Embryology lab – complete guide on IVF Lab techniques and procedures

Tomas Rieger, PhD
Head Embryologist

Embryo Implantation, Embryo Transfer

Embrylogy lab guide on IVF lab techniques and procedures #OnlinePatientMeetings
From this video you will find out:

In this Online Patient Meeting you will find answers to these questions:

  • what pre-selection method is optimal for me?
  • can Embryscope/Time Lapse help select an embryo?
  • can it help in terms of embryo development and quality?
  • what can you do in the IVF lab to help with the male factor?
  • does EmbryoGlue work?


Embryology lab – complete guide on IVF Lab techniques and procedures

Watch the recording from the online patient meeting with RNDr. Tomáš Rieger PhD, Head Embryologist at Gynem IVF, in the Czech Republic. Tomáš answered patients’ questions about procedures and techniques that are used in an IVF laboratory.

- Questions and Answers

What pre-selection method is optimal for me?

The most important thing is the quality of the semen sample, which we get from the patients on the day of fertilization. According to the quality of the sample, we can choose one of the four main researching methods which we have in our lab. Some of them are focused on sperm where we have more than a thousand million per millilitre like Fertile chip and MACS. Every research method has some logic behind it. Fertile chip system or like pre-selection method is focused on the motility of the sperm. When we conceive naturally – only the sperm with the best motility can get to the oocyte. The second method is MACS system, but again to use this, we need to have good quality sperm and many sperm in the sample. It is focused on dividing the sperm, which are design to apoptosis in the body because the body itself has few systems on how they can solve or see if the cells inside are good or bad. We have these systems, like all human. One of the systems is phosphatidylserine system which can be marked like the bad quality cells or sperm as well, but white cells can be somehow checked, and if they are not good enough – the white cells will make some extra mark on this cell. This mark is a small molecule of phosphatidylserine, and then other white cells which are going through the body, they are looking for some cells which are not good enough. When they see the cell marked with phosphatidylserine, it will destroy it. This system can be applied on the semen sample, and we can divide the ones with phosphatidylserine mark on it, and they can be put away. So only the sperm without this mark will be used for the fertilization process – this is the MACS system. There are two more systems methods, which can be used even with a lower quality of semen, a small number of sperm in the semen sample. One of them is called PICSI method, it is focused on the maturity of each sperm, and it can be done on a 100 sperm. PICSI method works with a drop of hyaluronic acid, which is quite a common molecule in our bodies. In the fertilization process, the surrounding cells of the oocytes are also covered in hyaluronic acid. In normal fertilization, the sperm has to be attached to the hyaluronic acid for the process of capacitation and then across embryo reaction and, when the sperm doesn’t have this receptor ready for the attachments to the hyaluronic acid, this sperm cannot fertilize the oocytes at all. This method can show which sperm has this receptor, and this can be then used for fertilization. But nothing is optimal in life, and there can be mistakes. Sometimes we can see that on this hyaluronic acid there is also the sperm that doesn’t look very well. In general, if the receptor is there, the sperm should be fine, but as I said nothing is hundred-per cent. This might be my least favourite method, but someone else might have good results, so we do offer it as well. The last method offered to patients is IMSI, which is focused on the morphology of each sperm. We can magnificate the sperm up to the 6000 more than normal which can allow us to see the inner quality of the cytoplasm in the head of the sperm, and we can see some problems in the shape of the head, some morphology issues in the neck, in the tail etc. When we have many sperms, and some of them have morphological problems, we can choose the best-looking sperm. If the sperm looks how it should look, hopefully, the DNA will be fine as well. Unfortunately, none of these methods can show us the genetic information itself. And it is the most important thing, but there is no method now in the field that can show us the genetic information and not destroy the sperm. Hopefully, in the future, a better method will be developed.

Can Embryscope/Time Lapse help select an embryo? Would it help in terms of embryo development and quality?

