How 'three-parent babies' can prevent mitochondrial diseases

Scientists have long searched for a way to help mothers with faulty mitochondria have healthy children, without passing on the risk of disease. That search led to a groundbreaking technique known as mitochondrial donation IVF, standing on the shoulders of fertility science up to this point, and applying it to mitochondrial research. So far, eight babies have been born in the UK using this technique. One of the researchers behind this work is Mary Herbert, a reproductive biologist at Newcastle University and Monash University…

Mary - The patient, once they have approval, will undergo ovarian stimulation and egg collection. We will freeze those eggs, and then once a suitable donor has an egg collection, we thaw the patient’s eggs, fertilise both sets of eggs with the partner’s sperm, and then perform pronuclear transfer six to eight hours after fertilisation. What you get after that is a reconstructed fertilised egg, which contains the nuclear DNA from the parents and predominantly the mitochondrial DNA from the egg donor.

James - In this way, the doctors have effectively decoupled the inheritance of the nuclear genome from the mitochondrial genome. But despite how simple Mary makes it sound, this is a very tricky procedure to pull off.

Mary - It is indeed tricky. It requires a high level of skill, all the more so because we do it about eight hours after fertilisation, usually between midnight and 2 a.m., so it’s a pretty tough calling. The reason we do it at that time is because, initially in our preclinical research, we came in the next morning to do the pronuclear transfer, but the eggs didn’t survive that very well. We realised this was because it was too close to the time of division to the two-cell stage. So we decided to radically change the protocol so that we removed the pronuclei as soon as they appeared. That makes it a small-hours-of-the-morning job.

James - Putting the all-nighter to one side for a moment, the key technical challenge of the pronuclear transfer is presented by the natural rigidity of human egg cells.

Mary - You have to inhibit that rigidity before you can take out the pronuclei. You use fine pipettes to do this to exactly the right diameter. Once you have placed your egg into these inhibitors, the cytoplasm becomes a bit more fluid, and you pinch off the pronuclei so they are surrounded by a little bit of cytoplasm and bounded by a fragment of the egg’s plasma membrane. That’s what we call the karyoplast. Like a little cell in itself, it contains the pronuclei surrounded by a small piece of cytoplasm from the egg and bounded by the plasma membrane. We take one pronucleus out at a time. There are therefore two of these karyoplasts per patient egg enucleated. We then give them a very brief exposure to a fusogen, an agent that enables them to fuse back with the enucleated donor egg, and they fuse very nicely. It’s really nice to see this in a movie, actually.

James - But as precise as these highly skilled surgeons may be, removing the patient’s nucleogenetic material carries some risk.

Mary - Human eggs are really packed full of mitochondria. So when we transplant the patient’s nuclear DNA, it’s almost inevitable that it will also contain some patient mitochondrial DNA. Of course, you have to consider what happens to that. If it’s preferentially amplified, then you may not be preventing the disease. In the research phase, we found that when we did this procedure, the embryos had very low levels once we optimised the process. But when we made embryonic stem cells, 20% of them reverted to the maternal mitochondrial genome. That told us that pronuclear transfer can reduce risk, but we cannot guarantee prevention.

James - All patients are rightly informed of the limitations of mitochondrial transfer, that it’s a risk-reduction strategy rather than a guarantee of prevention. But without letting the perfect be the enemy of the good, Mary and the team have been able to give families who thought they might never safely have children a wonderful opportunity.

Mary - We got the go-ahead to start treatment in 2018. In the paper we published in the middle of July, we reported on 19 patients who had the pronuclear transfer procedure. Of those, seven became pregnant, and so far there are eight babies with another one on the way.

James - But as to the crucial information: what levels of mutated mitochondrial DNA were found in these babies in follow-up testing?

Mary - Crucially indeed. Six of the eight have undetectable levels; five of the eight had undetectable levels at birth, and one had 5%. When they went back and looked at three months, that case was undetectable. That level dropped. The other two cases had 12% and 16%, but the important point is that these levels are far below the threshold for disease. In general, you don’t get severe symptoms until over 60%. The eggs that would have given rise to these babies, had we not done pronuclear transfer, had levels of mutated mitochondrial DNA ranging from 67% to 100%.

James - Bearing down on exactly why these mutated mitochondria are still present will therefore be the next step in the development of this hugely promising technique.

Mary - This resurgence of the maternal mitochondrial DNA has been a focus of research, and we’re still trying to understand what the drivers are and whether we can prevent it so that we bridge the gap between risk reduction and prevention.