In this webinar session, Dr Halyna Strelko, Chief physician, Reproductive specialist at IVMED, Kiev, Ukraine, has given an overview of fertility treatment options for women with a low ovarian reserve. Dr Strelko once again has shared an inspiring success story.
The 1st case presented by Dr Halyna Strelko described a 38-year-old patient who already had 1 child conceived naturally, and she tried to get pregnant for the second time, however, she had 5 early pregnancies that ended in spontaneous abortions.
The patient has done 2 IVF cycles at another clinic without success. During her 1st attempt she got 6 oocytes and only 2 embryos, she has done a fresh embryo transfer, unfortunately, the result was negative. On the second attempt at the same clinic, only 5 oocytes were retrieved. The doctor decided to cultivate till the blastocyst stage, but unfortunately, there were no blastocysts. Then, the couple came to IVMED, we performed all examinations, ultrasound showed 2-3 antral follicles in each ovary, the patient had also a low AMH level (0.3 ng/ml), but other parameters were normal. The general analysis was normal, coagulation tests were normal, hormonal tests were normal as well, and the partner’s sperm count and motility were normal.
We decided to try a modified protocol, we give anti-estrogens, in this case, we have used Letromara, which increases FSH level, and we have prescribed 150 UI of urinary FSH because in urinary FSH there normally is some LH activity. For poor prognosis patients, it may give a better response and better quality. Unfortunately, we didn’t get a good response, we got only 2 eggs, we fertilized these 2 eggs with ICSI, but we didn’t receive any blastocysts as it was in the previous clinic.
We investigated her husband a bit more thoroughly because we thought there is something with the sperm. We know that after 72 hours, male genome activity is increased, so we should see if something is wrong with sperm, and we proposed to do DNA fragmentation of sperm, FISH, and Karyotype. We found that DNA fragmentation was normal, the percentage of genetically abnormal sperm cells was in a normal range, and nothing special was found.
Therefore, we tried another stimulation with standard antagonist protocol and a higher dose of urinary FSH (225 IU), and the response was the same, we received only 2 eggs, and we fertilized the eggs. After discussion, we thought that there is some cytoplasmic factor issue, and we tried to improve the nucleus by putting them in a donor cytoplasm. We proposed to do a Pronuclear transfer. At the same time, we started the stimulation of the egg donor, we have done a Pronuclear Transfer, and we received much better results. We got good fertilization, then we had good development of the embryo, and in the end, we received a good quality blastocyst, which was genetically examined via PGT-A. It was a healthy female embryo. The patient was prepared with standard protocol, 6 milligrams of estrogens and 600 milligrams of micronized progesterone, we have done the transfer, and we received a good viable pregnancy.
Why did we decide to do a Pronuclear transfer? Most of the time, the strategy for poor responders is to try to increase the number of eggs, but this particular patient repeatedly had embryo development arrest. Why does it happen? We know that the percentage of aneuploidy increases with the age of women, but the source of this aneuploidy is not only maternal, there can also be some paternal meiotic problems or mitotic problems. If we analyse the percentage of those other issues in the genetic abnormality of the embryo, we can see that around 40% of this abnormality is not directly related to the eggs’ nucleus. It arrives later at the time when the embryo is developing. There are probably some other parts in the egg inside the cytoplasm, which may influence the final embryo quality. We know that poor egg quality is one of the main causes of repeated failure in assisted reproductive treatment. The quality of the egg is mainly determined by small organelles called mitochondria, which is a little organelle that produces energy for the egg. If we have some issues with mitochondria, we will also get an embryo development arrest, worse quality or failed fertilization, and so on.
We know that low concentrations of mitochondrial DNA were found in patients with low ovarian reserve, approximately 2-3 times less than in patients with good ovarian reserve, also in patients of late reproductive age. Mitochondria take part in most intracellular events, which means that the normal activity of mitochondria is needed for normal fertilization normal cleavage also apoptosis regulation and embryo development. A low mitochondrial level in the oocyte may decrease the infertility treatment result.
Mitochondria are mostly provided from the oocyte, mitochondria from sperm cells are quickly degraded, and that’s why we can take mitochondria mostly from eggs. On the next slide, the role of mitochondria was shown during ageing, during life, we have free radicals, and active oxygen radicals, and they may somehow destroy mitochondria, and it can result in mitochondria mutation, and with the ageing, the processes of cell death activate. We also know that mitochondria are like the power plant of the oocyte. This energy is produced by mitochondria called ATF molecules, and it is the main energy source of the cell.
