Complete Guide to IVF Lab Techniques - ICSI, IMSI, blastocyst culture, AH, PGS
The IVF journey consists of a number of steps. One of the most mysterious and fascinating of these occurs in the Embryology Lab. . In fact, the whole magic happens there and it’s the people who make the IVF laboratory what it is. It’s their knowledge which makes it all possible. During this webinar, Laura Van Os, an embryologist from IVF Spain, presents the Complete guide to the IVF Lab which includes techniques and procedures.
Sperm selection techniques
At the beginning of the presentation Laura reminds us of a crucial fact: eggs are always limited in number so basically it is only the valuable mature eggs which will be fertilised whereas every sperm sample has millions of sperm cells and in the end, only a few will be needed. For this reason, several sperm selection techniques have been developed in the latest years for different reasons: to find the best way to select the best sperm cells in the sample and to use this best sperm cells to fertilise the eggs. Usual and mandatory techniques used in the laboratory while processing a sperm sample are density gradients and the swim-up technique.
Laura pays attention to the fact that there are some patients who have some more specific problems, like a higher rate of apoptotic cells among the sample. That’s why they need specific techniques. One technique that can be used to overcome this problem is MACS (magnetic-activated cell sorting). Apoptotic cells have started the process of dying and no-one wants to fertilise the eggs with these sperm cells. With MACS , embryologists can remove these sperm cells from a sample. Another technique that is useful to overcome some molecular problems is piCSI or Spermslow. These two techniques they are based on the fact that mature sperm cells will be able to bind to hyaluronic acid whereas the sperm cells which have not undergone this maturation process will be moving around freely. It will give embryologists a hint to distinguish these two kind of cells and it help them to choose the best sperm cell for fertilisation in the laboratory.
Another extra technique to select sperm cells which have morphologic problems is IMSI. Basically you look at the sperm cells with a really big magnification – the sperm cell is more than 6000 times amplified. The theory says that thanks IMSI it is possible to select sperm cells which have a best morphology.
On the other hand, there are alternative sperm sources to main source which is obviously ejaculation. These alternative sources are the testicles and the epididymis. Sperm cells are produced in the testicles in a process that lasts around two and a half months. They then later mature in the epididymus. Sperm cells can be extracted from there by aspiration with a needle or by a little surgery. And this is indicated in case of patients for example who have undergone vasectomies, which might have difficulties collecting the sperm sample, there are some genetic mutations (like the absence of vas deferent in case of cystic fibrosis). There are also some cases in which the quality of the sperm in the testicles seems to be better than the quality of the sperm cells in the ejaculation.
Fertilisation process and ICSI
Once the eggs have matured and the sperm cells have been selected, the next step is fertilisation. It can happen by one of two main techniques. It can happen through ICSI. ICSI stands for intracytoplasmic sperm injection and through this technique, the sperm cell is directly microinjected into the egg. The other technique is conventional insemination (IVF). In this technique there are some droplets of media with sperm cells. The egg is placed in these droplets and the sperm cells will compete to naturally fertilise the eggs. IVF, despite seeming more natural and less invasive, has more risks because no one is really sure if cell reaches the place that it should reach. Usually there’s also a higher rate of abnormal fertilisation so viable eggs may be missed out. These are always the embryologists who decide which of the two techniques should be used depending on the egg and sperm quality as well as the previous history of the patient.
Artificial oocyte activation
Artificial oocyte activation is starting to be used more frequently with patients who have had recurrent fertilisation failure meaning that they has less than 1/3 of their mature eggs fertilised. This is sometimes due to a failure in oocyte activation, both due to egg and sperm factors. To provoke this artificial activation embryologists are using calcium ionofore and have very good results. In this protocol you basically have to culture embryos in droplets of calcium ionofore rich media for 15 or 10 minutes after ICSI. We apply this media in all 100% of eggs in case of case of previous complete fertilisation failure and if it’s just a low fertilisation rate we consider applying it only in half of the eggs to see if this is really helping.
If the egg is correctly fertilised, the embryo will start dividing: on day 2 it will have four cells, on day 3 – around eight cells, then 16 cells. At that point the embryo will turn into morula stage. A morula will start to expand expand until we find the blastocyst. The blastocyst is a very defined structure of an embryo. It’s composed of two parts there is some mass in the middle of the embryo this is called the inner cell mass and it will become the future baby. It is surrounded by a layer of cells this is called the trophectoderm and it will become the future placenta. At this point the embryo usually has around 1 hundred – 2 hundred cells. The shell surrounding the embryo becomes thinner at the blastocyst stage.
