Vitrification of embryos and oocytes

Jose Luis Pablo Franco, MSc
IVF Laboratory Director

Egg Freezing

Vitrification of embryos and oocytes
From this video you will find out:
  • Do the embryos lose any possibility to achieve a pregnancy after warming (thawing) them?
  • When is the best moment for fertility preservation?
  • What do you think about cumulative cycles of oocytes in poor responders?
  • In oocyte donation, what do you prefer, fresh oocytes, frozen oocytes or it doesn’t matter?


Egg and embryo vitrification

Watch the Online Patient Meeting with Jose Luis de Pablo Franco, the IVF Laboratory Director at Fertility Madrid and ART Vitoria in Spain. Jose Luis answered questions about the vitrification of eggs and embryos from the embryologist’s point of view.


Jose Luis de Pablo Franco has been working in the embryology field for 20 years and has explained the differences between the old technique called slow freezing and vitrification. When we’re talking about vitrification, we talk about a much higher cryo concentration of cryoprotectants, we’re talking about a much higher cooling rate, and that means that we’re not going to have ice crystal formation. That was the problem in the past. The issue with the slow freezing was the transformation of a liquid into a solid with the formation of ice. It is essential for the ice not to form inside the cell. It can damage the embryos, or it can lower their quality.

The time it takes to freeze the embryos with slow freezing was about 120 minutes, which means that it took a lot of time to freeze the embryos, while the vitrification technique takes only 12 minutes. The main and the most important difference between these two techniques is the survival rates. In the slow freezing technique, the survival rate was 60%, in oocyte verification it’s about 95%, and with embryo vitrification, we’re talking about almost 100% of survival rate.

I always say vitrification is the greatest discovery in the 21st century because it has allowed us to make the cycles more affordable. We’re going to have more embryo transfers if we have frozen embryos, with the slow freezing technique because of the survival rate, it was not possible.

The embryos are stored in the tanks with nitrogen, they are kept well organized, and straws have different colours to identify them easier. Luis also presented 2 published papers that he took part in as well. One of them talks about the deleterious effect of estradiol hormone stimulation on embryonic implantation. The other describes 2 groups of patients, one with fresh embryo transfer and the other with warmed embryos. The paper showed that there are better results with wormed embryos, not because of the vitrification itself, but because of the first paper mentioned. It turns out that increasing levels of estradiol hormone stimulation can affect the implantation rate because it directly affects the embryo. Therefore, at Fertility Madrid, fresh embryo transfers are not performed, the embryos are vitrified and then transferred. ‘Vitrify all’ approach seems to be improving IVF outcomes.

- Questions and Answers

How long can the embryos/oocytes be stored?

They can last forever. The thing is that the tanks that we use, they need maintenance, and I would need to check them every day, and we need to see the level of the liquid nitrogen, just to keep them safe and better, but they can be a stored for a long long time.

Do the embryos lose any possibility to achieve a pregnancy after warming (thawing) them?

First, we need to achieve a good survival rate. That’s the first thing that we need to do, that’s why I said we need a good protocol, we need good vitrification program to achieve results close to a 100% survival rate. After that, we’re talking about achieving pregnancy. So the answer to this question is we don’t lose any possibility because if the embryos survive, they have the same possibility to implant as the fresh embryos. When we have frozen embryos, we’re going to transfer them in an endometrium, that doesn’t have any effect of the stimulation.

When is the best moment for fertility preservation?

I mean now with this question, we’re going to talk more about oocytes. I would say the best moment for fertility preservation would be 25 years old. We all know that young women between 25, they don’t think about this kind of treatments or something like that. This is like the last thing that they’re going to think about. So I always say this should be a present from the grandmas to their granddaughters. The grandmas could pay for a treatment like this, for a fertility preservation treatment. The thing is that you need to see every case. Patients under 35 that could be a good age for fertility preservation, so if you’re thinking about doing it, you should do it before you’re 35 because so many patients at the age of 40 don’t have as many oocytes or the quality is diminishing. They wished they knew there is something like fertility preservation back then.

What do you think about cumulative cycles of oocytes in poor responders?

