In this webinar, Dr Uliana Dorofeyeva, Director of International Cooperations at IVMED, and a Medical Director at OVOGENE Egg Donor Bank, Ukraine, has talked about unexplained infertility – the diagnosis and prospects for patients.
Unexplained infertility is infertility that is idiopathic in the sense that its cause remains unknown even after an infertility work-up. However, as this is, as I said, an old definition, this work-up of infertility includes only:
Most clinicians, and physicians, define this as:
I really hope that you, being a patient, haven’t received this kind of diagnosis and also that you haven’t been advised that the cause remains unexplained after the negative IVF treatment or IVF attempt because this means, and I accept this definition, that unexplained infertility means that the physician was not able to find the real cause of your infertility, and we need to perform deeper testing and wider screening.
We need to understand that if it’s still unexplained, meaning that which tests that have been performed are not complete, probably some tests were not available in the clinic or the country.
We need to understand what the quality of those tests was who evaluated the tests, and also that the diagnostic work-up was incomplete or of poor quality.
There are some levels of what needs to be considered in the very beginning or during the initial stimulation cycle or initial embryo transfer as well as what needs to be added as additional testing or methods after we had already an IVF treatment, and it was negative.
In simpler terms, we need to have two main things:
However, this is not the complete question and answer of what else needs to be done.
These are immunological factors we discuss a lot with the professionals and some consider this as being important while some do not consider them as really important, but our practical experience says that this works or these could be genetic factors as an issue of why pregnancy was not achieved. These could also be factors related to an oocyte, sperm, or embryo itself, but the most significant for most of our patients the causes are multifactorial.
We should not stop in case we just received one cause, and we decide that this is an issue, and we need to treat infertility according to considering this case as a main one or the only one. If we started the treatment, and we did not achieve the pregnancy, we need to understand that, probably, they missed something, and we need to think wider and understand what else can play the role of why this was not achieved.
Another issue, starting from the very beginning, is the endometrium and the uterus. A normal endometrium is more than seven millimetres and a receptive endometrium is on the timing of implantation. Once we start the preparation of the patient for IVF, we need to consider the endometrium cavity and in case any abnormalities are found, for example, as endometrial polyps, we need to remove them, we need to recommend hysteroscopy to be performed and treatment to be provided.
Unfortunately, in some cases, considered by the doctors, a thick nice looking endometrium of 16 or 17 millimetres; however, we need to understand that this sick endometrium is hyperplastic. This is histologically a healthy endometrium, and also such status of the endometrial cavity needs to be treated. Moreover, the abnormal endometrium needs to be removed.
The reproductive surgeon needs to consult and treat patients with Asherman’s Syndrome. We need to understand how good would the cavity be after the treatment of Asherman’s syndrome and the synechiae will be performed. It should be a trial to treat this. If not, we need to recommend the surrogacy treatment; however, for most of the surgeons right now, there are technologies and possibilities to treat this successfully.
Myomas are different in their localization and sizes; however, also after the preliminary treatment, before the IVF cycle for many patients with myomas, with high-quality and euploid embryos, we are still receiving implantation. There is an issue which we need to control during the pregnancy care and the first and the second trimester during the pregnancy, and the whole pregnancy in general. However, as well as for the adenomyosis, with high-quality embryos, for most of the cases, pregnancy is still possible.
The protocols for endometrial preparation are different, and we need to achieve, an endometrium of 7 millimetres and more for the proper and successful implantation.
There is a natural cycle which requires endocrine monitoring ultrasound evaluation, and measurements of the LH levels very often. We don’t use natural cycles for the preparation of the endometrium, even not modified natural cycles, we prefer to use hormonal replacement protocols even for endometrial preparation with pituitary suppression once to prevent the indigenous surge of LH hormone causing luteinization and progesterone exposure. Therefore, we are more flexible, and the patient is more flexible.
There is a lot of statistical data saying that this kind of endometrial preparation gives higher implantation chances for the patients and is more successful.
What if, after the proper evaluated endometrial preparation, we are facing the syndrome of thin endometrium? Why does it happen? It usually happens due to:
Such syndrome needs to be treated, and we are treating this with 2 options, the growth factor for the treatment of this thin endometrium is recommended, and it showed also a highly successful evaluation and efficiency.
