Figure 1. Mitochondria structure (1).

Although most of the genetic material of eukaryotic cells are located inside the nucleus, mitochondria are organelles that also possess a certain amount of DNA. Mutations in mitochondrial DNA (mtDNA) or nuclear genes involved in mitochondrial function are a cause of infertility and diseases, not only in individuals but also in their offspring. In these cases, one of the solutions known to be efficient in order to conceive and give birth to a healthy child is the “three-parent in vitro fertilization” approach.

THE MITOCHONDRIAL GENOME IS MATERNALLY INHERITED

During fertilization, mitochondria from the sperm are normally eliminated by a ubiquitin-dependent mechanism. As a consequence, in case the father carries the mutation both his health and fertility could become affected, but never his offspring (2). By contrast, mitochondria in the oocyte must present a specific location and distribution pattern (which represents an actual sign of oocyte maturation), and they are solely inherited from the mother.

​WHY ARE MUTATIONS IN mtDNA OR IN NUCLEAR GENES INVOLVED IN MITOCHONDRIAL FUNCTIONS SO PROBLEMATIC?

Mitochondria provide energy to the cells through oxidative phosphorylation, and so mutations in their genome mainly affect structures form the nervous system, heart, skeletal muscle, pancreas, gonads, colon, blood, kidney or liver (3). Why these structures? The higher the energy demand is, the higher the need for more mitochondria in the cells (4). Also, cells that present a slower division process are more likely to present some kind of mtDNA mutation (5).

HOLOPLASMY vs. HETEROPLASMY

The situation in which all cells from an individual contain identical mtDNA (mutated or otherwise) is known as holoplasmy. By contrast, heteroplasmy is defined as the condition in which part of the mitochondria from the same individual present a DNA content that is different from the other. These cases are the most common among patients affected by mitochondrial DNA diseases (6).

WHY DOES HETEROPLASTY REPRESENT A PROBLEM?​

Even though heteroplasmy implies the presence of two different DNA contents in the cell, cells with great amounts of mutant (or affected by a specific condition) mitochondria respond to proliferate their entire DNA. This is why the percentage of mutant/affected mtDNA tends to increase in certain tissues (7).

THE “BOTTLENECK EFFECT” AND THE “THRESHOLD EFFECT”

During oogenesis, only a subset of molecules of mtDNA are eventually amplified and passed on to the offspring (8). This effect explains why it is possible to obtain homoplasmic individuals in just a few generations (2).

Previous reports on human diseases caused by an mtDNA mutation have shown that the mutation needs to be present at a certain percentage in order to manifest pathological effects. Typically, this percentage should be higher than 60-80% (8,9), although it also depends on age, affected tissues, type of mutations, etc. (4)

WHY APPLY THE “THREE-PARENT IN VITRO FERTILIZATION” APPROACH?

It might be reasonable to think of other possibilities to treat patients suffering from mitochondrial diseases in order to achieve pregnancy. Rather than prenatal diagnosis or preimplantation genetic diagnosis, the “three-parent in vitro fertilization” technique because (4):

In the first case:
1. It needs a uniform mtDNA distribution in the extra-embryonic and fetal tissues.
2. It needs the mutant DNA load to remain constant over time.
3. There must be a close relationship between the severity of the disease and the amount of mutant DNA.

As for preimplantation genetic diagnosis (6,9):
1. It is not applicable to patients with high levels of heteroplasmy.
2. It reduces but does not eliminate the risk of suffering from a mitochondrial disease-related condition.
3. The amount of tDNA found in blastomeres or the trophectoderm does not represent the whole embryo.
4. This approach is not an efficient diagnosis due to the combination of heteroplasmy and the “bottleneck effect”.

KNOWN TECHNIQUES TO BE USED FOR MITOCHONDRIAL REPLACEMENT

PRONUCLEAR TRANSFER

It involves the transfer of the two pronuclei from a zygote affected by diseased (or mutated) mitochondria into an enucleated zygote containing healthy mitochondria. Even though this technique has not yet been performed in humans, the efficiency of pronuclear transfer in mice has been adversely affected by descendants bearing high levels of carryover mtDNA (10).

Figure 2. Pronuclear transfer technique (10).

POLAR BODY TRANSFER

Since the polar body has a lower proportion of mitochondria around it, this is currently considered the best method for preventing the transmission of mutated mtDNA on to the next generation (9,10). Embryos derived from polar body transfer support normal fertilization and are capable of producing live offspring in the mouse. Polar body transfers leading to a minimal amount of affected mtDNA carryover have demonstrated the great potential of this technique for preventing inherited mitochondrial DNA diseases (9,10).

When applied in mice, this technique has shown the best success rate so far due to the transfer of mitochondria being lowered to a minimum (9).

Figure 3. Polar body transfer technique (10).

SPINDLE TRANSFER

This technique involves transferring the meiotic spindle along with the associated chromosomes, the spindle-chromosome complex (SCC), from an unfertilized oocyte with affected mitochondria into an enucleated healthy mitochondria-containing oocyte (11). This technique recently became popular when performed by Dr. John Zhang and his team, hitting the media within the latest weeks. However, potential problems could arise; just as for the previous techniques, the spindle is also surrounded by mitochondria, and so they could too be introduced into the ooplasm, thus causing heteroplasmy (12).

Figure 4. Spindle transfer technique (10).

​WHAT DOES LAW STATE REGARDING MITOCHONDRIAL REPLACEMENT?

So far, scientific societies are very skeptical about experimental techniques. Thus, this particular approach is specifically prohibited by the Food and Drug Administration (FDA) in the US. It can only be performed in countries such as Mexico, where legislation is more flexible, or in the UK, where it was approved for application in very specific cases (14).

CURRENT DATA ON MITOCHONDRIAL REPLACEMENT

As has been previously mentioned, cases of cytoplasm transfer have been performed. In fact, there have been around 30-50 live births from this technique. However, newborns presented certain genetic defects, and so this technique was banned and replaced by pronuclear transfer and meiotic spindle transfer (14). Such data demonstrate the potential damage that could be inflicted to the embryo when performing these techniques (12,15). In addition to this, ethical issues must also be taken into account, which means the sole possibility of successfully applying a specific procedure does not imply its moral appropriateness. In order to guarantee so, a committee of experts should pronounce their opinions and reach a consensus about it. On a related note, long-term effects derived from these procedures are still unknown, and so it would be necessary to monitor all babies born through these techniques.

 

This post has been published in the Scientists in Reproductive Technologies (SIRT) newsletter, a special interest group representing the scientific membership of The Fertility Society of Australia.

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REFERENCES

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