Pronuclear transfer (PNT) – process, indications and prospects

In this webinar, Elena Lloret, Biomedical Scientist at iGLS (Reproductive Genetics Laboratory – IVF Life Group), has explained what pronuclear transfer is what the process looks like and the prospects of these techniques.

It’s a very complex topic to understand the context of these techniques; therefore, Elena started by introducing the mitochondria: What is it? Why is it so important? What happens when they are not working properly? For this reason, she also explained mitochondrial diseases, the causes of these diseases, and inheritance patterns.

Afterwards, Elena discussed Mitochondrial Replacement Techniques, and more in depth, she explained Pronuclear transfer, the indications, the process itself, prospects of this technique.

Mitochondria as the powerhouse of the cell

Mitochondria are a double membrane organelle present in most eukaryotic cells. It’s important to know that eukaryotic cells are present in human bodies, and they also form a part of animals and some insects. An important aspect of them is that they’ve got their own genetic material, and they could double membrane.

There’s an important hypothesis that says that a mitochondrion back in time, ages ago, used to be an independent organism, like an independent bacterium, that was phagocyte by another cell and, therefore, they established a symbiotic relationship where the mitochondria were able to produce energy and then the other bigger cell would protect the mitochondria. Thus, in that way, the cell could live in different atmospheres and environments, and survive, which gave a really important evolutionary advantage.

The main function of the mitochondria

The main function of mitochondria is to provide energy in the form of ATP, which is a molecule. It also provides around 95% of the solar energy requirements. It has other functions such as:

• Thermogenesis
• Homeostasis
• Apoptosis, which is really important. It’s the programmed cell death.
• Produces reactive oxygen species.

It’s important in all sorts of processes, but here we’re going to be focusing on ATP production and; therefore, energy production.

Mitochondrial diseases

We’re going to look at mitochondrial diseases, which are a group of genetic pathologies that present functional mitochondria; therefore, there’s no appropriate production of energy and these conditions mainly affect tissues that have a high consumption of energy, such as the brain, muscles, liver, kidneys, these are like back muscles and big tissues that require a lot of energy.

Above you can see some mitochondrial diseases.

Regarding the diagnosis:

• Clinical recognition and biochemical screening (one of the main parts).
• Histopathology test through biopsies of the tissues.
• Molecular genetic tests (very important to mention). These tests imply whole genome analysis, exome sequencing, and all the tests.

How common are mitochondrial diseases?

As mentioned at the beginning, mitochondria have their own genetic material, which is known as empty DNA. It’s important to differentiate between mitochondrial DNA and nuclear DNA. In our cells, we’ve got our nucleus, that’s important, where we have all our genetic material that will synthesize proteins and lead to lots of processes in the cells. We’ve also got mitochondrial DNA because, remember when I said that they used to be independent organisms, so the thing here is that not every protein in the mitochondria is synthesized with this mitochondrial DNA. We also require nuclear DNA for the mitochondria to function properly.

Therefore, either mutations in the nuclear DNA or the mitochondrial DNA may lead to an unhealthy mitochondrion and, cellular alterations. Remember that we said that the mitochondria’s main functions were energy production, reactive oxygens, species production, homeostasis, and apoptosis, so if the mitochondria are not functioning properly, all these functions will be disrupted.

It’s really essential to differentiate between nuclear DNA and mitochondrial DNA because this will give rise to different conditions. If the mitochondrial disease is caused by mutations in both the nuclear DNA and mitochondrial DNA, these conditions will not be treated by the technique that we’re talking about today because we will only replace the mitochondrial DNA; therefore, the nuclear DNA that has a mutation will still be present, and the limitation will be passed through generations.

Let’s say we have a mutation. It’s really important to see where this mutation is placed. If it’s in the nuclear DNA, these will be inherited through normal mechanisms and, therefore, we’ll not be able to use these techniques. However, we’ll be able to use other techniques such as PGS or Amniocentesis to detect these abnormalities.

If we do have a mutation in the mitochondrial DNA, then these mutations might be treated by pronuclear transfer because we will be taking out the mutated mitochondria from the patient, and we will be replacing them with healthy mitochondria.

How is the mitochondrial disease inherited?

Three concepts are really important to keep in mind when we talk about the inheritance of mitochondrial diseases.

1. There’s more than one DNA molecule in each mitochondrion.

   This is called polyplasmia. If we do have a mutation, this mutation might not be present in every single DNA molecule. It might be present in only a few of them, so the fact that we’ve got a mutation doesn’t imply that we will present the disease or the condition because we may have a mixture of them, which is known as heteroplasmy.

2. The level of heteroplasmy is crucial for determining the extent of cellular dysfunction.

   This implies that the cell is not functioning properly, we’re not getting enough energy, we’re not having a homeostasis, it’s not being controlled, and other things that could happen. Therefore, it has been described that when heteroplasmy achieves a level over 60%, we’ve got more than 60% mutated mitochondria in a cell, meaning we are likely to present the disease. This is called the threshold level.

3. Not every cell and the body are the same.

   There will be cells that will be more effective than others. This is known as mosaicism. There will be tissues that may be more dysfunctional than others, so all these factors will determine the extent of affection, will determine how bad the disease is, or even if it is present or not.