One of the studies performed for the treatment of the thin endometrium included using the Granulosa Growth Factor.
The study included 30 patients. All of them had an endometrium of less than 7 millimetres. They were between the age of 30 to 48 years old. Only good quality embryos were selected for transfer based only on the morphological conditions, not including the genetic testing. At least two negative outcomes those patients had in the past. They had a resistant, thin endometrium. An intrauterine administration of 30 milligrams of Granulosa Growth Factor into the uterus was used, and it showed that the thickness of endometrium was higher for 2 millimetres in comparison to the previous protocols, the endometrial preparation, and the implantation was higher for this group of patients. However, for some patients, the growth factor was not working, so as an alternative blood and rich plasma PRP has been added as a treatment option for thin endometrium.
Another group of 38 patients was selected, those who had cancelled the cycle due to inappropriate endometrial thickness in their previous cycles, and also included those patients who had no reaction to the growth factor treatment. That group was randomly put into two groups. 20 were injected with the PRP and 18 were a control group.
Preparation of The platelet-rich plasma is always prepared from the patient’s blood. 15 millilitres of blood is used and centrifugation is performed and only the part of the PRP is selected. As soon as the material is prepared, 1 millilitre of it into the uterus is injected. Two injections on day 10 and/or 12 of estrogen administration are performed and also on day 1 of progesterone, and this thickness is measured on the day of the hCG after 48 hours, and on the day of the embryo transfer.
Criteria for evaluation:
We found that results were better in that group of patients as the increase of the sickness of the endometrium was 3 millimetres for the group of the pregnant women and 2.8 as average in the group of those women who were not pregnant, and we need to understand that also the embryos which were transferred they were not evaluated with the PGS testing, these were only morphologically selected good quality embryos. The implantation was 50% for those patients for whom PRP was injected, in comparison to 15 only in the group of patients with no PRP.
We now also know much more about the implantation, the interval of implantation, and then the embryo-maternal crosstalk. There are a lot of publications and there is a different reaction of the endometrium into good quality and poor-quality embryos.
This is additional proof of the fact that having those possibilities for the evaluation of our embryos:
That’s why in our practice we are using a thorough evaluation of the embryos, all new techniques which are possible for embryo investigation, and this gives the best results.
There is a specific time frame during which endometrium has maximum sensitivity to embryo implantation. It usually lasts for 48 hours, and we expect this to be six to eight days after the ovulation.
For hormonal replacement cycles, we consider this to be synchronic with the blastocyst development, meaning between day five to seven of the blastocyst development. This is once our blastocyst is well expanded, and they are ready to be transferred. The endometrium should also be ready, and we investigate this by checking the pinopodes which is a special tissue you can see in the grafts here, that they need to be mature and well-developed. They are not developed until the window of implantation is open, and they are already decreasing and going out once they are getting older and there is no function of the pinopodes anymore.
The endometrial receptive array is very similar in terms of understanding the receptivity. It’s based on the evaluation of a genetic chip of expression of 238 genes which are responsible for endometrium receptivity.
Both are used; however, electronic microscopy shows just the pinopodes and their maturation and development, therefore, this test is used more. It can be performed in our clinic, and it’s also cheaper. We perform:
Why do we move this further? So, six is the beginning because for most of the patients who have so-called moved windows of implantation, it moved into day 8 or 9 or 10, just for some, and this is less even than 5% of the patients who have moved this to be earlier than day six, but we can also see this if we see that the pinopodes are already decreasing and getting older on day six. Then, the scanning electronic microscopy of endometrial specimens was performed to assess the presence and maturity of the pinopodes.
In the photos below you can see that pinopodes are well-developed on day 8 of progesterone.
In this picture, for example, you can see that on day six there are just very few and very small pinopodes. On day eight they are just starting to grow, but they are still not mature, so this is not the timing of the proper implantation. On day 10 we only can see nice-looking pinopodes, so this means that this is the timing for the implantation. For example, if we consider that the patient and we are transferring her blastocyst on day six or seven of progesterone and the embryo will leave only for a maximum of 48 hours inside of our uterus and will have the potential to be implanted, but the pinopodes will start growing only on day 9 for this specific patient, so, of course, this will be asynchronization and we will no window of implantation.
