▎WuXi AppTec content team editor
In 2016, when the world's first "three-parent" baby was born, it attracted global attention. Genetically, the baby had three parents — in addition to inheriting most of the genomic information wrapped in chromosomes from the parents, his mitochondrial DNA was derived from an anonymous donor.
▲The world's first "three-parent" baby and its "founder" Dr. Zhang Jin (Image source: Reference [2])
The original intention of the Three-Parent Baby project was to interrupt the intergenerational transmission of hereditary deadly diseases. The baby's mother carries a genetic mutation in the mitochondria of the disease, and replacing the original mitochondria in the mother's egg cells with mitochondria from a third-party donor egg cell prevents the baby from inheriting the mitochondrial genes for these lesions. The therapy is now licensed in several countries, including the UK, Greece, Ukraine and Australia.
However, the "triple-parent" technique is not entirely reliable to prevent the intergenerational transmission of hereditary fatal diseases. One of the problems is the phenomenon of DNA "reversal," in which the proportion of maternal mitochondrial genes increases over time. This phenomenon has been observed in some babies born with the help of "three-parent" technology, and some children have mothers whose mitochondrial gene ratio has increased from less than 1% to more than 50%. Although neither of the babies' parents had a mitochondrial disease gene, so the phenomenon did not cause health problems, it alarmed scientists. Some scientists estimate that about one in five babies born using the "three-parent" technique will eventually inherit a large number of mitochondrial genes from their mothers. This can be catastrophic for babies whose mothers carry disease-causing gene mutations, as it can lead to potentially fatal disease. These findings have prompted some medical institutions to reconsider whether to use this technology to prevent mitochondrial diseases.
What is a "three-parent baby"
A three-parent infant is a "recombinant embryo" consisting of mitochondrial DNA from a donor and nuclear DNA from both parents to reproduce. This approach is often referred to as mitochondrial replacement therapy (MRT). Mitochondria are small "energy factories" in the cytoplasm that store mitochondrial DNA (mtDNA) that can only be passed from mother to child. When a mother's mitochondrial DNA undergoes a disease-causing mutation, all of her offspring will be affected, potentially leading to a range of health problems and sometimes life-threatening.
▲Schematic diagram of mitochondrial replacement therapy (Image source: Reference [3])
MRT technology has two different routes. A common route is to first remove the nuclei of two eggs (one from the mother and one from the donor) and then place the mother's nuclei into the donor's egg, which contains healthy mitochondrial DNA. Next, this "recombinant" egg combines with the father's sperm to produce an embryo with three genetic parents. Another route is to first let the germ cells of both parents complete the fertilization process, produce a fertilized egg, and then transfer the nucleus of this fertilized egg to another fertilized egg whose nucleus has been removed in advance, which also has three genetic parents and does not carry the mother's disease-causing mitochondrial DNA.
MRT technology is thought to be helpful in treating certain cases of unexplained infertility and improving the success rate of IVF, as it is thought that the mixture of proteins in the egg cytoplasm may cause couples to be unable to conceive. However, while this technique has shown potential in treating infertility, studies based on in vitro cell culture and animal models have shown that MRT may not completely prevent mitochondrial disease, which could have serious consequences if it occurs in humans.
The hidden dangers behind cell-to-cell "transactions"
When a technician retrieves the nucleus in an egg or fertilized egg, it is difficult to completely avoid passing some cytoplasm, which may include mtDNA. These mutated mtDNA enter the embryo that carries the baby with subsequent manipulation. Scientists have managed to limit the amount of mtDNA that may be incidental during the operation to less than 1% of the embryo's total mtDNA. Some scientists believe that the 1 percent of mtDNA that could be brought in during the operation is not a concern, because the other 99 percent of mtDNA is healthy. However, it seems that things are not that simple. Studies have found that this number increases over time, causing the mtDNA reversal phenomenon mentioned earlier. If the proportion of "bad" mitochondrial DNA is too high, it can lead to disease in offspring, even if those children were born through MRT technology.
To test whether this could happen in humans, a team of researchers used MRT technology in 25 couples who had all experienced 3 to 11 unsuccessful IVF cycles. All women were diagnosed with some form of infertility. Of these 25 couples, each wife first underwent a standard IVF procedure so that the doctor could collect a sufficient amount of eggs. The nuclei of these eggs are then removed and placed in the donor eggs that have been removed from the nucleus. The resulting "recombinant" egg is then combined with the husband's sperm to form an embryo. Once the embryo began to develop, scientists took several cells from it to see their mitochondrial genetic status. The vast majority of all embryos come from donors, with less than 1% coming from women in infertile couples.
