control follicular atresia and prevent oocyte development. Thus, their transfer during ICSI could prove to be detrimental to the oocyte. High-quality, autologous mitochondria must be used for energy augmentation of oocytes through mitochondrial transfer. The transfer of the same mtDNA obtained from oocyte precursor cells would reduce possible compromised oxidative phosphorylation function that could arise through mixing mtDNA genotypes. Due to the fact that these cells have differentiated to the oocyte lineage but have not spent years in a postmitotic state, these cells may be used as a source of tissue-specific, autologous mitochondrial DNA free of mutations and deletions.
Following donor ooplasmic transfer during ICSI, improvements were observed in embryo formation, implantation, and live births. Patient selection is not based solely upon maternal age but rather on those who had previously demonstrated poor embryo cleavage rates and morphological anomalies. These factors are considered to be representative of inadequate mitochondrial function. As live births of healthy children are achieved, this method was proposed to augment resident mitochondrial activity in compromised oocytes.
The benefits of ooplasmic transfer have been addressed and it was shown that the source of the donor cytoplasm is a major factor in successes or failures. Because of the close functional dependency of mitochondria on the nuclear genome the cell type used for donor transfer is of primary concern and should be closely related to the recipient cell for optimal coordination of mtDNA and nuclear DNA. Ovarian or oocyte-differentiated cells have been shown to yield the highest success rate in transferring donor cytoplasm containing healthy mitochondria into quality-compromised oocytes. Treatments to augment the healthy mitochondrial population through ooplasmic transfer from uncompromised cells would not only benefit the mothers to overcome fertility problems but also children of affected mothers who otherwise may inherit mitochondria with deficiencies and suboptimal function.
There are alternatives to mitochondrial donation. However, oocyte donation does not offer the opportunity to have a genetically related child and relies on a supply of donor eggs, and prenatal diagnosis and pre-implantation genetic diagnosis would have limited success for those with ooplasmic mitochondrial disease.