Once transferring embryos on day six, we would just lose those embryos and we will never get pregnant for such a patient without evaluating the window of implantation, transferring, even euploid embryos, highest quality embryos, checking all the other factors, we would say that cause is still not here. We still don’t know what the cause and we is will say ‘This is unexplained, this is idiopathic, there is no reason for this.’
Moreover, transfer concerning the window of implantation is called personalized embryo transfer.
We did a complex evaluation of those first analyzed embryo transfers starting from 2018 and we evaluated transfers of 114 women with one to six failed attempts before.
The age of the patients was between 28 to 46 years. We only selected good-quality embryos; however, we are not using any others in our practice. For those patients who are over 42, we use donor oocytes for the creation of the embryos. Pinopodes were obtained on days 6, 8, and 10 of progesterone in the hormonal trial cycle and the percentage of abnormalities in that group was 72% of the implantation window being asynchronic.
All patients underwent personalized transfer and pregnancy was obtained for 58 women in that group, so 70% of the patients with previous non-successful embryo transfers got pregnant after performing personalized embryo transfer, evaluation of their endometrium, and the receptivity of it.
Back in 1995, this was the first publication for immunological diagnosis, testing, and treatment for infertile patients. After that, it was the first book called “Is Your Body Baby Friendly?”
As I said, there is still a huge and very busy discussion regarding the immune response of the patient to NK cells which are different in the peripheral blood and the urine. They have different functions and if we are testing the immunological status of the patient by understanding the peripheral level and volume of the NK cells, we need to consider this as a patient to be dedicated to the IVIG treatment, for example.
However, in our practical experience and also the discussion is because many countries are not allowed to use IVIG for IVF. It is not written in the instructions, it is not a clinical experience. It needs to be confirmed and there is no evidence.
On another hand, IVF was also done in the way it was done almost 50 years ago, it was the first time done as a clinical experience and after that it was successful. Thus, we just need to understand what is working and we need to consider this for each and every patient.
For those patients who potentially could have or we consider their medical history as those which we need to think about immunological infertility, we would recommend testing:
This works well and there is high efficiency for those groups of patients.
Talking about the oocyte quality, we all know that the poor oocyte quality is one of the main causes of reproductive failures in assisted reproductive treatment. With regards to the quality of eggs, now we know this is also quite new knowledge, that the quality of the eggs is mainly determined by the small organelles named mitochondria which are also energy suppliers of the cell present in the cytoplasma of the cell.
We also know a lot about morphological, cellular, and molecular predictors of the oocytes. The main causes of the poor egg quality are:
Once we have an aged oocyte and mitochondrial dysfunction, we are going to have lower IVF success rates, lower fertilization, and higher aneuploid rates of the embryos. The main solution, a very easy and simple one, also very effective, is an oocyte donation.
We perform and, frankly speaking in my experience with the patients, this is probably the number one frequency of the IVF treatment cycles which we are performing. This is oocyte donation, also because our patients are in their age over 35 and up to 45, 46 even older as in Ukraine there is no upper limit for the IVF treatment; however, we still consider medical indications, medical risks, and ethical issues in terms of planning the IVF treatments.
Still, oocyte donation is very effective and being involved in the Ovogene egg bank which is an extended egg donor catalogue offering also oocytes being sent globally into different countries, specifically, this is very important in the era of COVID, that patients should not travel, we can transport biological materials. We need to consider this as a very effective treatment.
In our practical experience, for the clinic and for the bank, we perform a strict and very deep evaluation of morphological parameters of each oocyte from the donor, including all morphology which can be assessed right now and everything the science knows about the oocytes, also we are offering a unique technology of genetically certified oocytes.
What are genetically certified oocytes? This is an oocyte for which we do NGS-based aneuploid screening, also a carrier screening for donors, and the testing of a male partner in a couple to understand that the created embryo will have no genetic risks for the potential baby, at least for this number of the mutations and this is over 350 mutations which we are testing.
How is it performed? Why is it important to genetically screen the oocyte? The genetic structure of the oocyte directly affects the pregnancy success rate. The aneuploidy of the embryos will reduce the pregnancy success and will lead to chromosomal abnormalities.
