Post-Thaw Evaluation of Embryos

March 23, 2014Carole 146 Comments »

I received this email from a reader who wanted to know more about the process for thawing and assessment of embryos that have been cryopreserved.

This last cycle involved a FET of a day 5 and day 6 blastocyst from an earlier IVF that we were not able to complete a transfer for.  The embryos had been vitrified and when defrosted, we were told they did not expand, and remained shrunk.  The embryologist said there were a few cells in there that were still alive but their assessment was that these were not viable.  I haven’t found much on the web and I didn’t see it in your blog, about defrosting embryos, the process, and how an embryologist makes the call on a defrosted embryo, and how long the process takes, etc.  I did find a study that had compared transfer of blastocysts that had expanded and those that did not, and the pregnancy rate among those that did not was zero.  Thanks for considering this as a topic.

Taking a few steps back in the process, let’s start with the decision of which embryos to thaw. In speaking with other embryologists over the years, the majority or programs will as a matter of policy pick the embryos for transfer that scored the highest among the still living embryos. It is routine for some embryos to stop growing between fertilization and the time of transfer, probably due to genetic issues but not always. So, the remaining non-transferred embryos may have slightly lower quality based on having scored lower-assuming scoring is a good sign of quality–which is not always true. These possibly second tier embryos are frozen. Of course, if you have a large number of good quality embryos, you will be freezing high quality embryos too. The reason I say this is to explain that not all embryos start off equal at the time of freezing. Embryo quality is not improved by the cryopreservation process; the best outcome is no loss in viability.

The method used to cryopreserve the embryos makes a difference too in the quality (and hence the scores) of embryos post-thaw. In the bad old days, we used slow freezing which was relatively poor in preserving embryo quality. If better than half the cells in the embryo appeared clear and shiny after thaw- and thus alive- we called it a good outcome. Now, with vitrification, we expect to see the embryo reappear from the thaw looking as good as it did going in.

When preparing for a frozen embryo transfer cycle, the lab needs to coordinate well with the clinical staff so that the patient’s uterine lining is synchronized with the thawed embryo’s stage of development at the time of transfer. The embryo needs to be ready to implant and the uterus needs to be ready to receive the embryo. The “window of implantation” is only open during a few days in every menstrual or artificial cycle.

The clinical staff “runs” the cycle, educating the patient in how and when to take the medications that will make the uterine lining thick and ready for the embryo. Clinical staff will use test results from blood work and ultrasound visualization of the uterine lining to adjust medications and decide when the patient is ready for transfer. Clinical staff need to keep the lab apprised of the patient’s progress so that that embryo can be thawed at the right time. For instance, if the patient has embryos frozen at the zygote (fertilized egg stage), the embryo will have to be thawed several days in advance of the actual transfer to allow the embryo to continue to grow to the 8 cell stage (for a day 3 transfer) or the blastocyst stage (for a day 5 transfer). If the embryo was frozen as a blastocyst stage embryo, it will be thawed close to the time of transfer, at most, the afternoon before but most likely the morning of the transfer day.

When vitrification is used, the term “warming” is used instead of the term thawing. This naming convention probably arises from the fact that the process of vitrification bypasses ice crystal formation, moving the embryo right into a solid phase- glass-like -state. Ice thaws but glassified embryos warm from a colder state to a body temperature state.

In any case, the first step in removing any embryo from storage is verification of identity of patient embryos and physician  orders to warm the embryo. Every good clinic will have a process to ensure that the right patient’s embryos are removed from storage and among those embryos, that the right one or two embryos is warmed and readied for transfer. All storage systems have some method of identifying embryos that is physically adhered to the smallest storage unit- the straw or the vial. Often, a waterproof permanent marker is used to print patient name or and ID number or both with date of freeze or other info on the straw or vial. Newer methods use bar codes. In any case, the right straw is recovered from the cryostorage tank. A second pair of lab eyes must verify that the right cryostorage unit is removed or thawed at one or more stages of the process. At transfer, usually someone at the clinic and lab side verifies that the right patient is matched to the right straw.