Using 122 maternal eggs and 122 donor eggs, the team successfully fertilized 85 embryos with healthy mtDNA. In the study, a total of 24 embryos were developed healthy, 19 of which were transferred to the uterus to continue to develop, resulting in 7 successful conceptions and the birth of 6 healthy babies, all born between the end of 2019 and 2020.
The research team has been tracking the children's mtDNA levels since birth, and they found that the mothers of five of the babies still had low mtDNA levels of less than 1 percent. But something strange happened to the last child...
At the embryonic stage, less than 1 percent of the child's mtDNA came from its mother and more than 99 percent from a donor. But when he was born, that balance was upset—30 to 60 percent of the body's mtDNA came from his mother, almost as much as a donor's mtDNA. The findings were published in the journal Fertility and Sterility.
Following the publication of the study, a second similar case was reported at the Fertility Center in Kiev, Ukraine. Dr. Pavlo Mazur, an embryologist at the center, said in a 2020 online conference that a baby boy also showed a reversal. The baby was one of 10 babies born in the center's MRT infertility pilot trial. They took a slightly different technological path, starting with the creation of a fertilized egg, then removing the nucleus and then performing cytoplasmic replacement.
The baby boy, born in 2019, is the second child of a woman who has been treated for MRT twice. Her first child, a girl born in 2017, showed no reversal, and her mother-derived mtDNA levels remained below 1 percent. But her little brother wasn't so lucky—despite having the same team using the same woman's eggs and performing the same procedures at the same clinic, about 72 percent of his mtDNA came from his mother.
Dr. Mazur says they discovered the phenomenon before the journal Fertility and Sterility reported cases of mtDNA "reversal," but never published it.
What is the risk of disease associated with mitochondrial DNA reversal?
So what are the consequences of mitochondrial DNA reversal, and how many babies born based on MRT technology will be affected?
Professor Dagan Wells of the University of Oxford, one of the lead authors of the report, said it was difficult to predict exactly how many babies might be affected by the reversal, and that the current sample size was too small to reflect the probability of such a scenario. Another author of the paper, Dr. Shoukhrat Mitalipov of Oregon Health and Science University, based on current research and previous work in cell and animal models, judged that the frequency of reversal occurs is about 20%, in other words, if the original intention of MRT technology is to avoid the transmission of mutated mtDNA between generations. Babies born with this technique then have a one-in-five chance of inheriting the mutated mtDNA and progressing further to pathogenic levels. In his opinion, this is not unusual.
For infertile couples with no history of mitochondrial disease, the risks of using this technique appear to be low. But for couples who use MRT to prevent mitochondrial disease, the risk can result in the birth of babies with serious health problems. A medical ethicist at Ghent University said it was important to consider what intended parents would choose if the technology wasn't available. If these couples were to try to have children anyway, an 80 percent reduction in the risk of passing on disease-causing mtDNA might be acceptable. Some people may therefore consider using donor eggs to conceive or adopt a child, as the risk is unbearable.
Putting human considerations aside, some scholars believe that the 20% risk of mtDNA reversal is already very high, which a mitochondrial geneticist at the University of Oxford describes as "very worrying." In her opinion, certain low-level mutations in the human body (such as 15%) can cause disease problems, so the ultimate risk of mtDNA reversal may be much greater.
In addition, differences in gene expression in different tissues complicate the problem. In the case of MRT infants, pathogenic mtDNA is low in the blood, but they may be expressed at high levels in brain or muscle tissue. This phenomenon can also be observed in monkeys born using MRT technology.
To complicate matters further, levels of these pathogenic mtDNA may change over time. The consequences of disease-causing gene mutations do not manifest themselves at first, but accumulate over the life cycle and do not show symptoms until much later. All of this makes it very difficult to predict how many babies born with MRT technology will be at risk of serious disease.
What is the future of MRT?
For now, researchers plan to continue trials to figure out the risks of reversing the phenomenon, but some caution that MRT should be suspended, at least for now, and that research can be done in people without mitochondrial disease, such as infertility patients.
For those who really "need" MRT therapy, such as couples who are determined to have a genetic connection to their children, experts suggest that medical institutions undergoing MRT treatment need to update the information they provide so that people know the real risks they face when undergoing MRT treatment.
The development of any new technology is accompanied by doubts and twists and turns, and we should keep an open mind for the development of MRT technology. At the same time, it also gives scientists a little more confidence and patience that they will eventually find a solution to the mtDNA reversal phenomenon.