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How do the nuclear transfer techniques work and is it the future of IVF?

Pavlo Mazur
Laboratory Director, IVMED

Category:
IVF laboratory

nuclear-transfer-techniques-IVMED
From this video you will find out:
  • What are possible indications for NT (nuclear transfer & how is it performed?
  • What is germinal vesicle transfer (GVT), how is it performed, and when can this be indicated? What are its main advantages and disadvantages?
  • How is Maternal Spindle Transfer (MST) performed?
  • What is a Pronuclear Transfer (PNT) and what does the process look like?

How do the nuclear transfer techniques work and is it the future of IVF?

In this webinar, Pavlo Mazur, Laboratory Director at IVMED Fertility Center, discussed nuclear transfer techniques, how they are performed, their indications and outcomes.

Pavlo Mazur started by explaining that we can have different sets of chromosomes. If we have only one set, the organism is haploid, while two sets make it diploid, three sets make it triploid, and so on, up to infinity. Speaking of humans, we are diploid organisms, which means we inherit one set of chromosomes from the mother and another set from the father. One set is from the mother’s side, and it should be haploid.

The structures or pictures in question can be referred to as chromatids, although this term is chosen randomly. When there is only one chromatid, we can call it “C,” with the number of Cs representing the number of chromatids. The whole process of meiosis, or reductional division, involves dividing by 2. Starting from the immature GV oocyte on the left, which contains 4 chromatids of each chromosome, it goes through an immature stage and then a mature stage, where it contains only 2 chromatids. This division by 2 is the essence of the meiosis process.
As deployed organisms, we inherit one set of chromosomes (haploid) from the mother and another set from the father. In the mature oocyte, which is a normal, mature human oocyte, we have 2 Cs within the cytoplasm. The second division by 2, resulting in the haploid number of maternal chromosomes, occurs after fertilization. This process restores the deployed state in the future embryo, leading to the first cleavage and subsequent development into a blastocyst, which consists of over 100 cells. The blastocyst can then be implanted into the uterus for further development.

By adding polarizing microscopy to normal or common microscopy, we can visualize the spindle, a tiny structure that holds all the chromatids. This spindle contains the genetic material that can be manipulated, replaced, or removed. After reconstitution, we achieve normal fertilization, where the sperm with its haploid set of chromosomes enters the cell. This initiates the second division, dividing the 2C structure into one C from the maternal chromosomes and the other C from the sperm.
The process is not overly complicated. Starting with 4 chromatids, we go through maturation, ending with 2 chromatids. The only step remaining between this cell and the embryo is fertilization by the sperm, which triggers the mechanism of division, the reductional division, resulting in the normal deployed type within the embryo.

Indications for nuclear transplantations

The whole story of nuclear transplantations in humans began with the prevention of mitochondrial diseases. Interestingly, there was recent news about the birth of the first babies in Great Britain after pronuclear transplantation, specifically for the prevention of mitochondrial diseases. However, mitochondrial inheritance can be somewhat random, leading to a phenomenon called reversion in embryos. When the genetic material is transferred from a patient cell into a donor cell, a small number of maternal mitochondria around the genetic material may persist, repopulating the entire cytoplasm and restoring the maternal mitochondria type. This can potentially cause mitochondrial diseases.

While there are more instances and information about this, further investigation is needed to understand if the complete removal of maternal mitochondria from the transferred genetic material is possible. There is still a risk involved in preventing mitochondrial diseases, and we need more research to find definitive answers.

The technique is quite fresh for humans, and the children that were born are still young, we don’t know how their mitochondria or maternal mitochondrial distribution will occur within their tissues and cells. It remains a question mark.

Looking at the embryo arrest indication, nuclear transplantation works well for this type of pathology. If we have young patients with normal fertilization rates and no developing embryos until the blastocyst stage, we can change the cytoplasm, which restores the ability of the genetic material to be realized and functional within the new cytoplasm. This results in the production of normal embryos that can be transferred, leading to successful pregnancies and the birth of babies. Many babies have been born after applying different types of nuclear transplantations, and the primary indication has been embryo arrest at different stages, whether before or after fertilization. The cytoplasmic factors that prevent the maturation of all sides can be overcome by replacing the cytoplasm, allowing for normal maturation and subsequent fertilization.

Another indication is repeated implantation failure of euploid embryos. Although debatable, in some cases where nothing else has helped, nuclear transplantation becomes an option worth considering. In common IVF, some patients recurrently experience immature oocytes with each protocol, resulting in the absence of mature oocytes. In such cases, nuclear transplantations can be performed on the early immature stage to restore the cells’ ability to mature and produce normal embryos.
In cases where fertilization occurs, but the zygotes exhibit abnormalities with more than two pronuclei, nuclear transplantation before fertilization can yield normal zygotes. Similarly, when embryos do not divide on day 2 and fail to reach the blastocyst stage, nuclear transplantations can be beneficial.

