PGT-A, or preimplantation genetic testing for aneuploidy, is a test that can be performed on embryos to see if they have the correct number of chromosomes. These embryos, also known as “euploids,” are more likely to lead to a successful pregnancy, which makes PGT-A a useful selection technique.
PGT-A involves biopsying a tiny piece of a blastocyst, freezing the embryo, and sending the biopsy to a lab to have its DNA tested. Once the results are in, the embryo is thawed and transferred to the uterus.
The decision to do PGT-A is usually made before an IVF cycle starts, but sometimes people may want to perform PGT-A after the embryos are frozen. This can happen in situations where multiple transfers have been made using frozen (untested) embryos, without any success.
But does thawing a frozen embryo to perform a biopsy, and then refreezing it and potentially thawing it again, compromise the embryo in any way?
In short, we don’t know for sure but it’s possible.
How embryos are frozen
These days, embryos are frozen using a process called vitrification. Vitrification is a rapid freezing technique used for embryos that prevents the formation of ice crystals, which can be sharp and can damage the embryo.
Embryos are frozen rapidly using liquid nitrogen, which is extremely cold. Once vitrified, the embryos are stored in liquid nitrogen until they’re ready to be thawed for a transfer, or for PGT-A in this case.
It’s possible that subjecting an embryo to a second round of freezing and thawing might compromise the embryo, particularly if it’s followed by an invasive procedure like an embryo biopsy.
There have been a number of studies looking at whether or not thawing embryos for PGT-A leads to worse outcomes. Let's look at a couple of them.
Should you do genetic testing on frozen embryos? Here's what the research says
In one study, researchers transferred a single euploid embryo that was either biopsied while the embryo was fresh or biopsied after the embryo was frozen and thawed. After the biopsy, both embryos were frozen. So the fresh embryo was frozen once and the frozen embryo was frozen twice (or refrozen).
They found that both of these groups of euploids had the same survival rates when thawed. This is good news because it shows that refreezing didn't kill the embryos.
As far as pregnancy, live births, and miscarriage rates go, there were no differences between the groups. You can see the results below:
Let's talk about this graph for a minute. Even though it seems like there are differences (the once-frozen miscarriage rate is about twice that of the twice-frozen group), none of these differences were statistically significant. This means that these differences were due to chance, and not due to the refreezing process.
There were 105 babies born from this study (45 from the once-frozen group and 60 from the twice-frozen group), and they found no differences in neonatal outcomes, such as preterm births, low birth weights, or birth defects.
This study didn't find much of a difference, so that's good news! But with science, there are a lot of variables to consider, and sometimes another research group may get different results.
For example, another study found a decrease in clinical pregnancy rates with twice-frozen euploids, but no difference in miscarriage or live birth rates.
This is confusing, I know, but common. Especially in studies that are small and not well-designed. Many labs just want to publish their data without much effort (or expense), which leads to an abundance of lower-quality studies.
Larger and well-designed studies hold more weight because the quality of the evidence is better. Unfortunately, for the particular topic of thawing embryos for PGT-A, there isn't much in terms of high-quality studies.
We can switch gears a bit and look at a more high-quality study but with a slightly different topic.
Does refreezing embryos, without the use of PGT-A, lead to worse outcomes?
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For this, we’ll look at what's called a "meta-analysis." This is a type of study that combines the results of multiple studies to see what the overall results are. These are typically higher quality because they include multiple studies.
One study did a meta-analysis on the subject of refreezing embryos. They combined the results of 14 studies that included 4,525 transfers (with over 1,000 using embryos that were refrozen).
This study included both slow freezing (an old way of freezing) and vitrification. Here's the results for when they only looked at the studies that used vitrification (based on 5 studies):
No difference in embryo survival rates.
No difference in clinical pregnancy rates.
40% reduced odds of live birth.
Unfortunately, there was quite a lot of variability between these 5 studies, as some included different embryo stages (blastocyst or cleavage stage, or both), or blastocysts from days 5, 6, or 7. This can all affect the results. Furthermore, 2 of the 5 studies found no difference.
All to say that drawing conclusions based on this meta-analysis isn’t easy either! That’s because it uses low-quality studies (garbage in, garbage out). So once again, bigger and better studies are needed to see what impact refreezing has on outcomes.
Furthermore, this meta-analysis involved the transfer of embryos that were refrozen and didn’t include any PGT-A cycles. So, the data from the meta-analysis may not apply to our original question of thawing frozen embryos for PGT-A.
The biopsy itself is a stressful process for the embryo, and it’s possible that an embryo biopsy combined with multiple freeze/thaw cycles may further stress the embryo and lower outcomes. But without good-quality data, it’s hard to know for sure.
As always, it’s best to consult with your doctor, as well as a genetic counselor, so they can review the available data in detail to make an appropriate decision.
Sean Lauber a.k.a. "Embryoman" is a former embryologist and creator of Remembryo.com, where he provides weekly summaries of the latest IVF research. You can also follow him on Facebook, Instagram and TikTok.