This is a really good strategy in our daily routine. From the economic point of view, we’re going to have more oocytes to get embryos in the end. In our clinic, we don’t recommend this. If, patients are over 38, or 39 their quality of the embryos can be compromised already. So if we vitrify them, the only thing that we can be doing is decreasing their quality so damaging them, so that’s why we don’t recommend it. Sometimes, they don’t have a partner, and they are working really hard, or they just don’t think about having a baby now, and the thing is that you need to tell them that at the age of 39, it’s not a good technique to do because instead of preserving those oocytes we’re damaging them. To answer this question, that is a really good strategy, but you have to know the age of the patient, and you have to explain to them the problems if they are over 38-39 that we can find. Perhaps, we will vitrify 3 oocytes, and in another cycle, we’ll vitrify 3 oocytes as well, so we’ll have 6 but maybe what we have been doing is that those 3 if we used them as they are fresh, maybe we could have some embryos. By vitrifying them, we could be losing the possibility of having some embryos.

In oocyte donation, what do you prefer, fresh oocytes, frozen oocytes or it doesn’t matter?

When we’re talking about oocytes donation, we prefer to use fresh oocytes, we have our own donors, and we use fresh oocytes as we prefer them. In cases, that we have a particular phenotype requirement, we need to buy them in a bank, but we prefer to have our own oocytes, that way we can perform ICSI with fresh oocytes. I prefer fresh oocytes because you need fewer oocytes to achieve a pregnancy. When you freeze the oocytes, you always need more oocytes to have the same results.

What are the chances of success with frozen eggs with slow freezing versus vitrification?

I’m not sure if today there is somebody that still uses the slow freezing method. For sure, the results are much better with vitrification. Oocytes are more difficult to vitrify than the embryos because of the size of the cell. We’re talking about one big cell, and the thing is that we need to dehydrate the cell, so all the water needs to go out and what needs to go in, is a cryoprotectant. That’s the problem with this low freezing, with the slow freezing we’re not able to take all the water out. The problem is the ice crystal formation inside the oocytes, and that can damage the oocytes, and so the survival rate will be much lower.

You mentioned that eggs/ embryos can be frozen indefinitely. Is there any medical evidence that shows this? In the UK we are trying to get the law changed beyond the current 10 years freezing. My 10 years have expired, and I am trying to build medical evidence to support a case to use my eggs.

I mean vitrification is not an old technique, and that’s true that we don’t have embryos vitrified for a long time, but I know that there are reported cases where with this slow freezing, they have a healthy baby born after a lot of years of that freezing.

Unfortunately, we have gone through 10 x IVF failures, 8 with my own and 2 with an egg donor always resulting in miscarriages. With our next donor (and this time surrogate) my husband and I would like to use his slow frozen sperm from 2007 (in Australia) as he was 42 then. My problem now is the clinics in Ukraine won’t accept this sperm because they didn’t do an infectious diseases test within 2-3 months of that particular sample which, Ukraine requires. The closest report is 7 months from the time of that sample. As we had done 6 treatments previously they did not do this every time. My husband has had done many infectious diseases tests since then, which proves he doesn’t have any syphilis etc., but they still won’t accept this sample. Unfortunately back then it was slow frozen. Is it possible to have this sample tested? We have also had his sperm analyses by Professor Don Evenson who invented the DNA Fragmentation test, and he said to only use this sample in comparison to another 2 samples we have. The reason we don’t want to use a fresh sample is that older men’s sperm might result in autism, dwarfism. 

The thing is that we make like newest sperm every 72 days, so that’s not really a problem. When it comes to the oocytes and the age, it is a great problem, but when it comes to sperm, it is a lot different. I don’t know why they don’t want to accept the sperm that you have frozen, because I am sure when they’ve frozen the sperm back in Australia, they performed all the tests for the infectious diseases. But even if your husband is 50, I don’t see a problem with using a new sperm sample. When we’re talking about sperm we can test the chromosomes, we can test the DNA fragmentation, but we don’t really see if we have any infectious disease in that sperm. Once you have your sperm frozen, the quality of that sperm is going to be damaged, so you better not do too many things because you’re not going to have enough sample for ICSI or other technique that we’re going to use.