For around 3% of young patients who experience recurrent embryo arrest, nuclear transplantations have been successful when no other interventions have helped. Although we know that several genes may be involved in these processes, the reasons behind these conditions remain largely unknown. Genetic panels for testing have been offered by some companies to identify patients carrying genes that may result in the absence of maturation, cleavage, or implantation. However, in most cases, the exact causes are still unidentified.

There was an interesting case in my practice where a patient’s embryos were unable to develop beyond the zygote stage, experiencing fragmentation and failed implantation. Pronuclear transplantation was performed, resulting in a normal blastocyst that was transferred, leading to a successful pregnancy and the birth of a healthy baby boy. Subsequently, the patient conceived naturally. This case demonstrates that nuclear transplantations can be effective in treating infertility in certain situations.

In summary, nuclear transplantation techniques can be divided into two main groups: those performed before fertilization on very immature cells or mature cells, and those performed after fertilization. Before fertilization, techniques such as spindle transfer and polar body transfer have been successful in producing normal embryos. After fertilization, pronuclear transplantation is an option, although it is less commonly performed.

Embryos can be derived from a single cell, and this method works just as well for polar body transfer. By using the first polar body as another spindle of a mature cell, we can multiply the number of embryos from a single cell. It may seem like magic, but it’s simply a matter of dividing by two at each step. This technique is particularly useful for mitochondrial diseases, as polar bodies typically contain a minimal amount or no mitochondria at all. This reduces the risk of heteroplasmy compared to spindle transfer.

The most challenging aspect is the second polar body. It is extruded right after the fertilization process, marking the final stage of maturation in human oocytes. At this point, there is only one C from the female or mother inside the cytoplasm, and the second one comes from the sperm, resulting in a diploid normal embryo. The second meiotic division leads to the extrusion of the second polar body, which contains a haploid set of chromosomes. This polar body can be collected and inserted into an activated, nucleated donor oocyte. By doing so, the diploidy of the cells can be restored, and an asynchronous zygote can be obtained. The pronucleus from the sperm will advance, while the pronucleus from the polar body will lag. However, if synchronization is achieved, a normal zygote can be formed, leading to the development of a normal blastocyst that can be transferred after testing.

Working with human oocytes can be challenging because the second polar body is extremely fragile and thin, making its removal from an activated cell difficult but still possible.

Conclusions

In conclusion, I want to emphasize that nuclear transplantations are not the future of IVF. These techniques should only be used in cases of infertility where no other options are available. They have strict indications and are considered highly experimental. Only a small group of patients can benefit from these techniques with good results. However, as we continue to advance our knowledge and experience, more and more children will be born after applying different types of nuclear transplantations. In cases where the genetic cause of infertility, such as the TBB-8 mutation, is known, these techniques can provide significant help and hope.’

- Questions and Answers

What about the use of nuclear transfer to reverse increased aneuploidy with increasing female age?

From my experience, we cannot guarantee an increase in chances of euploidy through nuclear transplantations. For advanced maternal-age patients, the only option we can offer is reverse reconstitution, where we use the patient’s cytoplasm and genetic material from a donor. However, the phenomenon of mitochondrial distribution reversal makes it uncertain whether all the mitochondria in the resulting embryo or baby will be from the patient. Therefore, we cannot cure aneuploidies with nuclear transplantations at this time.

After how many attempts can we talk about repeated implantation failure and consider using nuclear transfer?

The number of attempts that define repeated implantation failure and the suitability of nuclear transfer can vary depending on the clinic and country. However, it should be at least several attempts. If a patient has only had 2 tries without any embryos, it may not provide sufficient information since various factors, including laboratory conditions and stimulation protocols, can affect results. It is best to consult with your doctor about this rather than an embryologist.

Can you provide information on clinics that offer this service?

I’m not sure if I’m allowed to advertise specific clinics, but all the information you need is included in the presentation slides.

What is the difference between nuclear transplantation and cytoplasmic transplantation?

The terms used can be confusing, as they often mean the same thing. Nuclear transplantation and cytoplasmic transplantation are essentially synonyms. It depends on the point of view. From one perspective, we can say we perform nuclear transplantation by removing the nucleus from one cell and placing it into another cell. From another perspective, we can say we change the entire cytoplasm, which includes mitochondria, organelles, factors, proteins, and the molecular machinery within the cell. However, they refer to the same technique with different names.

Is the genetic material of the donor used, resulting in embryos not having the genetic material of the parents?

This is another instance of confusion caused by terminology. In the press, the term “mitochondrial replacement” is often used, but in reality, we do not replace mitochondria alone. We replace the entire cytoplasm, which includes mitochondria, organelles, and other components. The patient’s genetic material is used, usually with a small amount of cytoplasm around the nucleus or spindles, and it is placed into a donor oocyte that provides a larger cytoplasmic environment.

Is the procedure legal, and what paperwork should be signed? What about the costs?

The legality of the procedure varies between countries. In the UK, it is legal but only for mitochondrial diseases. However, I personally believe that infertility should be the main indication for nuclear transplantations, not mitochondrial diseases. Each country has its own regulations, so it’s essential to check the legality of the procedure in the specific country of interest. As for paperwork and costs, I cannot provide specific information as it varies between clinics and countries. It is best to inquire directly with the clinics performing the procedure.