Embryo culture till day 5
Laura admits that it is more and more common to leave embryos in culture until the day five of development. The point is that even if the embryo looks nice on day 3, it does not mean that it will become a blastocyst. Not all eggs are programmed to do so and it’s like a natural selection process . Embryologists tend to leave embryos to day 5 because if you transfer embryos from day 3 and you’re not becoming pregnant you might be wasting your time because maybe they are not even able to reach the blastocyst stage. Laura highlights that quantity is not the same as quality. Some patients have many eggs but very few of them turn into blastocysts of good quality, the others may have less eggs but most of their eggs will turn to good quality blastocysts.
Embryo monitoring – time-lapse
Laura says there is a very powerful tool to evaluate embryo development called time-lapse. In this process, the camera inside the incubators takes pictures of the embryos every five minutes. This allows embryologists to have a continuous recording of the embryos without disturbing their stable culture conditions. In addition, morphokinetic analysis of the embryo can be obtained and this information can be used to predict implantation and to select the best embryo for transfer.
Embryo selection via PGT / PGS
Another way to select which embryo to transfer is PGT (pre-implantation genetic testing). It is based on the fact that some embryos have genetic errors. In the laboratory, when the embryo which is the blastocyst stage, the embryologists remove a few cells, extract a genetic material from these cells, amplify it and we sequence it. In this way they are able to know if the embryo has the correct number of chromosomes. PGT is used in order to transfer only the genetically normal embryos and thus, increase the implantation rate per transfer. As the percentage of abnormal embryos is linked to maternal age, PGT is especially recommended for older patients.
Embryo transfer – increasing the implantation rate
When it is decided which embryo to transfer, there are also some techniques used to increase the implantation rate of these particular embryos. The embryos can only implant when they have broken the shell around them. This happens at the blastocyst stage around day 5 or day 6 of the embryo development. In the laboratory this process can be assisted. According to Laura, embryologists find little benefit in performing assisted hatching in fresh embryo transfers. However, the implantation potential increases when assisted hatching is performed before transferring frozen embryo. Assisted hatching is performed by the use of chemical or mechanical ways, like with a little laser that is very easy to control and very safe for the embryos.
After a blastocyst or an embryo is transferred it will come out of the shell, contact the endometrium (the wall of the uterus), adhere to it and finally invade the endometrium. This process implies complex molecular interactions between the embryo and the endometrium. The stage of endometrium when it is capable of performing these interactions is called the implantation window. In other words, it is the point at which the endometrium is receptive and ready to accept the embryo. That’s why some women perform biopsies to check when exactly their implantation window is.
Last but not least, Laura mentions one more technique which is related to embryo transfer: embryoglue. It is basically a media containing hyaluronic acid that makes the media little bit more viscous and thus, helps the embryo stick to the endometrium. So it does make some scientific sense to think that this media could slightly improve the adherence of the embryo to the endometrium.
Questions and Answers from the event
Are there lab techniques, excluding ICSI, that can help with fertilisation? I had a complete fertilisation failure in IVF this month.
This would be the kind of patient for which we could consider to apply calcium ionophore. We would need to know a little bit more about your case, for example how the sperm sample is. We’ve had some patients which had extremely bad sperm samples where nothing was moving. I remember a patient in the same situation, we’ve retrieved sperm cells from the testicles through testicular sperm extraction. We actually managed to have a higher fertilisation rate and the patient is pregnant and they’ve had a baby. So I would need to know your specific case and maybe it would also be a patient candidate for calcium ionophore treatment.
Do you prefer one step media or sequential media?
I don’t think it makes a very big difference if the media you’re using is good. If this media has been tested for a long culture, it should have sufficient nutrients to hold for the six days. I think if the cultural media is good, I don’t think it will make a big difference as far as I know.
Can we really trust PGT-A? What about mosaicism?