This testing will give us a big advantage in avoiding mitochondrial mutation risks while we will be fertilizing only euploid oocytes. The advantage of the polar body, NGS testing from the patient’s perspective is the fact that the oocyte donation program mostly brings financial responsibilities for patients and each oocyte looks like a potential embryo from the patient’s perspective. Once we provide euploid oocytes, we will have the highest chance of achieving a high-quality blastocyst. We need to understand the sperm factor as well, however, in the general population in the number, we would have the highest possible blastocyst outcome.
Most of the oocyte donation programs in different locations do not require NGS or PGS testing of the oocyte or the embryo before the embryo transfer, but we would recommend doing this to maximize the chances of the patient. We also provide and we recommend to perform NGS testing of the oocytes for our patients, not only for the donors.
Why did we start to recommend this? After the evaluation and after the polar body biopsy of the 1978 oocytes, almost 2000 oocytes, and we vitrified them and the results of the NGS testing were that 75 almost per cent of those oocytes were euploid, so 25% of those oocytes coming from the cohort of young and healthy women were aneuploid.
We need to understand that we are dividing our patients into two groups, one group is those for whom we potentially can receive mature and healthy oocyte with the healthy nucleus. We understand that this group will be in their advanced maternal age, potentially, but still, we can work with this genetic material and that’s why oocyte donation really has an alternative.
The other group would be a group of patients who are not producing oocytes already, and we just need to accept this. Of course, we will recommend the best stimulation protocol, and we will talk and evaluate previous stimulation protocols, medications which were recommended, and dosage, for example, for all patients in their advanced maternal age we need to combine medications with the LH as well because this works well for the receptors and will give us higher results in terms of the recruitment of the follicles and also their growth and the maturation.
The alternatives are the following:
The alternatives we’re offering right now are a spindle transfer and a pronuclear transfer.
The spindle is the structure inside of the cytoplasma which helps the cell chromosome to be shared equally. It has a very important role in the cell division and for the genetic potential of the oocyte.
We can determine the spindle only by special software and a polarized system on the microscope. There are a very limited number of clinics using this in the world. The system needs the experience of the embryologist and high investment.
We are doing spindle transfer by the replacement of the entire cytoplasm of poor-quality oocytes. We transfer the spindle which consists of the genetic material from the egg of the older woman into the donor eggs with the healthy cytoplasm and also the spindle removed. The eggs which are resulting from this procedure with the repaired cytoplasm can be inseminated by conventional technique, which is ICSI, with the sperm of the patient’s partner and will give the intended parents a chance of having a child genetically related to them.
Advantages:
The pronuclear transfer consists of performing in vitro fertilization using the oocytes of the affected women with mitochondria that contaminated the DNA, and the sperm of the future father. After the fertilization is performed, we extract the pronucleus on day 1 of development and transfer them into the cytoplasm of the donor’s oocyte which has been enucleated before. So, we took the pronuclei from the donor eggs and we put pronuclear from the contained nuclear of the mother and the father.
Moreover, the hybrid zygote is then developed in vitro until it reaches the stage of the blastocyst.
Advantages:
The results achieved were:
We need to consider this for the patients in their advanced maternal age with the potential, without these procedures, to provide embryos not more than 20%. So, this is the difference.
This is the rate of the genetic abnormalities we consider. We had this published for many years until we started to practice the mitochondrial and pronuclear transfers.
For patients over 35 to 39, we would expect 50% of genetic abnormalities, mainly due to mitochondrial DNA. We understand right now that between 40 to 42, 70%, and over 42, 85% of the embryos would be aneuploid.
The transferring of aneuploid embryos gives false hope to our patients that there is a potential for a successful outcome because there is not.
The options we would recommend and do in our practical experience are:
These are very limited techniques. There was allowance in the UK to perform these kinds of techniques, and also not every clinic, if we are talking about all these issues, including implantation potential and testing of the window of implantation, IVIG treatment, all kinds of alternatives for oocyte donation and even oocyte donation itself.