I am 33 years old and have a low ovarian reserve of 0.6, I have done an IVF with 5 follicles, all fertilized but with qualities C and D. Three days before the puncture the size was 26, 21, 18 and 17, it could be that the quality was compromised by waiting too long for a puncture? Were they very large? What is the right size to schedule a puncture at 3 days?

I would say the one that was 26 maybe is a little bit big. We normally plan 2-3 days before the oocytes retrieval. We like to have not more than 21-22 follicles, depending on the number of follicles that you have. Sometimes, you need to lose one, like this one f. e. sometimes you need to lose that one. I guess you have 5 follicles and 5 oocytes, 26 one is a little bit too much, but 21, 18, 17 they’re correct. The question on if the quality was compromised by waiting too long, I don’t think so, sometimes with this size, you can not even retrieve it, you just lose it. In this case, as you are 33, it is very good, and the problem is that your ovarian reserve is low, and the quality is not good because it is C and D, we don’t use the D for transfer, we could only use the C, but I don’t think this was the problem. With just one cycle, we cannot say that neither the quality was bad nor the follicles were too big, and that’s why you have Bad quality embryos.

What is the difference in the success rate of achieving a birth with vitrified oocytes versus fresh oocytes?

Like I said, here we prefer to use fresh oocytes. A lot of clinics, prefer to bye the oocytes from the banks because having own donors is always a lot of work, you have to do a lot of things to have a good donor program. We always prefer that because as I’ve mentioned you will always need more vitrified oocytes to achieve pregnancy. The success rates are about the same with fresh oocytes. The only difference is that we need more oocytes.

Is a 37-year old frozen egg better or a 40-year-old fresh egg in terms of quality?

If you are 40 years old woman and you have a very good response, you can get good quality embryos. I would always prefer the 37-year-old with frozen eggs than a 40-year-old. Because of the chromosomal abnormalities that you’re going to find when you are over the 39. This is the problem with the age of the oocytes which means the number of chromosomal abnormalities that you’re going to have in the embryos as a result. Most of our patients are over 38, so that’s why we perform the PGD which analyzes the embryos because we have a lot of embryos with chromosomal abnormalities and that results in no pregnancy, or even if you get a pregnancy, you can have a miscarriage more easily.

What is the best time oocytes freezing after egg collection?

We perform the oocytes vitrification 2 hours after the oocytes retrieval. We prepare all the media, we prepare everything in our cabin, and we accumulate the oocytes for 1 hour and a half, and in 2 hours they should be already verified. Once you want to use those oocytes, we warm the oocytes, and we wait 3 hours after warming to perform the ICSI. We do that because that way the oocytes can recover and the quality of the embryos is better.

Just to clarify if I managed to transfer my eggs over this year to Spain I would hope to get the treatment done asap (dependant on travel restrictions being uplifted into Spain. I certainly would aim to do before I am 50 in July 2021 but if the first transfer isn’t successful and embryos are still there then can I use them until successful?

That depends on the clinic. We cannot perform embryo transfer over 50, but like I said if you do a transfer and you achieve a pregnancy, and we’re talking about the next year, I don’t know that depends not really on the embryologist but more on the gynaecologists because they are the ones that see the problems when you get pregnant over 50, we’re talking about obstetric problems etc.

How many more oocytes, in general, do you need to achieve a birth with vitrified oocytes versus fresh oocytes?

In general, you need like 7-8 oocytes to achieve a pregnancy, and when we’re talking about vitrified oocytes, you need like 12 depending on the age. If we’re talking about oocytes from a donor we can say maybe 6 fresh oocytes, then 8-9 vitrified oocytes.

Is there a certain kind of food that he can eat for him to have a good quality sperm?

It is something that a lot of patients ask, and my answer is no, not really. They are always talking about the DHEA, folic acid that you can find in Fish, or there are some pills that you can take, but you don’t really increase the quality of your sperm. I mean you can try it because this is something that is is not bad for you or your partner.

I’m 44 do you think I can use my own eggs for IVF?

