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.
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.’