The joint research group of the University of Pécs and Semmelweis University identified a marker affecting the success of artificial insemination. We talked about the success to immunologist Júlia Szekeres, who in her study published in Nature Scientific Reports in January, also have drawn attention to the fact that parenthood is put off to later and later points in time in developed countries, therefore there is an increasing demand for artificial insemination.
How big work preceded the success?
It is the result of about two year's research, but it also needed a good idea. It is known that every cell produces extracellular vesicles (vesicles surrounded by lipid membranes), which can contain various molecules – cytokines, nucleic acids, hormones, enzymes. These vesicles get out of the cell and get to the next cell which takes them. With a help of controlling mechanisms, for example, a vesicle can transfer information from a cell of the endometrium to the embryo or vice versa, or maybe from a lymphocyte to another. It occurred whether there are such vesicles in the culture medium of in vitro embryos during artificial insemination. It seemed as a wild idea, because it was questionable whether a sole cell would be able to produce a detectable amount of vesicles.
How big is a vesicle in comparison with a cell?
Its size can extend from 50-100 nanometers up to one thousand. The cell is about 10 micrometers. That is, vesicles are minuscule compared to the size of the cell, just like a tiny little bubble next to the cell. Éva Pálinger from the Institute of Genetics, Cellular and Immune Biologe of the Semmelweis University was involved in the research, who is the member of an internationally appreciated research group studying extracellular vesicles. They checked the idea, and it turned out that there were such vesicles in the culture medium of the embryo. Then it had to be proved that they really were extracellular vesicles. The evidence is that the lipid membrane of the vehicle dissolve in detergents and the vehicles disappear. These vehicles originate from the embryo, since there were significantly more vesicles in the culture medium of the embryo than in such culture mediums in which no embryos were bred. Later on it was also found that if there is nucleic acid in the extracellular vesicles, it means that the embryo does not feel well. We made use of this phenomenon to identify “right” embryos, suitable for implantation.
I suppose 'test-tube babies' are not bred in test-tubes any more.
Indeed, after insemination, they are bred in plastic culture vessels at 37 degrees in an appropriate culture medium that contains amino acids, salts and other nutrients required for development and provides for an appropriate pH level.
What amount is this medium?
An embryo is bred in 40 microliter of culture medium. It is a small drop. By the end of the fourth day the embryo looks like a raspberry, then it turns into a blastocyst on the fifth day; this blastocyst is implanted, but before that the embryo is transferred into fresh medium. Thus we can examine the number of extracellular vesicles containing nucleic acids in the medium in which the embryo has been bred for days.
Excuse me, but how?
There is a special paint – propidium iodide – which colours only nucleic acids, particularly the DNA, which becomes fluorescent red after painting. Flow cytometry is able to differentiate between vesicles of different colours and count them. We got the medium cultures of already implanted embryos from the IVF laboratory of the Gynaecological and Obstretical Clinic of Pécs, thus we were able to observe the correlation between the lab results and the success of the implantations. For instance, if the implantation of three embryos in the same woman resulted in a singleton pregnancy, then the number of extracellular vesicles containing nucleic acid was low in the culture medium of one embryo, while in the other two it was high. In another case, the implantation of two embryos resulted in twin pregnancy. Here the number of extracellular vesicles containing nucleic acid was low in case of both implanted embryos. Only one embryo is rarely implanted, and if this value is low, it is almost always followed by pregnancy. This is how we reached the conclusion that those are the “right” embryos in case of which the number of extracellular vesicles containing nucleic acid is low.
A research project also needs to be proved; how did that happen?
Only particular implantations are evidential, and these are rare in Hungary. However, we managed to obtain 20 such samples. There was only one among them which did not show correlation between the number of extracellular vesicles with successful pregnancy. Based on this, we can predict with a 95% probability, which embryos are suitable for implantation. However, the embryo identified with the above method is only one condition of a successful pregnancy. A suitably healthy maternal side is also necessary; that is, an endometrium which is capable of accommodating the embryo.
In district V., Budapest, the average age of women having their first babies is 40 years. To what extent is it a disadvantage?
It is tragic. It leads to the fact that many women are not able to get pregnant on their own. The rate of chromosomically abnormal embryos increases with the age of the mother. At the age of 44, 90% of embryos are abnormal. At 35, this rate is nearly 40%. Fortunately, the majority of abnormal embryos are not able to implant; this is partly the reason behind increasing female infertility. Biologically speaking, it would be optimal for women to give birth between the ages of 20-25, because it is the period with the best chance of producing the genetically least problematic embryos.