The process used to freeze the embryo dictates the process that must be used to thaw the embryo. That is why when frozen embryos are shipped between labs, thawing or warming instructions must accompany the embryo so that the receiving lab can recover the embryos successfully.

No matter the method used, the embryo is thawed or warmed and transitioned from being immersed in media that is incompatible with life and growth to being moved into a nutritive, warm, proper pH culture medium for a period of hours or days to recover and then grow. In the case of the day 5 or day 6 embryo, the embryo would typically be thawed in the morning of the day of transfer, especially in the case of a day 6 embryo because day 6 embryos really can’t tolerate much more culture time- they need to be moved to a uterus. If you look at pregnancy rates for transfer of day 5,6 and even day 7 embryos, by day 7 the pregnancy rates have dropped to unacceptably low levels (less than 10%). Whether this is due to the fact that culture conditions aren’t optimized for what the day 7 embryo needs or because there was something wrong with an embryo that was growing so slowly that it needed 7 days in culture to become a blastocyst isn’t entirely clear. But I think most clinics would agree that when you thaw a day 5 or 6 embryo, you want to give it a few hours to recover and re-expand, then transfer quickly.

Blastocysts have a large space in the middle of the embryo that fills with fluid. This fluid-filled space or blastocoel is not helpful for vitrification and embryologists have found that if you make a tiny hole in between two cells to let the fluid leak out- collapsing the embryo- the embryo will vitrify more effectively.  Blastocyst stage embryos are often “collapsed” on purpose before freezing to allow the cryopotectant agents to infiltrate the cells in the embryo more effectively.

After warming, we expect to see the embryo re-expand and re-fill that space with liquid. Re-expansion is an active process that requires energy to pump fluid into the cavity and requires that the embryo be alive. So failure to re-expand is of concern. What is not clear is how long should we give an embryo to re-expand before we consider it to be dead? Often times, because we want to thaw and transfer quickly, we might only allow the embryo a very short time, an hour or less to recover in culture medium –and this may not always be enough for an embryos to re-expand. The re-expansion of a live embryo will happen in the uterus after transfer if the embryo is alive but we don’t get to see and document that.  I am not sure that anyone has ever determined what the average time for human blastocyst re-expansion is.  But a failure to re-expand by the time of scoring–perhaps just one scoring event right before transfer-  may or may not indicate that the embryo is non-viable.

For a good idea of some of the factors that effect the outcomes of thaws, you can read this article by clinicians at the Cleveland Clinic’s IVF program, which covers many of these issues in greater detail.

The scoring methods used to assess embryos after thaw are the same ones used to assess fresh embryos. I describe the Gardner method with pictures for scoring blastocysts in this previous blog post. The problem with blastocyst scoring of post-thaw embryos is that the scoring  includes an assessment of the size of the blastocoel. A blastocyst with an collapsed blastocoel will score more poorly and it might not be obvious whether this will be corrected by full expansion in just a few more hours- or not. So scoring post-thaw is problematic.

What embryologists like to see in an embryo that is reviving from cryopreservation are shiny clear cells, not dark opaque cells. Dark opaque cells are dead. Clear shiny cells are usually alive. This sign of life, while not as definitive as active re-expansion, might be all that can be scored immediately after thaw or warming, when the transfer procedure is imminent. Also, the time taken to score an embryo is a matter of seconds. Scoring is accomplished by a quick visual of an embryo that is rotated in culture under a microscope by the technician using a sterile pipette so that all sides are visible and takes less than a minute. The new real time continuous imaging of embryos in culture from fertilization to blastocyst stage using systems like Eeva, will change many of our procedures. Visual manual scoring techniques may be replaced one day by algorithmic scoring of real time embryos but most clinics currently use the old inexpensive methods.

For now, your embryologist will likely still need to make a visual assessment and make a decision based on past experience to decide which embryos survived thaw and are suitable for transfer. More often than not, this will be enough to pick viable embryos.

 

 

© 2014, Carole. All rights reserved.