In the whole IVF world, it’s very limited and we are lucky to practice in Ukraine. We used to receive many patients from all over the globe being treated in Ukraine. This was until a year ago, and now we are in the process of the COVID limitations, still, patients are coming even right now in Ukraine because we are trying to limit the number of our visits, to a maximum of two. For many patients for different kinds of treatments, this is one visit and a very short period of staying in Ukraine. We are doing a lot of our communications online, but so far telemedicine cannot provide embryo transfer or, hopefully, so that’s why it is still possible to be consulted, to get a second opinion, or to get treated with us in Ukraine.
It’s also important to understand those limits. Even before that, starting in 2008, in my practical experience there was an opportunity and we did this successfully to transfer embryos, oocytes or sperm from different countries to us, into our facility to provide those treatments needed to get donor oocytes, for example, or create embryos and send them back into the clinics. You can see those locations in the picture.
Last year, we also added Australia so there are no geographical limits right now, not for our donor oocytes and we are compliant with all regulations and requirements for the embryos as well as for nuclear transfer we can receive zygotes from your clinic. We can do pro-nuclear transfer for those frozen zygotes there and then you can come just for the embryo transfer in Ukraine.
Transportations are allowed. There are very clear, international regulations for the transport of cryopreserved materials. This can be done either with the supervision of the medical courier for some countries, even right now, or for some, this can be cargo, but there are rules for this, and this is really safe and we practice this a lot.
If you are thinking to stop, there are very limited possibilities for this. This is a medical consideration or to stop trying something and to change the treatment protocol, for example:
These should be medical considerations and it’s better to get a second opinion about this.
There is an age limit for different countries.
I really like this publication of an Israeli group. This is a unique country which is covering all treatments until the family has three children born. They published data that mentioned if there were unlimited, free IVF cycles, 95.5%of couples would get pregnant, and this is very promising. The issue is that, unfortunately, not all of us and our patients have unlimited, free IVF attempts.
Other data coming from the UK, for more than 150,000 women, for more than 250,000 IVF cycles, you can also see the statistics of how the potential of pregnancy increases with the number of trials, but also if we would think about the cause, if we would work to avoid this cause or multiple causes, we would get closer and faster, and we would be more effective in terms of receiving our clinical pregnancy.
I agree you probably were not touching every step, but I believe that these were the most important steps for those cases when we can say that this is an unexplained factor. This is additional to wider your knowledge of what else we can think about or what else can be recommended, but I agree absolutely that, quite well known in the US, Dr Norbert Gleicher that unexplained infertility generally means that the physician failed to find the true cause of your infertility. In our clinic, no patient receives that kind of treatment, and I would not like you to receive this as well.
If you are listening to this webinar and haven’t tried all the mentioned above, you should still keep trying and we will do everything to think differently to gather all decisions together and the previous experience and to achieve a pregnancy in the first cycle or as soon as possible by recommending something else and thinking wider.
Related reading:
A quality blastocyst can be achieved on day-5 or day-6; however, many publications show that day-6 blastocysts have lower potential in comparison to day-5 blastocysts. I was a co-author on one of these, quite a big, data, and our clinic also participated in the oocyte donation. So, day 5 has the best potential for implantation, and there is a grading system by a, b, and c, and also the grading by 1, 2, 3, 4, and 5 by Gardner.
There are different evaluation methods, but the Gardner is the most commonly used internationally. Day 5 is the day of the advanced and high-quality blastocyst, also grade A is the best. Some clinics would consider putting 2a, meaning A is the best, and B is also high quality; however, there are some disorders already, but they are awesome. Different embryologists would consider this differently. That’s why there is a lot of machine learning and artificial intelligence is starting to be used to evaluate those embryos. However, if this is the 5aa and advanced blastocyst, meaning grade 3 or 4, this is the best embryo.
It’s great to evaluate the endometrial lining during the follicular phase, then during the ovulation, and the luteal phase. We can do this in a natural cycle and the best lining and right change would be that we would see a triple lining, nicely growing until the ovulation time, and during the ovulation time, it’s already over at least seven millimetres or wider and it’s triple. So, we can see a very clear white line inside the endometrium, we call this triple line endometrium. As soon as we start luteinization, so corpus luteum starts to perform LH surge and then progesterone rises, we would see the lining being white and more homogeneous, thus it changes into the second phase, and it keeps being wider.
Also, it is very important that, since the ovulation is in a natural cycle or the administration of progesterone, we would not have this short luteal phase, meaning that implantation may fail because there would be not enough time for the proper implantation. That’s why there should be progesterone supplementation at least 14 days after the ovulation.