How long have you been applying this technique, and has it helped in cases where embryos developed until day 5 but experienced implantation failures? I have a previous history of endometrial cysts, and my doctor says the cytoplasm quality may be a problem.

I have been performing this technique since 2014, but please note that I am not a doctor and cannot provide medical advice. It is best to consult with a doctor regarding your specific case, as they can recommend the appropriate procedure. Regarding the history of infertility, it is essential to discuss it with a doctor to determine the cause. Our role as embryologists is to perform the recommended procedure.

Regarding the donated cytoplasm’s duration of effectiveness in the embryo, particularly at day five when there are over 100 cells, does each cell utilize its cytoplasm?

I apologize for the confusion earlier. Allow me to explain. Cells consist of two main parts: the nucleus, which contains the genetic material, and the cytoplasm, which encompasses everything between the nucleus and the cell’s outer wall. In the case of oocytes, the area between the nucleus and the oolemma is considered the cytoplasm. During nuclear transplantation, we replace the entire cytoplasm of the cell, including its organelles. The cytoplasm plays a crucial role as it contains numerous factors and interactions that affect embryo development. At the blastocyst stage on day five, the cytoplasm of each cell contributes to the overall embryo development. The source of the cytoplasm can make a difference in certain cases, where problems may arise either within the DNA or the cytoplasm itself. Hence, cytoplasmic quality matters in embryo development.

If I am 42 years old and my husband is 43, can nuclear transfer help us if we also do a genetic check?

Unfortunately, there seems to be a misunderstanding. Somatic cells, which make up our bodies, and gametes, such as oocytes and sperm, are fundamentally different. Even if you do not have any genetic mutations or issues in your somatic cells, it does not guarantee that your oocytes will have the same DNA quality. Oocytes have a unique process, and even healthy individuals can have limitations or impaired DNA within their oocytes. This is nature’s way, and it is unrelated to lifestyle or other factors.

Is there a minimum requirement for the number of oocytes to be used in order to achieve positive results?

Yes, there was a table presented that indicated the number of oocytes required to achieve positive results for each age group. The number of oocytes needed varies depending on the age group, with different ages requiring different quantities to increase the chances of achieving a successful pregnancy and obtaining euploid embryos. You should keep in mind that not all normal embryos implant due to nature and biology. Even if you have euploid embryos, it doesn’t guarantee implantation. However, approximately half of them usually implant and result in children. I cannot provide a specific minimum requirement for the number of embryos because it depends on various factors.

What if we take the GV nucleus from a 45-year-old woman who has IVM with no HCG trigger, presumably GV oocyte will be euploid. If that GV nucleus is transferred to a young egg donor’s egg, and then perform IVM and ICSI and culture to blastocyst, and do the PGT-A, presumably, the 45-year-old will have her own DNA but may now be euploid. What do you think?

The GV stage for a patient of advanced maternal age might be too late to perform nuclear transplantation. By that stage, the DNA is already damaged, and there could be issues between the chromatids. The cell’s apparatus, which has been inside the body for a long time, has not undergone effective repairment, leading to impaired DNA and improper spindle formation. Unfortunately, from my experience, nuclear transplantations for advanced maternal-age patients have not been successful in increasing the chances of obtaining euploid embryos. Even if we managed to have embryos, all of them were aneuploid. Conversely, when using cytoplasm from a young donor carrier, we observed euploid embryos. Thus, it is not solely about the cytoplasm but primarily about the DNA. We cannot repair it for these women, at least not with current techniques. Perhaps future techniques could focus on somatic cells and stem cells to address the meiotic process.

I started IVF at 35, and the theory of my clinic is that the problem has always been the quality of the cytoplasm, which cannot be improved. Is it possible this technique could help?

I cannot make any specific promises or claims regarding this case. The chances of obtaining euploid embryos decrease with age. There is a curve, showing the relationship between age and the number of euploid embryos. After the age of 37, the chances decrease significantly. Therefore, in cases where patients are above 35 years old, oocyte donation becomes a viable option.
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Authors
Pavlo Mazur

Pavlo Mazur

Pavlo Mazur is the director of an embryology laboratory in IVMED FERTILITY CENTER. He has a Master's Degree in General and Molecular Genetics. He is a member of the Ukrainian Association of Reproductive Medicine (UARM). His priorities in work are innovative techniques and technologies in human embryology.
Event Moderator
Caroline Kulczycka

Caroline Kulczycka

Caroline Kulczycka is managing MyIVFAnswers.com and has been hosting IVFWEBINARS dedicated to patients struggling with infertility since 2020. She's highly motivated and believes that educating patients so that they can make informed decisions is essential in their IVF journey. In the past, she has been working as an International Patient Coordinator, where she was helping and directing patients on their right path. She also worked in the tourism industry, and dealt with international customers on a daily basis, including working abroad. In her free time, you’ll find her travelling, biking, learning new things, or spending time outdoors.
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