146 Responses to this entry

  • Carole Says:

    Hi Elizabeth,
    The blastocyst has two working parts- the inner cell mass which is a group of cells which give rise to all the cells of the baby and the trophectoderm cells which surround the inner cell mass and give rise to the placental cells. For an embryo to continue, it must have sufficient cells in each category to survive. It is hard to know what the minimum number of cells are. I wish you MUCH GOOD LUCK for your transfer. Carole

  • Amanda Says:

    Hi Carole

    I just had 2 frozen 5d blastocysts transfered. They don’t look anything like my last embies I transfered. Can I send you photos to get your opinion of why they look the way they do?

  • Carole Says:

    HI Amanda,
    I really don’t feel it is fair to judge photos because in the lab, I can roll the embryo around and get a look at it from all sides. You can’t do that to a photo of an embryo. Your embryologists should be able to discuss this with you. Please ask them for the fullest, most accurate assessment. Good luck on your transfer! Carole

  • toniann Says:

    Hi there-
    Does it mean anything if a blastocyst is graded poorer than when it was originally frozen?
    IE- the blastocyst was 4AA when frozen and then 3AA when defrosted and transferred.
    Thanks

  • Carole Says:

    Hi toniann,
    Not necessarily. After thawing, embryos slowly regain their full expansion so a slightly lower score may reflect an earlier stage of expansion.
    I wouldn’t worry about that too much. Good Luck!! Carole

  • Zuza Says:

    Hello Carole,

    We are doing IVF in Czech and we have one frozen embryo aCGH tested and graded as HB/2.
    What do you think about this aCGH testing (I am a little suspicious because the price was very low).
    Also, what HB/2 means? Does HB stand for hatching blastocyst? What about 2?
    My coordinator is not very responsive and I am loosing my mind trying to figure out if we have good embryo…

    Thank you!
    Zuza

  • Adrienne Says:

    We decided to freeze embryos left over from our fresh transfer. The Doctor suggested to let them grow till day 6, to see if they got to blastocysts stage. They did, one was 5AA and the other 6AA. My wonder is will the already hatched embryo survive the freezing and thawing?? your thoughts?

  • Carole Says:

    Hi Adrienne,
    Yes, they can survive vitrification (one type of freezing) very well. They need to be handled very gently during the embryo transfer to avoid splitting can cell loss. Good Luck!
    Carole

  • Carole Says:

    Hi Zuza, HB may mean Hatched Blastocyst but you will need to get the embryologist to speak with you if possible so that you can better understand the clinics grading system- it may be unique to that clinic. Good Luck!! Carole

  • Nana Says:

    Hello Carole,

    My doctor decided to thaw our day 3 embryos (7 & 8 cells grade 2) to blastocyst stage before transfer. Is this advisable as I only have 3 embryos from 2 IVFs (egg quality problem)? I am panicking that I may turn up at the clinic with no blastocyst to transfer.

    Thank you.

  • Carole Says:

    Hi Nana,
    You don’t say how many embryos were thawed. Thawing and culturing to day 5 is done for several reasons, 1) to check and see if they are capable of continuing to grow (viable), 2) to select the best among many (too many to transfer) or to 3) biopsy some cells and run a genetic test before transfer. If you want to be absolutely sure of a transfer, you would transfer them immediately upon thaw, but run the risk of transferring non-viable embryos. I hope you did have some survivors/growers to day 5 and I wish you all the best!! Carole

  • Catherine Says:

    I have one frozen fully hatched blast left from my last ivf that was done in May, 2016. I got 5 blast and transferred 2 fresh one of which became my son. I transferred two this past September and one took but I lost it at 8 weeks. I now have the one blast that was fully hatched when it was frozen. I’m wondering if the blast was already fully hatched when frozen, if it will be ok to transfer once thawed? Or should I just do another fresh cycle because my last fresh cycle resulted in my son?