We perform Pinopodia testing by electronic microscopy. We do this always in the hormonal replacement cycle, meaning that the next cycle if we follow the same protocol. So, we do agonist injection before the menstrual cycle, then we start menstruation, we start estrogen supplementation, we continue this for 10 to 12 days until we have proper thickness of endometrium, and then we start progesterone and, on the same day on day 6, 8 and 10, we do a pipelle biopsy, and we send this directly to the laboratory to perform the scanning, and we see we are getting the pictures which I showed during my presentation and there is no level of the pinopodes. There is just their presence or absence, or they are too young, or they are mature, or they are too old. By evaluating these, we understand when, specifically, we have a window of implantation, and we do the testing in the cycle before. We do an embryo transfer because we are inside the uterus we are doing the pipelle, so we cannot transfer embryos in the same cycle.
I will start with the guarantee. We have a guarantee of pregnancy in case we are creating blastocyst in our facility; however, for the TESA sperm, we do not consider this. For the sperm factor, we have criteria regarding the total motile count and the morphology percentage of normal morphology sperm, and also DNA fragmentation level for these guarantees of live births. Unfortunately, the TESA sperm would not be there for the life birth guarantee or clinical pregnancy guarantee because we can guarantee life births in the surrogacy treatment, but in egg donation, we can guarantee only clinical pregnancy because the older woman, if there is a pregnancy period, there are other risks related to infertility, mostly they are related to the somatic status of the patient. That’s why we do not guarantee live births if the pregnant woman is the patient, and mainly if this is an older patient.
TESA sperm can be accepted in our laboratory, but we need to communicate before this and to understand the number of the sperms inside the quality, the reason etc., however, it would not be with the guarantee, but all those complicated cases we communicate together with our laboratory manager and me, for example, and we would evaluate this specific case, and we will give our conclusion, what would be the best in this case.
Also, embryos can be sent for surrogacy from Australia and, I would say that right now, this is the most common option that now not patients are coming to Ukraine for treatment, unfortunately, but embryos are coming.
Yes, we do. We do the testing in the laboratories. We have immunogram. We have all kinds of antibodies because this is a basis for us to make a decision regarding IVIG treatment.
Likewise, we do not have intra lipids in Ukraine being registered, but we have great IVIG. This is a Ukrainian producer, a company which also produces blood factors, even being exported outside, and we already have been working with them since 2010. This is when they started the factory in Ukraine. We were checking this very carefully at the very beginning, but now we trust absolutely.
In terms of the costs of Ukrainian IVIG, in comparison to the cost for example in Ireland or in the UK, it’s absolutely different.
We recommend a maximum age of 43; however, in our practical experience, we have now a patient 46 years old being pregnant after a pronuclear transfer. It’s very individual, I would say, so we need to evaluate all the parameters, of course AMH is the most important, and we would expect AMH to be present. This is complicated, but we even had a patient with AMH 0.3, and we produced few oocytes in several cycles, so we did 3 or even 4 because one was stimulation in the follicular and the luteal phase, and we received 1 to 2 oocytes every cycle, but she was insisting on this, so we spent a lot of time, but we ended up with 1 euploid blastocyst after pronuclear transfer, and she is successfully pregnant now. But mainly this is up to 43, and AMH over 0.5, but again this is to the discussion.
To do a pronuclear transfer, we need to have a zygote, or we need to have an oocyte. We will fertilize the oocyte and on day-1 we would get a zygote and the pronuclei, and we will transfer them into the cytoplasm of the donor oocytes, so we can do an embryo but only on the zygote stage and of course on the oocytes.
In our practical experience, we have been doing pronuclear and spindle transfers since 2018, so this was the very first cycle. More have been performed during 2019 and 2020, of course, this is the busiest year right now, and we successfully continue this now.
Our clinic is the second clinic in Ukraine that practised this, so the first was another clinic working for this with a group of Chinese doctors from the US. Again, in China, I would consider this as a location where many sciences are developed, but due to regulations, they cannot practice there so they are looking for practical experience somewhere else, but they were the first, and they have pregnancies since, also, 2018. Why? Probably because, I don’t know why, like we are talking a lot already about the pronuclear transfer, so you can discover this together with us, maybe with some clinics from the UK, but I think that their practical experience is still quite limited, but this is a sign for me that we need to talk even more about this.