Yes, the EmbryoScope or any time-laps operating systems can get us useful information about the development of the embryo. In the classic cultivation, f.e. we have 6-7 embryos in one well, and they are still developing all these five days, and after these five days we see 3 or 4 good-looking blastocysts, we will choose one of the best one for transfer, and other ones for freezing. In EmbryoScope or other time-lapse monitoring systems – all the embryos are cultivating in separate wells, so we can perfectly tell after these five days of cultivation how this special embryo develops throughout the whole time. It can happen that, let’s say on the 5-day embryos, three of them look pretty much the same, they have good quality and when we have classical cultivation, we only select one, which I think is the best at the moment of the transfer. When we see the time-lapse footage of each embryo, this system takes a photo every 10 minutes of the embryo development. After these 5 days, we have a whole movie of embryo development. That way, I can see which day-3 embryo was the fastest one etc. , so I can select the perfect embryo. I will be able to see the time, speed of splitting etc. , all the steps of embryo development will be recorded.

What can you do in the IVF lab if Male factor is involved? What’s the difference between techniques like IMSI, PICSI, MACS etc.?

It depends on the quality of the semen sample for sure. When we say male factor, it means that the sperm is not in the normal range. The norm is 15 million per millimetre, so more than 40% should be motile, there should be less than 96% of the pathology of the sperms. When we’re talking about male factor, it usually means semen samples with only 100 thousand, perhaps few million, and in this cases, we use IMSI and PICSI procedures. We can combine them as well, but keep in mind that in patients with male factor issues, it is important to understand that sometimes we cannot find the sperm in the sample at all even if it has the best quality. We may find the best-looking sperm from the sample but, when we compare it to normospermia, it might not be good enough and but there might not be any better sperm in the sample. Our goal is to find the less defected sperm, less damaged sperm from the whole sample and wait to see if they will be good enough to do the fertilization. We can also repeat the cycle or do 3 cycles, but sometimes it can still mean that sperm donation would be a better option. We don’t have any methods to improve the sperm, and we have to work with what we have, then choose the best and see how it works.

There’s a lot of information on the forum about how EmbryoGlue helps patients. What’s your opinion about it or similar products?

Embryoglue contains hyaluronic acid, and one of the advantages of this molecule is that it is sticky. With EmbryoGlue we cultivate the embryo, two hours before the transfer we put it in the embryo glue medium, and it will cover the zona pellucida, which is protecting the embryo from the outside world. This embryo glue will be stuck on the zona pellucida from the outside then it is transferred to the uterus, and it should prevent moving the embryo to the cervix or fallopian tubes in the opposite direction. It will decrease the chance of embryos falling to the cervix. On the other hand, when you see the lining of the uterus under the microscope, it looks like there are big tunnels where the embryo is placed, and there is almost no movement there, the cells are sticky. Another thing is that when the embryo is hatching, which is an optimal phase for transfer, it can be helped with laser-assisted hatching. If patients ask us what we can to improve the transfer – I would recommend more using the laser-assisted hatching. When the embryo is going out from the zona pellucida, the cells are sticky by itself, so there is no need for them to be even more sticky. I’m not a fan of EmbryoGlue, that’s my opinion. When we check our statistics, and we are using the EmbryoGlue from 2013, and we never had better results because of this. At times, perhaps it was 2% more, another year it was 2% less, so it is not something I strongly recommend.

Is AH (Assisted Hatching) anything I should consider? There are clinics Rusing AH for frozen embryos, and there are also clinics using AH for fresh embryos. What’s your opinion? 

I work in this field for more than 10 years. I started in 2009, and when I was starting working in this field, the assistant hatching was quite a new method. And at that time everybody hoped that the assisted hatching will increase the success rates. Thanks to this method we open the zona pellucida, we can make the hole in it by the laser, and when the embryo is inside the zona pellucida, we can get it out without any problems. On the other hand, truth is that if the embryo will be out, and if it will be successful outside of it, this is a different question. In 2012 a worldwide study was done which showed that the results weren’t somehow better. So after this study, many IVF labs stopped offering this method. Here, at our clinic, we do perform it. I recommend more to do it on the frozen embryos. The vitrification is not really a natural process, it’s something that the embryos are not used to, and the vitrification process can affect the chemical compounds and chemical hardness of the zona pellucida itself. It can happen that after freezing, the zona can be a little bit harder than in the fresh one, and in this frozen cycles, we are sometimes helping the embryos to get out from the zona pellucida by making the hole for the embryo. On the other hand, the statistics in our lab or from abroad are not showing any big differences. In the fresh transfers, I believe that the embryo has many steps before that to become a successful pregnancy, and I can help embryo with the first step to open it, but I believe the embryo should be strong enough to do it on its own. Therefore, in the fresh transfer, I am leaving this on embryos, we’ve seen the embryos that were hatching on their own many times, at the time of transfer which is around 120 hours after fertilization. Around 30-40 % of embryos are hatching on its own, and if not I am leaving it with the fresh transfers.