  • Carole Says:

    Hi Catherine,
    I might want to see what the already hatched blastocyst can do, but there are some things to consider. If the blastocyst is fully hatched, it has lost its shell, which makes it a little more vulnerable in handling and it might not do as well or it might split, increasing the risk of identical twins. I would discuss this in detail with your doctor to see what their experience has been with these sort of embryos. We have had implantations and live births from these but it is less than ideal for handling, but on the other hand, this embryo has demonstrated good viability by getting to hatching stage. Again, I would ask your clinic about there experience. IF they are not comfortable with handling these, a fresh cycle might be best. Carole

  • CuriousIVFer Says:

    How common is it for a blast to increase in grade after thaw? We had a frozen 2bc day 5 blast that became a 3bc by the time we transferred. (+ hpt then – beta)
    Now hoping for next fet the frozen 2ab to become a 3ab….is this common?

  • Suhani Says:

    I have 2 expanded blastcyst frozen with 4BB, 4CC grade (i.e. Poor Quality embryo). No AA Quality embryo available for transfer….. Is there any chance of pregnancy with this poor quality embryo……

  • holly Says:

    Hi!

    I just did our last FET transfer. It’s a PGS male.

    He is blastocyst and they said he is fully, 100% hatched…is this bad? Are these way more fragile or are the success rates similar to hatching ones? (my last was starting to hatch and he took)

  • Holly benson Says:

    Sorry if this is a duplicate.

    On 12-15 we rransferret a healthy pgs tested 5 day blast that was 100 percent hatched.. is that bad he was fully hatched? She didn’t seem concerned but it seems like that might have been not ideal?

  • Carole Says:

    Hi Holly,
    Don’t worry. Fully hatched is fine. They are a little more delicate to handle but they are just as viable. Good luck!! Carole

  • Carole Says:

    Hi Suhani,
    There is always a chance. The first positive pregnancy –which produced a beautiful child!- from an embryo in the lab was with an embryo that received the lowest possible score. The appearance of an embryo is much less important than the chromosomal quality of the embryo. If you are under 35, you have a better chance, even if the embryo receives a lower appearance score. The fact that they made it to expanded blastocyst is a very good sign. Good Luck!! Carole

  • Carole Says:

    Hi CUriousIVFer,
    You will need to ask the lab to explain the scoring system they use. The 3 means fully expanded vs a partially expanded 2- assuming they are using the Gardner scale :https://www.advancedfertility.com/blastocystimages.htm
    That’s why it’s important to ask the clinic or lab to decode the system they are using for you. Good Luck! Carole

  • Jessica Says:

    Hello,

    I have 3 embrios frozen together. Grade AB, BB and a blastocyst. When they are thawed and let’s say only 1 or 2 are transferred, will the remaining one be damaged from the thaw and Re-freezing it?

    Thank you!?

  • Carole Says:

    Jessica,
    If they were cryopreserved using vitrification- which is the method most commonly used now- they tend to tolerate re-freezing very well. The older slow freeze methods killed a percentage of the embryos cells with each freeze, thaw and re-freeze. Good Luck! Carole

  • Becky Says:

    On my second FET this month, I had three 4AA embryos fail to thaw successfully before a 4th thawed to its original 4AA grade and was transferred. (FET #1 was also a successful thaw of a 4AA embryo, but didn’t result in pregnancy.) I’m fairly certain that they’re using a vitrification process, and so this failure rate was a big shock. My doctor said she’s only seen this once before, in another patient with PCOS. Any thoughts on why this might have happened? Is it possible that the lab screwed up (either in the freezing or the thawing)? Or is it just bad luck? I’m really worried because I have only 2 4BB, day 6 embryos left, and now I’m concerned that neither of them will make it through the thaw, and I’ll have to go through another IVF cyclel, which, I was hoping I wouldn’t need to, since I started out with 5 4AA and 2 4BB embryos frozen.

  • Carole Says:

    Hi Becky,
    I am sorry you are having such a hard time. It is impossible to dissect what might have gone wrong from this perspective–could be any number of things. I would ask your doctor to do the analysis and ask them what they will do differently next time to try to prevent the same outcome. On the positive side, you still have two embryos to transfer so I would still be hopeful. Good Luck!! Carole

  • The Biology Behind the Fertility Clinic Meltdown | PLOS Blogs Network Says:

    […] the temperature slowly rises, and after a few hours at body temp, embryos can be implanted if they look okay – they’ve refilled the fluid-filled space at their centers and the cells are clear and not […]

  • Jay Says:

    Hi there,
    This is a fascinating thread!
    I have a very quick question…My clinic thawed 2 of my 5 day embryos last Thursday afternoon and transferred both on Friday lunchtime.