It will be a nasty question, but due to social conditions, will your research be a salable product?
Absolutely. Everywhere in the world the goal is to transfer only one embryo, but it requires the identification of the one embryo which has the most chance for implantation. Ideally transfer should work in a way that on the fifth day, right before transfer, the single embryo with the biggest chance for implantation could be chosen by a simple examination. Our method is quick and simple. Professor Gábor Kovács L.'s research group has identified a marker reflecting the differences in the metabolism of “right” and “wrong” embryos, and the development of a chip-based quick test is in progress as well. If the two methods were applied at the same time, and both markers showed that this is the right embryo, we could make the method of choosing the right embryo much more safe.
What is the next step? How can your method be applied?
On the fifth day, there are 3-4 embryos available – several ones are bred from the same woman. On the day of the transfer the culture medium has to be examined with our method, it only takes 15 minutes. The examiner then can tell which embryo has to be used out of the four; let's say, egg no. 1.
What happens to the other eggs then?
We transfer one of the good eggs, while the others can be frozen, because we have to be happy about a good embryo, and by next time the mother will be even older. In other countries only one embryo can be transferred; in Hungary more. In the USA, doctors inducing twin pregnancies face a malpractice lawsuit, because twin pregnancies have more health risks.
How is it possible that no business has been made of taking the eggs as early as possible?
There has. For example, Apple offered their young employees to pay for taking and storing their eggs – this is called social freezing. When the woman feels it is time for having a baby, the embryo will be transferred. But a good egg is not enough for 100% success, because the endometrium ages as well, and hormonal conditions change, too.
Where can you put this achievement in your research career?
I find it important because it has the promise of practical implementation. Of course, chance also played a part in this discovery. Research is like a game. We head for something, and more and more new things open up. We manage to see beyond phenomena. Then we search for points where we can intervene in mechanisms. It is very exciting.
What is your main field of research?
The influence of hormonal changes during pregnancy on the immune system. The genetically half foreign embryo is not rejected because the operation of the immune system changes during pregnancy. The immune system does wire-dancing. It has to prevent the rejection of the embryo, but meanwhile it also has to protect the body from external infections. The hormone progesterone has a key role in maintaining pregnancy and in the immunological changes during pregnancy as well. It was us who first showed that there are specific progesterone bonding nodes in the lymphocytes of pregnant women. Pregnancy means a constant stimulation for the maternal body. The maternal immune system recognizes the “alien” embryo; it activates lymphocytes and develops progesterone receptors which are able to bond this hormone. The effect of progesterone is like a hit-and-run. When the effect of the hormone occurs, progesterone itself is not even present any more. Bonding to its receptor, progesterone reaches the nucleus of the cell and induces genes; new proteins are produced. Such a newly produced protein, PIBF – progesterone induced blocking factor – transmits the effects of progesterone on the function of the immune system. But I could talk a lot more about that.
And now, after the big success, what is next, what will you research? Does research need a grant, or rather a good idea?
A proper research environment and a little money. Unfortunately, this environment is not granted nowadays, not in the country, and unfortunately I must say, not even at the university. During research, new aspects emerge constantly, due to which I would need to buy a new reagent for a couple of hundred thousand Forints, but right now. In research, you cannot plan at the beginning of the year what problems will emerge half a year later. The proper procedure would be to order the reagent directly from the producing company that could deliver it in the shortest time possible, and I could pay it from the research budget. This is the practice in other countries, and it used to be in Hungary, too. But what is happening now? I have to ask for quotations, write justifications and wait for signatures for months. Considering that an intermediate authority performs ordering, even the delivery address is often wrong and I have to find where the parcel has been sent. It is difficult to do research this way. Primarily, this is not the fault of the university. The way of spending money is determined by people who have no idea how research is performed, but they also have no idea about teaching and patient care either. Unfortunately, similarly uninformed people deal with research budgets at the university as well. Administration is excessive, and the system is inflexible. It could be helpful if administrators occasionally took study trips to the workplaces the work of which they are supposed to help, in order to get acquainted with the processes and emerging problems.
Robert Balogh, UnivPécs International