How do we choose embryos for transfer?

Sometimes, after a five days cultivation, we have 1 or 2 blastocysts, so there is nothing to choose from, and the patient wants only one from those two, so I will choose the faster one. Usually, the most important thing about embryo development is speed. When I see that one of the embryos is bigger, so it has more cells, it grows faster – I will choose this embryo. We are grading the embryos in three ways: we check the size, speed of the development and the quality of the inner cell mass and trophectoderm cells. If I have two embryos or more embryos, I choose the one that has the best potential. The benefit of transferring the blastocyst is when we have them in the time-lapse monitoring system where if I have 2, 3, or 4 same looking embryos on the day of transfer, I can play a movie of their development, and I can check the morpho-kinetics parameters. In the Embryoscope we have the artificial intelligence which helps us to choose embryos, it usually chooses the same embryo as we do in the lab, but if somebody is not experienced enough, this can help.

How do we freeze embryos and oocytes for future usage?

When I started working as an embryologist, there was only slow freezing method available, for embryo development, and this method was of good quite good for pronucleus which means zygotes, for cleavage embryos up to the morula stage. At the blastocyst stage, it wasn’t working well at all. It was not possible to freeze the oocytes with this method. When the vitrification, which is a fast-freezing method was developed we were able to freeze the blastocyst, oocytes. You need to remember, the embryologist needs to be experienced because the vitrification process is quite demanding when it comes to hand skills. After 2 years of doing vitrification, I can say that I’m good at it, it takes many years of repeating it, to be sure you are doing this in the best possible way. If you have an experienced embryologist, the survival rate is 99%. The outcomes from the frozen transfers are the same. In our clinic, during these recent years, our results are the same as the fresh and frozen transfers. So the vitrification method is very important in the IVF field. Some clinics start to do only frozen transfers because it seems that the outside stimulation process can affect the quality of the lining of the uterus, and the whole process of implantation can be shifted by it. In our lab, we offer fresh cycles as well, but when somebody is not feeling well after the stimulation and egg- retrieval, it is better to freeze the embryos and do a frozen transfer. In recent years, social freezing has become very popular, which is understandable, and it gives women a chance to have better outcomes when they decide to have a baby a bit later in life.

How can we tell that the donor oocytes are the quality ones?

The age of the donor is one of the most important things. The younger the donor is, the better quality oocytes she will have. But it is still biology, so let’s say everything works fine, we have a young donor, 10 donor oocytes were fertilized, and from 10 embryos – we got like 8 blastocysts, and the outcome was successful. And yet next time with the same donor, and same good quality-semen etc. the result is different. Fortunately, we have in our lab, a new possibility to see if oocytes are ready for fertilization. It’s called a spindle observation system. Sometimes under a standard microscope, we can see the oocytes with a polar body, they look like they’re mature and ready for fertilization. But when you can see the inner quality of the position of the spindle, it might be possible that the process of maturation of the oocytes isn’t completed yet. So you can inject sperm into oocytes because you think they are mature, but when you have the spindle observation system, you can see that the oocytes will be ready for implantation f.e. in two hours so this system can be very helpful in all cycles. Thanks to this, we can find the most precise time for the fertilization.

What is beneficial in your fertility centre in comparison to other ones.

I’ve worked previously in two different labs, two different IVF clinics, and both of them were bigger than our Gynem clinic. I can see the main difference, is that in those bigger clinics, there is not much time for communicating with each patient. We are really focused on communication with patients, so the patient has time for the questions on the day of fertilization, their oocyte pick up, then again they can ask whatever they want to know during developing time, throughout those 5-days, and on the day of transfer, we meet them personally once again. We discuss the results, what we are recommending. What I hear from the patients is that they appreciate a lot this kind of approach, many of them had a bad experience with some of the bigger clinics. It is crucial as the whole IVF process can be very stressful, and if you are well-informed and updated, you feel calmer, and that can also provide better results in the end.