I would like to tell you, of course, use them. I mean we have to see what is your ovarian reserve. If you can have a good number of oocytes, you can try, but the possibilities to get pregnant are very low. In your case, I would use the PDT-A to study the chromosomal abnormalities in those embryos, and the thing is that we have different possibilities. After the oocytes retrieval, we need to have good quality embryos, after that, we need to perform the biopsy PGT-A, so pre-implantation genetic diagnosis and see which embryos are normal or which ones are abnormal. The first possibility is if we analyze 3-4 embryos if none of them is normal, we cannot have an embryo transfer. The second possibility is more common, we will analyze the embryos, and the possibility to have some normal embryos is very low too, and if so, we’ll transfer a normal embryo.

Should I still use the frozen eggs even if they were frozen by the slow freezing method?

Yes, I mean you have them there, so you have to try even though if they were frozen with an old technique, you should try and you see what you have. I mean you don’t lose anything trying to do it.

Is there a significant difference in transferring vitrified embryos in the clinic where the embryos were vitrified versus a transport and transfer of the same embryos in another clinic? Can transport and defrosting procedure in another clinic harm the embryos?

The transport shouldn’t affect the quality of those embryos or the possibility of those embryos to implant. If we’re talking about warming, defrosting procedure, we’re talking about different things. You have to make sure that in that clinic, they use the same protocol or even the same media because we have a lot of different media from different brands. You have to make sure that they know how to use that procedure in that clinic because that could harm the embryos for sure. With the transfer, I don’t see any problem because if you have a good tank for transport and you have liquid nitrogen inside, this shouldn’t be a problem.

What do you mean by different media being used?

I mean we’re using different brands, every media has a different protocol. If you bring your embryos to my clinic, if I don’t use that media, I’m not sure if I’m going to be able to do it correctly because of the different protocol.

How many times is safe to take IVF injections and not be worried about the risk of ovarian cancer?

This is more of a question for the gynaecologists, but as far as I know, there is no risk for ovarian cancer. We normally use the amount of hormones that are not too high. In the past, it used to be higher, but now it’s much lower, and I wouldn’t worry about it.

What is a good number of eggs for 38 years old?

This is a difficult question to answer because sometimes you only need 1 egg you know with that egg, you have a really good quality embryo, and you don’t need anything else. Sometimes, you need more depending on the quality of the eggs and then the resulting embryo. It’s not something that I can really answer. Sometimes, even younger patients with 20 oocytes, they didn’t achieve the pregnancy. In general, a good number of eggs it would be 12, that’s a good number, in general for all the patients. We always aim for a good quality embryo, not a good number of eggs.

How are vitrified embryos stored, is it in straws like sperm?

The devices that we use for vitrification are not really like in sperm, they have like a plastic at the end, where we place the embryos, and then we put it right away in liquid nitrogen. So they’re not in the straws like the sperm. The way we do it nowadays, so with vitrification, the results are better because you’re putting the embryos in contact with the nitrogen.

We have vitrified embryos in a clinic in Spain and would like to change the clinic. We are not satisfied with this clinic, we are struggling with transferring the embryos to another clinic. The clinic does not want to collaborate. Are they not obliged to collaborate, do they have some duties? Who is responsible for organizing the transport? We were told we cannot order transport being private persons, only the clinics can do it.

This is something that we normally do. The thing is it cannot be transported by a private person, it has to be done between clinics. They have to collaborate with this. The only thing you have to do is to go to that clinic and say that you want to take my embryos and I want my embryo to be transferred to another clinic. You have to sign the consent for that and then once you have signed, we talked to each other, and we’re talking between labs and try to organize the transport. This is something that we do quite regularly so they should collaborate for sure. It doesn’t make sense that they are collaborating with this.
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Jose Luis Pablo Franco, MSc

Jose Luis Pablo Franco, MSc

Jose Luis de Pablo Franco is the IVF Laboratory Director at Fertility Madrid and ART Vitoria in Spain. He holds a Degree in Biology and Biochemistry from the University of Navarra and a Master’s degree in theoretical practice and procedures in Assisted Reproduction laboratory techniques from the University of Valencia. Jose Luis has Senior Clinical Embryologist Certification from the European Society of Human Reproduction and Embryology (ESHRE) and ASEBIR certification in clinical embryology. Jose Luis de Pablo Franco has more than 15 years of experience managing In Vitro fertilization laboratories (IVI Bilbao, Quirón Bilbao, and ART Vitoria).
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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.
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