It is a good question regarding the mosaicism. Sometimes we have a patient’s results showing that some embryos are normal and other embryos are clearly abnormal and we also have one result of PGS which is a mosaic. We have found different cells in the embryo. But depending on which chromosomes are affected, the mosaic embryos can be transferred, at least at our clinic. If the patient decides to transfer mosaic embryos, he’s able to transfer them. It is true that in a small percentage of cases, it could be that the placenta is not representative of the fetus, but I think the same happens with a later screening. It’s a very small percentage of cases. In cases where PGS is indicated, I think it’s more important to perform the PGS than to look at this little percentage.
What is your practice of using media in a time-lapse? Do you use media up to day 5 without any change or do you prefer to change media on day 3, even though it’s a single step media?
We have seen that we have very good results with the single step media. If you use sequential media, you need to take the embryos out, change the embryos from dish to dish, this takes some time so it’s a little bit of extra manipulation. You would really need to make sure that it’s worth it, that there is an added value in changing the media. It’s my experience that single step media, which is good media, will give good results. I’m skeptical that sequential media will improve greatly the results, unless that particular media is very good. I think there’s a difference between one media and another media but as far as I know I don’t think there’s a big difference between single step media and sequential media.
I am going to IVF Spain for IVF with PGS in October. Is it possible to have a biopsy for the implantation window ahead of a frozen transfer scheduled for the end of November?
I would not dare answer this question because it would be more up to the doctor. You have to follow a certain medication plan to perform the biopsy and also for the stimulation and the PGS and everything. I think it’s more a question for the doctor to see how much it lasts to do all this endometrial preparation. I think we should leave this question to for the doctor later. I think all the questions will be forwarded to the medical team so you will get an answer.
It’s a follow up to my first question regarding the use of alternate lab techniques instead of ICSI. I have had three failed IUIs and now a complete failed fertilisation of eggs with ICSI this month. I used donor sperm which was good, so sperm is not the issue.
Well, it could be a failure of egg activation and this is not always due to the sperm. So calcium ionophore could be an alternative. It can also be due to bad egg quality. Right now I think that the only thing that could solve it is the calcium ionophore. Or otherwise the egg quality is so bad that it’s preventing the fertilisation.
What’s your opinion on group culture and single embryo culture methods?
I guess what do you mean by this is that the wells in the dish might be connected so the embryos in the dish might be sharing the media or the the wells in the dish might be separated so the embryos might not be sharing the media. I think when these dishes how have so much media at least the ones that we’re using, that I think there are plenty of nutrients for the embryo. The molecules the embryos might produce are quite diluted. So looking at the good results, I’m skeptical that it’s having bad influence on the embryos. I think there are many studies about this and it’s definitely worth giving a look into it. It’s an interesting idea.
In one of our last IVF we tried out PICSI. The spermiogram of my husband was 17 M/ml. After the binding to hyaluron, they didn’t find a single sperm to use for ICSI. Does this mean my husband’s sperm are all immature and what test could we do to find out?
It’s amazing that out of 17 million per millilitre, no mature sperm cells could be found. There are some tests that you can perform to evaluate the molecular quality of the sperm cells. You can perform for example apoptosis test or DNA fragmentation tests that might give you some additional information on what is going on molecularly inside the sperm cells. When you’re at the moment of a cycle and you have to find sperm cells, you’re running against a clock. You have to fertilise the eggs before a certain time, for example, you cannot do it at 8 o’clock at night if the egg retrieval was early. It has to be around 4 hours after the egg retrieval. So probably the laboratory haven’t had time to inspect 17 million sperm cells. They could have processed the sperm sample and looked into it several times. But maybe when you’re not expecting this, as it’s very unusual, you do not prepare enough time in advance. And having more time in advance could make it possible to find some sperm cell which is mature. It might be that the sperm cell is not molecularly correct, it might have been damaged at some point of the process. So maybe you could also consider using an alternative source at some point of the process. It’s a very interesting case and I would need to give it a long thought to know what to do.
My question was about time-lapse vs. single step media. Some labs have a practice of changing media on day 3 even in a time-lapse, because of the belief that endotoxins build-up over time. What is your take on that?
I don’t think there must be endotoxins floating around affecting the embryo development because we have good results. We are not changing the medium on day 3. My guess is since there is so much media in the dish, these endotoxins are really diluted. I’m guessing there are lots of studies about this and well if it’s the case, it could be an addition to the practice.
In your opinion, what is the hardest step in embryology?