Does the camera in the time-lapse incubator of the embryos taking so many pictures cause any stress to them?

That was one of the negative things at the beginning, if we are taking photos with flash, will it do some damage to the embryos. When it was counted how much light they get, it turned out it is only a few microseconds. Compared to classical incubation, it is indeed a lot less harmful to the embryos.

How long should we thaw blastocysts before transferring?

It’s recommended that the incubation should be at least two hours before the transfer. This is optimal we are doing it a little bit for a little bit more than 2 hours but not less. Because some blastocysts in the freezing process are shrunk by itself. Some will be fine after half an hour, while some other ones will be good after 2 hours. Most of them are fine after 2 hours, but some need 4 or 6 hours to expand. But there was no big significance in the statistics when we did check the groups fully expanded and shrunk.

Some clinics still have frozen embryos by slow vitrification, but unfortunately not in a cleavage state, only in a blastocyst one. Which chances have such embryos to survive after thawing in a %?

When I was working in the lab for the first time, there was only a slow freezing method available. We counted that it was around 30 -40 per cent of survival rate. Then from these survived embryos, only around 30% led to successful implantation. With the vitrification method, the survival rate is more than 95%, and around 45% of these embryos are successful. A blastocyst 6BB has already been hatched? Does it make sense to transfer such an embryo? As I’ve said, the optimal faze for the transfer is hatching. Naturally, the embryo is going through the fallopian tubes and falls into the uterus, where it has to hatch by itself, and then only the hatched cells can do the implantation. When we have two embryos in the lab, and one of them is fully hatched – number six means it is fully hatched, and let’s say the second is 4AA so it’s of better quality, but it is not hatched – I would choose the 6BB because it will have a higher chance of success.

If EmbryoGen is used until Day 3, does BlastGen need to be used to culture up to Day 5? What is the difference between both culture mediums? I am asking because in a recent cycle, the clinic only had EmbryoGen (not BlastGen) and none of the embryos we had at Day 3 made it to Day 5.

EmbryoGen is focused on the cleavage stage, and BlastGen is focused blastocyst stage. I don’t really think that the reason the embryo stopped developing from day-3 to day-5 was because they don’t have the BlastGen. EmbryoGen medium should be fine enough for reaching the blastocyst stage as well. So, BlastGen could help the embryos, but I’m not sure it will definitely help to reach the blastocyst. Possibly, it was a different problem, because it is known that on day 3-4, the genetic information from the sperm is involved in the process of development, and sometimes that may cause for the embryo to stop developing if the semen was of poor quality f.e.

If I choose an African donor. Can it happen by mistake that a different, Caucasian donor will be used by mistake?

It should never happen. Here, in the Czech Republic, we have very strict rules in quality management in the lab. We have many witnessing systems, how we should be sure that this mistake will not happen. In the lab, there are machines, but of course, there is always this human-error possible. There was one of such situations in the Czech Republic where the donors were switched in IVF treatment, so I can’t say there is 100% guarantee something like this will not happen, but I know I never experienced it. Keep in mind, that if a good clinic would make such mistake, it could be the end of such clinic, therefore, it is very important to follow all the steps, have everything signed and checked twice.

The best sample of my husband sperm is from 12 years ago (which was slow vitrified) when he was younger and will be used with 24yrs old young donor. Will their sperm be a problem because it was slowly vitrified as we don’t want to use recent frozen samples as the quality is not as good.

The sperm is much less complicated cell than oocyte, so the slow-freezing still works fine, we also still use this method in our lab. If I should compare a fresh sample with the frozen sample with the same quality, the fresh will be always better because the freezing is not something natural. But if the quality of the sperm was much better 12 years ago, the chances for good results will be better. After 12 years of frozen sperm, in terms of time, it is like 12 minutes later. If they were better quality then, it’s better to use it than the fresh one.

How many hours after IVF insemination do you transfer the inseminated eggs in the time-lapse dish?