A lot of scientific works prove that the amount of mitochondria is related to the age of women and decreases dramatically. The amount of mitochondrial is also related to fertilization rate and aneuploidy rate, as well as the possibility of implantation. We can see that the main cytoplasmic factor of the oocyte is mitochondria, and when mitochondria are not working well, or the quantity of mitochondria is not enough, we can expect a poor prognosis.
What can we do? How can we donate this mitochondrion and increase the number inside a cell? There are 2 different ways: Spindle Transfer – the spindle is the nucleus, which is put in the donor cytoplasm; Pronuclear Transfer is when we fertilize the egg and then put it in the donor cytoplasm.
From a technical point of view, for the Pronuclear Transfer, we should do fertilization of women’s egg and donor egg, donor’s eggs should be fresh, not frozen. The donor’s cytoplasm should be fresh and not damaged by preserving methods. We fertilize the woman and the donor egg, wait for the stage of pronucleus formation, and the next day, we take off the pronucleus from the woman’s egg and put it in the empty cytoplasm of the donor egg, and the pronucleus from the donor egg is taken out. The photo presented in the slide showed two pronuclei where the embryologists with the pipette take out the pronucleus, and then it will be put it inside the donor’s cytoplasm.
What is the indication for mitochondria donation? The indication is: decreased ovarian reserve because, in most cases, we have low mitochondria numbers, advanced maternal age, some PCOS patients with poor quality of oocytes, some genetic factors, endometriosis and mitochondrial diseases.
At IVMED, there were currently 38 patients that are undergoing either Pronuclear or Spindle Transfers. The total number of oocytes which were reconstructed with this technique is 177, it is a big number. We have quite a nice blastulation rate, with Pronuclear Transfer, we have a better blastulation rate at 74%, with Spindle Transfer, it is a bit less than 67%, but it is still a good percentage. Eighteen patients had an embryo transfer with these techniques, the clinical pregnancy number is 9, and we had 4 live births, and we still have 5 ongoing pregnancies, we are waiting for the delivery. We already have some experience with these techniques, and in most cases, this technique is used in patients after 4- 6 attempts with very poor embryo quality or embryo arrest.
When we are talking about the future of fertility treatment, different kinds of mitochondria donation like Spindle Transfer or Pronuclear Transfer, Transfer of Donor’s or autology mitochondria are going to become standard techniques. Another interesting direction is stem cell activation, the formation of new oocytes. Nowadays, there are a lot of works in the world done on this, but from a practical point of view, there are not a lot of successful results so far. Another thing is regulation of antral follicle atresia, and we try to do that for some patients, we’re prescribing them Replacement Hormonal Therapy (RHT), and in some cases after 3 months, we can see a couple of small follicles, and it may be also helpful. There are other methods of egg formation from somatic cells, so in a couple of years, we will see the new directions and new possibilities.
The main indication for pronuclear transfer is not to be in very advanced reproductive age like 45-46, it’s better to do it before 42-43. You are 40, your AMH level is quite good, so we can expect to receive 6-8 eggs, we can do even 2 stimulations in one cycle and receive more eggs. Normally we stimulate a woman, receive eggs, fertilize and do the freezing for the pronucleus stage. We can stimulate the egg donor at the same time, although it is not mandatory, we can do it a bit later when we have all pronucleus.
For this technique, we should have very good quality donor eggs, the phenotype of this donor is not important because genetic material mostly comes from the parents. The second point is that embryologists should have time for this technique, and it shouldn’t be a very busy day, so we can plan this thoroughly, we can do that during one or two menstrual cycles, and donor eggs pick up a little later. Then we were doing this technique and then waiting for blastocyst formation, and normally, in most cases, we are doing NGS diagnostic to be sure that genetics are okay.
n Ukraine, there are no very strict legal issues related to IVF treatments, a lot of things are not prohibited, so we can do them. For this technique, there is no restriction that we should do it only after 5 or 6 attempts. We can do that even without any attempts, but the indication for it is to do that if we have, for example, arrest of development, we have fertilization but after 72 hours or 96 hours embryo does not arrive in the blastocyst stage. This is one of the best indications for this technique, and in most cases, we receive the blastulation after pronuclear transfer.
The costs are much less than in the United States, you can email me, and I’ll get back to you with the costs
It is still experimental. On one hand, we have done quite a lot of pronuclear transfers and spindle transfers, but on the other hand, it is not thousands, so we don’t have big statistics.