I guess when you’re starting to learn a new technique, that technique is the hardest. But when some time goes by, everything seems to be easy. Actually I would say that the hardest job for an embryologist is talking to patients. It’s very difficult to put yourself in their place. Doctors have a lot of experience in talking with patients, they do so every day and it’s easier for them. But we are not really educated to do so. Patients sometimes have really devastating stories behind them and the news that you are giving them is very sensitive. I think for us this is really the hardest part of our job.
What is then the hardest step during the IVF from the patient perspective? Which step is the most critical?
For example, fertilisation is a very critical step. First it’s your expectation about how many eggs you are going to retrieve. Maybe you were seeing a number of follicles but not all of the follicles were mature. So there might be a change between number of eggs that you were expecting a number of eggs that are mature. The second cleavage point would be the fertilisation. This is also where some eggs can fall out, especially in IVF. Once the embryos start to develop, they usually do not block on day 3. The next critical point would be the development from day 3 to day 5. This is the biggest point where you lose a lot of embryos. Many embryos block from day 3 to day 5. They grow a lot, they grow from 8 cells to hundreds of cells. It’s like teenagers in their growth spurt ,it requires a lot of energy. It’s also the point where many embryos fall off. I think this is the most critical point where you can find the biggest decrease in numbers.
Some clinics use only donors that have children already. What difference does this make to the type of donor eggs produced?
Donors who already had children would be called donors with a proven fertility. You know they are fertile, they are able to have babies. But this doesn’t mean that they will be good donors. It could be that such a donor had a baby e.g. five years ago and her fertility has decreased a lot since then. So just because you have a donor with a proven fertility, it does not mean that she will be a good donor. On the other hand, now women in Spain are not having babies when they’re 18. So if you only took donors that have already had babies, you would lose a lot of good donors. Donors with proven fertility are definitely very valuable. But you would probably lose good donors if you put this as a rule to only take on donors who have already had babies.
What’s your opinion on group culture and single embryo?
I think we already came across a very similar question to this. I think it depends on the dish you’re using and how much media is on that dish. I think it’s different to have five embryos in 200 microlitres of media than to have only one embryo in 20 microlitres of media. Maybe there you’re culturing them in a single drop but they still have less nutrients then if they were sharing. I think it depends. But it might be a good idea.
Can you tell us what kind of training and experience a good embryologist needs to have? Do they have Masters as a minimum?
I definitely think it would be helpful if they had a theoretical specialisation in assisted reproduction because they need to understand what they are doing. What are the consequences that their decisions may have to the embryos, what are ethics opinions, what are endotoxins, etc. I think it’s important to have theoretical background to know what you were doing. And also the practical background of having worked in a real clinic with real patients.
In your opinion, are younger donors without children better than slightly older donors who have children? Does the age of a donor (a few years) make a big difference to the quality of eggs?
It depends on the age. If we’re talking about 24 and 22, then it would be better to take the donor who already has children. In any case, it would make very little difference. If we’re speaking about 30 and 26 , then I think it would be better to choose 26-year old donor, at least in terms of euploidy rate. But that said, some woman at the age of 26 might have very low ovarian reserve whereas others, at 30, might have super high ovarian reserve and maybe better quality eggs. So it’s something very intrinsic for every person. These are little things that we can look at and they will give us a hint. But it also really depends on the particular donor, her ovarian reserve, her hormones and so on.
I have 2 fertilised embryos. Would you go for blastocysts or transfer day 2 or 3?
To a certain extent, it’s also a personal decision. Scientifically speaking, I think the best option would be the blastocysts stage. Because if the embryo does not reach the blastocyst stage in the laboratory, it will also not reach the blastocyst stage inside of you. At least, you will know if your embryos are able to reach the blastocyst stage or not. This information is sometimes important because it helps you to know how to go on. We do see some patients who have done lots of transfers on day 2 and day 3 and they never became pregnant. Maybe if they have left their embryos to day 5, they would have known that there embryos didn’t have enough quality to reach the blastocyst stage and a viable pregnancy. But some people prefer to transfer blindly. Some people say: ‘Psychologically I do not want to see if it reaches the blastocyst stage or not, I just want to transfer it.’ We can respect that but yet personally I think it would be better to leave it to blastocyst stage. Like I said: if it does not reach the blastocyst stage in the laboratory, it will not reach the blastocyst stage inside of you.
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