    So would embryos be day 6 at the moment of transfer? Or does the re-expansion process cancel out the thawing time?

    Confused!
    Thank you 🙂

  • Carole Says:

    Hi Jay,
    The thawing time is usually just a few hours for thaw and re-expansion, then the embryo continues on it’s merry way. While frozen, the embryo is in full metabolic stop so there’s no metabolism, no growth. If your thaw of day 5 frozen embryos was in the afternoon last Thursday, the embryos would have started being fully metabolically active and continuing to grow in a few hours (less than 3 hours probably), so it is fair to say they were day 6 embryos about 24 hours later when they were transferred. While my lab preferred to transfer day 5 embryos, many labs report as good or nearly as good results with day 5 and day 6. It may be due to differences in the preferred culture medias in each lab. Good Luck!!!
    Carole

  • Rachel Says:

    Hi Carole, your website is decimating! Thanks for doing this. My question is about the changed quality of frozen embryos. Our only embryo was a day 6 3AB and when they thawed it and transferred it, it had become a 4BC. They said it was pulsing. AB seems a lot better than BC and I’m comcetmed it didn’t survive the thaw properly. Is this common?

  • Carole Says:

    HI Rachel,
    This previous post might be helpful: http://fertilitylabinsider.com/2011/12/understanding-the-gardner-blastocyst-grading-scale/
    There was a loss of cells moving from a 6 to a 4, but if the embryo is viable, it can recover, after transfer and implantation. The first IVF pregnancy we had was with an embryo that scored extremely poorly, but the baby was beautiful and healthy. This taught me an important lesson about embryo scoring–it is not absolutely predictive of outcome. Good Luck!!
    Carole

  • Erika Says:

    Hi,
    I did a FET on May 14th. I previously talked to my embryologist because we thawed all 6 of our embryos and performed PGS testing on 6 day embryos. 4 came back genetically normal and 2 were undiagnosed. They all survived the process of thaw and refreeze but the 2 undiagnosed were of poorer quality and the embryologist didn’t seem confident in their success for a FET.
    We transferred our best embryo back but when they gave me the picture it looked completely different than my previous transfer. It was not very expanded and appeared compacted and darker in color. I spoke to the embryologist and he said he had not concerns but did explain sometimes they can collapse and re-expand. I am not sure if he saw it fully expanded and then it collapsed or if the embryo simply wasn’t fully expanded at the time the picture was taken.
    Any thoughts?

  • Erika Says:

    UPDATE: I am pregnant from the embryo that had collapsed post thaw. This was an embryo that was frozen, thawed, biopsied, re-frozen, and re-thawed for transfer. This little guy is a tough embryo to go through all of that.
    Thank you for this thread and being so reassuring the collapsing and re-expansion is a normal part of this process. Hopefully he will turn into a beautiful baby boy in 9 months.

  • Erika Says:

    2nd Update: It was a chemical pregnancy with Beta confirmation. I do not know the statistical chances of this happening but the embryo obviously implanted but failed. Could this have been due to environment or embryo?

  • Christy Says:

    Hi. I did my first FET today with donor 5 day blastocyst. Doctor said 95% of cells survived the thaw. Embryo was an expanded AA at vitrification. Can you explain what it means that 95% of the cells survived, and does that reduce our chances for a healthy pregnancy and a healthy child?

  • Carole Says:

    Hi Christy,
    95% cell survival is fine. You should be in pretty good shape, as far as the thaw went. Good Luck!!
    Carole

  • Carole Says:

    Wonderful news Erika!! You made my day!! I wish you and your lovely embryo all the best in the months to come!!
    Carole

  • Laura Says:

    Hello. I just did my first FET and have some concerns. The frozen embryo that we transferred was a grade 3 8 cell embryo frozen on day 3. When they thawed the embryo the grade remained the same, however the cells dropped from 8 to 3. Does this dramatically reduce the chances of a viable pregnancy? Should the transfer have even been performed?