We don’t see a lot of issues with this technique. The main disadvantage is that we never know if this technique will be helpful or probably will not change a lot if the main problem is inside the nucleus of a woman’s egg. This technique probably will not be so helpful because the nucleus will be the same, and we cannot do some analysis or something to know that before, so we should try and see if it will be helpful. This is probably the main disadvantage, that we cannot exactly predict that in such case it will work or not. The main indication is poor egg quality, however, the egg should fertilize, and when a woman is in late reproductive age, we should have at least 2,3 or 4 eggs to be able to go ahead with the pronuclear transfer.
We have no such problem. Our embryologists choose the best eggs, which are correctly fertilized. Sometimes they put a fertilized donor nucleus into the patient’s cytoplasm. It lets us see if this donor’s nucleus inside the patient’s cytoplasm after arrest will not give us a blastocyst, and normal pronuclear transfer gives us a blastocyst, which means that this patient has a cytoplasmic issue and not a nuclear issue. We can only see that after we have done this technique, not before.
In the egg donation, we take the eggs from another woman, put them inside the sperm cell of the husband of the woman who would like to become pregnant, and we receive an embryo containing the genetic material of the husband, but it won’t contain the genetic material of the woman. After that, we put this embryo in the uterus of a wife, and she becomes pregnant, but genetically there is no relation with this baby.
The mitochondria donation all these techniques, pronuclear, spindle transfers, we can call mitochondrial donation. In pronuclear transfer, we take the fertilized nucleus from own cytoplasm from own eggs and put it in empty donor cytoplasm, it means that genetic material is from a woman and her husband, and only a little amount of mitochondria is inside this egg and future embryo. Genetically, future babies will have a relationship with both parents, not only from the men like in egg donation.
In some cycles, we are using own follicles and hormones of women for preparation, we can follow in the follicle growing and see when the uterus will be ready for transfer and put embryo. In most cases, the cryo transfer is done without your own hormones. If you have no period and no hormonal activity, we normally start with giving the replacement hormonal therapy. It is Estradiol Valerate or Estradiol Hemihydrate, normally, in dosage of 6 or 8 milligrams per day, there are various preparations we can use it for 7, 8 or 9 days.
After that, we will measure endometrium thickness and check the endometrium by ultrasound, it should be trilaminar, and it should be more than 7-8 millimetres, it’s better if your endometrium will be like 9-11 millimetres. As soon as we receive such thickness, we will add progesterone, we give around 600 or 800 milligrams per day. After a couple of days, we are measuring your progesterone level, which should be more than 10, and if we have such a condition after 5 days of progesterone, we will do the embryo transfer. It corresponds to the hormonal situation if you have ovulation and your embryo becomes a blastocyst.
Unfortunately, this happens quite often. It depends on the egg quality if we receive more or less good egg quality, even if it is 1 per cycle, we can receive 2-3 doing a double stimulation in one cycle. We can try to receive more and freeze a couple of times, then thaw one more time, fertilize, etc. It is time-consuming, it is very difficult from the emotional side as well. From time to time, it is possible to receive a good quality egg, it’s possible to try for 2-4 months and try to receive 3-4 additional oocytes and try to work with them.
With egg donation, you would probably achieve pregnancy much faster. The decision depends on your emotions and if you can wait for that.
There are a lot of supplements, for example, CoQ10, vitamin E, also replacement hormonal therapy can increase the number of follicles and eggs, not mitochondria directly. It is difficult to prove that these supplements improve mitochondria function directly. The egg development lasts 12 weeks, which means that we should take them for around 3-6 months, but after that, we don’t know what is working better.
I’m not sure, but I will try to search for it, and I can give you the answer via mail. I know that some clinics in Spain are doing it. In the United States, it is not done probably due to legal issues, it is quite complicated.
In this case, unfortunately, the only possibility is surrogacy. We can stimulate the ovary, receive eggs, fertilize with the husband’s sperm, create embryos and then prepare the surrogate mother, transfer the embryo, and then she will carry the pregnancy.
There are also some clinics, mostly in the north of Europe, where they try to do uterus transplantation, but it is quite complicated. It is necessary to receive medication that suppresses immunity, so for me, the easier option is surrogacy.
In Ukraine, the law is quite simple for surrogacy, husband, and wife should be officially married, we cannot do that for a non-married couple, only heterosexual couples with a medical indication. It should be described in some medical documents like, for example, document certified that hysterectomy was performed, or something like that. Then there should be an official notary and agreement with a surrogate mother, then from the legal point of view, we can do that.