MRT (Mitochondrial Replacement Techniques – when?

Mitochondrial replacement techniques are IVF techniques. What is done is a transfer of the nuclear DNA from the egg with mutated mitochondria to another cytoplasm with a healthy mitochondrion.

I really want to emphasize this because it’s really important, if the mutation is at the nuclear DNA, then this mutation will still be present even if we transfer the pronucleus to another egg. We will only do this technique when the mutation is present in the mitochondrial DNA.

There are different types of mitochondrial replacement techniques, such as Pronuclear Transfer or Maternal Spindle Transfer. There is also Germline Vesicle Transfer, these techniques are only indicated for cases where the mitochondrial disease is caused by a mutation in the mitochondrial DNA.

In the Pronuclear transfer technique, we’ll take two eggs, one from the patient and then one from a donor. The donor oocyte will have healthy mitochondria, whereas the patient’s oocyte will present mutated mitochondria. We’ll fertilize both eggs with a sperm cell from the patient, in that case, we will have two zygotes.

A zygote is an initial state of embryo development, so we’ll have two zygotes. As you can see in orange, we’ll have two pronucleus and that’s what you’d see under the microscope. So, one from the mother and the other one from the father, and then in green you can see the donor oocyte is being fertilized as well. Then, you’ll have one parenthood from the donor and then the other one from the father. After that, we will nucleate both embryos and, in the case of the orange embryo, which is the patient’s embryo, we’ll take out the pronucleus taken in the minimal amount of cytoplasm that we can get. This is a difficult technique because we have to be careful not to take any mitochondria, otherwise this would be pointless. We’ll discard the rest of the cytoplasm, contained in the mitochondria and other organelles.

Then, we’ll do the same process for the donor embryo. We’ll nucleate the embryo; however, here we’ll discard two pronucleus, and we’ll keep the cytoplasm and the rest of the embryo. After that, we’ll introduce the two pronucleus from the patient inside the cytoplasm that was created with the donor’s oocyte. Then, we’ll have this embryo that contains healthy mitochondria and the genetic material from the two patients.

There’s a lot of controversy about this term, but it’s sometimes called the 3-parent baby. If we look at it from the scientific point of view, you’ll have the DNA from the mother, which is one of the pronucleus, DNA from the father, which is the other one that will mingle, and then you’ll have like a normal embryo, the genetic material, and then you do have the mitochondrial DNA and the mitochondria from the donor.

Pronuclear transfer – prospects

There are very different opinions about this technique; however, if we look at the scientific data, Gorman et al., in 2015, suggested that these mitochondrial replacement techniques, may prevent 150 cases of mitochondrial diseases per year in the UK, and 780 cases per year in the USA, which are quite a lot of cases, so it’s important to consider the prospects of these techniques. Nevertheless, we should not forget about the ethical considerations.

The UK was the first country to allow these techniques, which also was in 2015 as well, and there have been a lot of opinions. The first baby from pronuclear transfer was born in Mexico by Dr. San and his team. The BBC News published the article in 2016.

Pronuclear Transfer (PNT) – ethical considerations

As mentioned before, there are some ethical considerations to take into account:

1. Use of human embryos and research.
   This is not legal in Europe. We have to be conscious about it and it’s a touchy topic. We should think about it because science sometimes goes fast, and we have to think of the consequences of our actions.
2. We’re throwing away an embryo that was compatible with life.
   The embryo was created with the donor egg and the patient’s spermatozoa.
3. Safety issues with pronuclear transfer.
   This is a really neat technique, and we don’t really know what will happen in the future, so I really recommend studying these cases and following them because it would be really interesting to see the effects in the long run of these techniques.

You need to be also aware of other available options:

• Adoption.
• Gestation with just a donor egg: We don’t need to do all these techniques.
• Pre-implantation genetic diagnosis: Techniques that may be recommended for cases where the mutation is placed at the nuclear DNA because sometimes, as I was saying before, the mutation is not at the mitochondria, so the mutation will still be passed to the embryo. Therefore, we can do pre-implantation genetic testing to see what embryos are affected and what embryos are not affected to select healthy embryos.
• Prenatal diagnostic tests: Such as amniotic biopsy or NIPT.

Final thoughts

• The pathophysiology of mitochondrial diseases is complex, so we have to consider a lot of things, it’s not just A, B, and C.
• Mitochondrial diseases might be caused by mutations in either nuclear DNA or mitochondrial DNA, so in those cases where the mutation is placed in the mitochondrial DNA, pronuclear transfer techniques might be helpful to prevent the conditions. This is the indication for these techniques when the mutation is placed in the mitochondrial DNA. However, the fact that we’ve got a mutation does not necessarily imply that we suffer the disease.

There are three important things to consider:

• Heteroplasmity: The fact that maybe we do have a mutation in the mitochondria in a cell, but that may not imply that we suffer the condition. The cell might be able to function normally.
• Mosaicism throughout the body: We do have different cells, so some tissues might be more affected than others, and then the extent of the expression of the condition might not be as severe.
• Threshold level: up to 60% they start to have more effect and, therefore, the disease might be more present, more severe.

In those cases where the mutation is placed in the nuclear DNA, there are other options such as egg donation, adoption, and pre-implantation genetic testing. If you’re interested in the topic please read, and do some research.