Success rates for the oocyte donation treatment with fresh or vitrified donor oocytes are not different at all in our centre. Frankly speaking, we are using more and more oocytes which have been vitrified from our donor database, and we see the benefits of the vitrified oocytes because there is no synchronization and there is no risk of asynchronization between the endometrium preparation of the patient and the growth of the donor oocytes. So, we should not look for a solution in case the endometrium is already ready, and the donor is not ready yet.
That’s why, in our hands, the survival rate of the oocytes is over 95% in our clinic from the practical experience of sending the materials to different clinics. Frankly speaking, the survival rate is different, but it will rise between 85 to 95%.
This experience of vitrifying the oocytes and thawing them started being widely used in 2013, only a few clinics have been practising this, and we needed to get knowledge in this practical experience, and more people vitrified and saw better results they are receiving. All clinics which are working with us, they adapted the protocols for thawing, and we work with them regarding the survival rate, so they are all doing fine, and there is no difference in terms of the fertilization rate, the blastulation rate, the implantation rate at all.
Vitrification is a very safe procedure for the oocytes and also for the embryos, but for embryos, this works already for almost 25 years and for oocytes only 10, so this is a difference.
For the own eggs, thawing rates are different. It’s lower, but it’s lower depending on the age of the oocytes which were vitrified. Also, vitrification and thawing are like a test method for their potential, mostly if the material does not survive after the thawing means that this was something with the material. If it was not tested before, so it’s like an additional testing method for the potential. So, again, the survival rate for won eggs would depend on the quality of the eggs which were vitrified.
In our laboratory experience, we use frozen eggs also for pronuclear transfer; however, results are better using fresh. If there is a possibility to use fresh, we would recommend using them fresh. If there is no possibility, we would recommend, that if there is a created zygote and then a vitrified zygote, then this is sent to us, if we are talking about sending some kind of materials or why we should use frozen eggs for the donor because we always, for pronuclear transfer, we are using donor materials because we need to get a cytoplasm. All those donor materials are fresh.
Because those numbers are very limited, like numbers of those women over 45-46, using their own eggs and also having a possibility to using their own eggs, because we understand that after evaluation of the patient and her ovarian potential and AMH, and after starting the stimulation and after selecting the best protocol, if you’re ending up with no follicular growth, or we are ending up with one follicle, but then it is the empty follicle, there is no further treatment. We need to, frankly speaking, for many of those patients over 43, even if we are starting the stimulation protocols or the treatment methods, we need to stop them in some cases because they are not working.
But still, there are patients and, mainly those are patients with previous PCOS, with the ovaries having multiple follicles, they still are producing some oocytes, and they are good responders or good candidates for this pronuclear transfers in their 43, 45, 46, but I cannot tell anything about statistics because these are individual cases. I can talk about the cases.
We are very transparent with all our patients and if we see that we tried the best stimulation protocol, we select the medication, we even for some of those patients did an ovarian rejuvenation before the stimulation, and if it’s not working we just say like ‘listen’, and this is a very clear and transparent discussion with every patient that, and we like them to know this and to understand this and to be involved into the process. It’s easier than to make a decision. There is no discussion, there is an agreement that the next steps should be that, and many patients who even started this or were thinking about pronuclear transfer ended up with the oocyte donation treatment, but at least they tried something different, tried an alternative, and for many of them it worked, but for some it doesn’t.
5 is already a blastocyst which is hatched, so the zona pellucida, which is a bit ruptured, and the cytoplasm and the structures of the blastocyst are getting out. This is a very sensitive embryo in terms of how all procedures with this embryo should be performed, either freezing or thawing or even how they get it into the catheter for the embryo transfer, or even how the doctor is doing the embryo transfer, that this is not a rapid injection, that they do not stick this into the wall of the uterus, etc.