  • Alexandra Says:

    Hi there,
    I’ve just done a FET, 5 day Blast.(Grade A, B?) Before the procedure I looked at the picture of my Embyro and it looked as though is was hatching out of the shell. So I asked if it were hatching, and they said no. That it was a hole where they put the needle in so the fluid would drain out. Q: IS THIS NORMAL process in lab that every Embryo thats vitrified gets a needle poke or just a one off they did on my blastocyst? I don’t know how I feel about this.

  • Carole Says:

    HI Alexandra,
    Yes, it is common to artificially collapse the blastocyst before vitrification. An ICSI needle or laser can be used. Read more about it here: https://www.liebertpub.com/doi/pdf/10.1089/cpt.2008.0007
    It helps the vitrification process be more thorough, and the cells do better. I think you are in very good shape with a day 5 blastocyst, grade A or B. Good Luck!! Carole

  • Lucy Says:

    Hi Carole,
    I have day 5 frozen embryos. I wish to cryoship them overseas for thawing, biopsy for PGS testing, then re-freeze, and hopefully do FET of healthy embryo later..This means transferring frozen embryos to a different lab overseas, and then they will be subjected to a double freeze thaw (they are non-hatched blasts, so will need thawing, expansion, and
    ?hatching, and biopsy/PGS)…Do you think this is too risky????

  • Carole Says:

    Hi Lucy,
    This plan has a lot of interventions. While re-vitrifying after thaw, culture and biopsy is not uncommon–and in a skilled lab, can be done without apparent detriment to the embryo– the shipping step adds yet another opportunity for a problem to arise. I would do it if I know I could do a fresh cycle again later if something happens. In the fresh cycle, you could plan to do the PGD up-front when they get to day 5, then freeze them once, if you need to because PGD results are pending. That would be ideal. I am not saying your plan will fail, it’s just a lot of steps. Good luck!! Carole

  • Lucy Says:

    Thanks carol. Is there any way to assess the skill of the lab?
    Are there different methods of vitrification between labs which might affect the method of thaw?
    Thank you.

  • Luis Says:

    Hi Carole, my wife had a FET of a 5AA blast about a week ago. Today we recieved a report from the lab updating the grading to 3AA. The photo pre – freeze shows that it had started to hatch. However, the photo post warming and pre – transfer is considerably different – it’s entirely enclosed, it doesn’t look as though it ever started to hatch. My wife is afraid the lab has frozen and tfrd the wrong embryo (we also had a 3ab but after pgs was determined non viable). Have you come across this significant change in appearance of the embroyo?

  • Carole Says:

    HI Luis,
    The embryo picture can look different before freezing and after thaw- especially if it hasn’t finished re-expanding. I wouldn’t worry about the appearance too much. The embryo can continue expanding -even after transfer– when no one is looking. Good Luck! Carole

  • Carole Says:

    Hi Lucy,
    You can ask the lab if they have published their pregnancy rates for vitrified embryo transfer. You can also see frozen embryo transfer results from US labs at http://www.sart.org. Most labs are doing vitrification – but labs vary in their preferences when it comes to their freezing mediaum, containers, tools etc , but chemically/physically vitrification works the same. Good Luck! Carole

  • Mariya Says:

    Dear Carole, I had one frozen embryo top quality 6ab, frozen after PGD. After defrosting, there was a totally different form like half of the embryo was missing. there was also no outline shape that is visible on the picture of the embryo before it was frozen. they told us it’s no vital and there was no transfer. Is this possible to happen?

  • Marie Says:

    Hi carol had a top quality 5 day blastocyst thawed today they said over 60% survived and was multiplying and taking on fluid well.. I hadn’t expected this I thought it was alive or dead . So I don’t know how to feel. It was transferred to me straight away. But no I’m searching, if it is successful will can there be DNA problems or other issues? Or does it simply regenerate as new inside me ???

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