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About rehydration times after OPS vitrification

Again a question from an embryologist: why do we suggest 5 + 5 min washing/rehydration in Human Holding Medium after OPS vitrification (and warming in 1M, and initial incubation in 0.5 M sucrose)? Some other companies with a similar cryoprotectant composition and concentration suggest only 5 + 1 min rehydration times in the last two sugar-free media.

Well, we are always ready to learn from others. In this case, however, the given company was the one that learned the composition and concentration of vitrification media from our OPS vitrification published twenty three years ago. They also kept learning from us the safe storage method, i.e. wrapping their devices in pre-cooled 0.5 ml straws after immersion into LN2. Unfortunately, they reported this solution as the product of their creativity - not mentioning that precisely the same double-straw technique was published by us 15 years earlier and used in many labs worldwide subsequently before the respected company "invented" it.

In this case, although we understand that time is essential in a busy IVF unit, we do not want to follow the newcomers' modification. Our reason is quite simple.

DMSO and EG are the least toxic permeable cryoprotectants used in embryology; still, they are not entirely harmless. The required intracytoplasmic concentration is high, although much lower than in the initial vitrification methods, and - in contrast to the common belief - not higher than that during traditional slow-rate freezing. After warming, this concentration is present in the cytoplasm (and in the blastocoel of blastocysts) and requires time to leave, mostly passively, during rehydration.

At the same time, oocytes and embryos have a seriously compromised metabolic state, and regeneration also requires time. Not just minutes, most probably several hours. If traces of DMSO and EG are present in the cytoplasm during this period, the regeneration is delayed, resulting in cell death, or compromised subsequent development.

I realized the need for thorough washing after cloning and activation. After exposition to DMAP, we wash reconstructed cattle embryos three times for 5 min in a holding medium before culturing in vitro. This step usually happens late at night, and after a hard day's work, embryologists want to go home and sleep. So, the last 5 min washing was sometimes shortened or even skipped for some embryos. Fortunately, we cultured embryos individually or in small groups, and the difference between the first and last series was quite shocking. After a few fiascos, we had to realize - tired, hungry, sleepy or not; we HAVE to do washing thoroughly. Otherwise, your whole day's work may be lost.

If this happens with DMAP, it may happen with DMSO and EG, as well. I started to focus on the situation after vitrification and warming - and found out some unexpected issues.

It has been revealed that thorough washing is essential. The lack of it may not lead to dramatic differences immediately, but the outcome's consistency will be lower. In case you skip the last 5 min, embryos will be more sensitive to the situation after vitrification. If you culture them in small drops individually; or in large volume, but in groups, close to each other, the survival will be compromised. Also, the anecdotal but most probably existing differences in sensitivity between developmental stages will have their effect. Superficial rehydration of oocytes may be more harmful to oocytes than to advanced stage embryos. Still, blastocysts and expanded blastocysts may not be very happy with your impatience, either.

One may refer to the direct transfer situation, when embryos are put into the uterus minutes after warming, in sucrose solution (I mean in cattle). Well, that is an entirely different issue; in that case, we are fortunate. Unlike the Petri dishes, the uterus provides an excellent environment for safe and efficient rehydration, with proper solution replacement. Also, the process seems to be consistent and slow - no hurry, no stopwatch, no schedule. And in humans, with the patient, with the operation theatre, this precise timing arrangement is quite demanding and risky.

In our labs, there are hundreds (underestimation!) of factors that may cause inconsistent outcomes. We have to do our best to decrease the risks. You work with human embryos - you risk human lives. To create these embryos, a concentrated effort of many people (prospective parents and their families, doctors, embryologists, nurses, administrative staff, etc.) was required. We are talking about 4 minutes. To eliminate even a tiny chance for failure - probably worth waiting. Results may not be stunningly different, but you may have some improvement in the consistency in the long term.

And - your conscience will be clearer.