Regarding the BA or AB, as I said, if we show even one embryo to 10 different embryologists, 5 of them would grade this differently from those five others. This is the fact of the human being, and they are grading. If there are two letters, they usually grade cytoplasm and the zona pellucida differently because they are both very important and play the role of the implantation, but in general, we would consider 5BA and 5AB as a good quality, advanced blastocyst, but if there was no PGS testing of this embryo, we don’t know everything about it, so there is still additional potential to get information about the embryo potential by doing a biopsy and genetic testing which is quite widely used now. Still, there is a discussion of whether should we do this for all. Or just for some? For aged blastocyst to avoid aneuploidies? Do not give a false positive, post negative expectations, etc., but it’s for every case. It’s individual and we also are discussing this.
Yes, it’s also a good-quality blastocyst. As we said, blastocysts 1 and 2 are so-called early, and everything is 3, and 4 these are advanced blastocysts, so they are good quality, but they are not hatched yet.
5 is an already hatched blastocyst. Usually, hatching should be performed inside the uterus, so this is the very first process before the implantation and, usually, embryologists like to work with three and four because working with five, as I said before, is very tricky in terms of that we need to be very careful with this embryo.
We are using a random start of the stimulation quite widely, both for our patients and even for our oocyte donors. From physiology, we understand that as soon as we have a follicle, there is a potential for this follicle to produce an oocyte until a new cycle starts.
We all understand that each cycle, we are given the specific volume of the follicles which may potentially produce the oocyte and only once the cycle is ended, those follicles are gone. We will not use them any time in the future, so we just lost them completely. Starting stimulation either in the follicular phase or in the luteal phase for good responders, there is no difference since we are giving additional FSH and since granulosa in the oocyte is recruited by additional FSH, which is normally produced only in the follicular phase. That’s why we were stimulating patients before only in the follicular phase because we were following the natural conception, the natural cycle, and in the natural cycle, the pituitary hypothesis is producing FSH in the higher level, only in the follicular phase. That’s why we decided to provide stimulations only in the follicular phase, but now we understand that even until the next cycle starts, if we turn on the FSH there will be a production of the oocyte, there is no difference for good responders.
For poor responders, we have the ovary-producing follicles constantly until they are there, so until the AMH, we still have some level of the AMH, and there are different stages of premature follicles, and they are getting matured. Even two weeks later, there is a difference that new follicles are coming from the very premature stage into the stage being already responsive to FSH, and if we are giving in the luteal phase additional FSH, we are bringing those small follicles from the premature stage into the early mature being responsive to the FSH. They are also receiving this additional FSH, they are getting mature, they are growing, and they can ovulate, so we can get oocytes from them.
Indeed, we are also using follicular and luteal stimulation for different cases. For good responders, as I said, quite widely. For poor responders, if we have no time, our time is very limited. For example, this is an advanced maternal-age patient who said like, ‘I have only three months and I would like to get as many follicles and oocytes as possible,’ so, of course, if there are no contraindications like cysts, etc., we would perform stimulations both in the follicular phase and in the luteal phase.
The slight difference is that in the luteal phase, we need to give a little bit more FSH and LH, so medication volume would be a little bit higher than in the follicular phase because of the natural reduced production, but it still works. This is our experience.
There is the issue of the nuclear DNA, so still nuclear DNA in the aged oocyte can be changed, meaning can be aneuploid, and even if we are helping with the spindle or pronuclear transfer to get the embryo into the blastocyst stage, it is recommended to do NGS testing or PGS testing of the embryo because we need to be sure that the DNA was not affected anyhow, there were no chromosomal abnormalities there. In comparison to that fact, if we are talking about oocyte donation, and we remember this table with the lines and the colours, the probability of having a euploid blastocyst from this material is higher than still getting blastocysts from the older oocytes, even changing the mitochondrial DNA, we need to check the nuclear DNA.
Pronuclear transfer and spindle transfer are more expensive in comparison to oocyte donation treatment. You can get in touch, I will forward you to our coordinators, and they will provide all the details.
I need additional information. If that patient is over 35 or 38 years old, I would not recommend pure FSH in any of these dosages. For these patients, there should be FSH plus LH, and there would be no critical difference between the dosage of 300 units or 350, but if that would be FSH plus LH, there would be a more significant difference because LH plays a role for getting receptors, being prepared to receive FSH. That’s why for all older women, over 38, we need to recommend these kinds of medications. So, if it was FSH and LH and over 38, there would be no significant difference between those two stimulations.
