Post-Thaw Evaluation of Embryos

March 23, 2014Carole 109 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.

109 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.

  • 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!

  • 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 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

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