For the IVF insemination, there have to be cumulus cells around the oocytes because without these – sperm is not incapacitating. So in these cases, we are doing this after 18 hours. We are trying to do it as soon as possible, but it can happen that we are missing the pronucleus in these IVF procedures. The truth is that in our clinic we are doing 99% of ICSI. I had a few issues with some patients who had quite good quality sperm, many oocytes, and they chose to do IVF fertilization, but the result was that from 15 oocytes – 3 of them were fertilized. We have a fertilization rate when it comes to ICSI at 85%. I know that when I put the good-looking sperm into 15 oocytes, I will have at least 10 embryos from the ICSI procedure. I always recommend patients who are undergoing this IVF treatment, when they realize they need this artificial help, to do it properly and maximize the chances.  I believe we did the IVF insemination in time-lapse once, and we placed the embryos the next day, after 15 hours.

Do mosaic embryos happen often? What is your opinion about such embryos?

Mosaicism happens quite often in an embryo. This is quite normal phenomena, and this is one thing which I don’t really like about the PGT screening. I recommend doing the PGT screening, in cases of some monogenic or inherited diseases. If there are some structural problems, like translocation, but just for checking the aneuploidy. Sometimes, we have only mosaic embryos, and it doesn’t mean that the embryos are bad or good from this result, so this is a field which is still not examined. There is lots of research done on this, and I believe that possibly in the future, the PGT-A screening and will be used less because the information we get from this can be confusing at times, we can throw away embryos that are perfectly fine. It’s very important to know why you need this. If you are 42 years old, then, of course, it is a good reason to do it or f you had some miscarriages, again it is a good reason. But if a patient is only 32 years old and wants to know to be sure that the embryos will not be aneuploid, I don’t recommend them to do the genetic testing.

Do you have known donors and is surrogacy legal in the Czech Republic?

In the Czech Republic, there is only anonymous egg donation, and surrogacy is not illegal, but it is not legal at the same time. It’s like a grey field, but because it’s not illegal, it can be done, so we are doing this even in our clinic sometimes. There has to be a strong reason for it, I know in some other clinics they are more open to doing the surrogacy, we are not that keen on this. About known donors – there is a big discussion on this in the whole EU, and possibly in a couple of years, the egg donation will be known in whole countries, even in the Czech Republic.

Do you have guarantee live birth programs? What about using a surrogate who are screened by your clinic?

We do not have live birth programs at our clinic. 60% of our patients are coming from abroad, most of them stay in the Czech Republic during the treatment, and they go back home. So let’s say a miscarriage happens, we cannot monitor such patients there, they would need to stay in the Czech Republic for 9 months. It the patients come to our clinic with the surrogate mother they found somewhere else, we do not recruit surrogate mothers ourselves. We will screen her, we will do the ultrasounds, check the uterus, and if all is fine, we can do the transfer to surrogate mothers.
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Tomas Rieger, PhD

Tomas Rieger, PhD

RNDr. Tomáš Rieger PhD has operated in the field of embryology since 2009, working alongside and under some of the finest embryologists in the Czech Republic. A qualified biologist with a Master of Science (MSc) degree from Charles University of Prague, and a Doctor of Philosophy (PhD) degree. RNDr. Tomáš Rieger PhD. is a certified clinical embryologist through the European Society of Human Reproduction and Embryology (ESHRE) in London and has attained high proficiency in an extensive array of laboratory techniques, including intra-cytoplasmic sperm injection (ICSI), embryo and oocyte vitrification, and laser application in pre-implantation genetic diagnosis (PGD) and pre-implantation genetic screening (PGS). Presently, Tomáš leads a team of four in the laboratory where, as lab head, he is responsible for ensuring practices are up to date and in keeping with state regulations. Amongst his personal interests are travelling, nature, cooking, tennis and golf. He understands German and speaks English fluently.
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Caroline Kulczycka

Caroline Kulczycka

Caroline Kulczycka is managing MyIVFAnswers.com and has been hosting IVFWEBINARS dedicated to patients struggling with infertility since 2020. She's highly motivated and believes that educating patients so that they can make informed decisions is essential in their IVF journey. In the past, she has been working as an International Patient Coordinator, where she was helping and directing patients on their right path. She also worked in the tourism industry, and dealt with international customers on a daily basis, including working abroad. In her free time, you’ll find her travelling, biking, learning new things, or spending time outdoors.