Quick Tips for perfect Vitrification

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Cryopreservation of oocytes or embryos is a critical step in maximizing the efficiency of any IVF cycle. Achieving clinical success with cryopreservation is highly variable from laboratory to laboratory, and may depend on many factors including patient age and stimulation protocol, quality of embryos selected for freezing, developmental stage at freezing, media formulation including type of method used, parameters of cooling and warming, and type and quality control of programmable freezing unit employed. The latter problem, however, occurs only at slow cooling, as vitrification does not require any fancy equipment.

All through our time in and outside of the laboratory, during discussions, we have heard vitrification is so difficult. It sometimes makes us remember, old stories foretold to us by our elders coming through generations, which may or may not always apply same way today. We do continue to listen and follow most of these stories but not all. Similarly, it is not necessary to listen and follow every story told to you. If you believe it without trying,  you will lose the battle even before it starts. Believe us, vitrification, is not difficult. Having said this, yes, we agree that there are certain critical steps you need to focus on and keep in mind and the rest just follows through. Vitrification is a much newer concept in embryology as against the conventional slow freezing technique. Not only is slow freezing a relatively long process, but, research has also shown the newer vitrification technique has a higher survival rate as against slow freezing. The preparation needed for slow freezing are also a handful as against the newer vitrification technique. From a learning perspective, we will urge everyone to know how it was done since it will add value to your understanding of vitrification as well. For instance, the importance of avoiding vapour exposure before direct contact with liquid nitrogen. The fundamentals still continue to remain the same and will until some research proves what we have been doing for all these years could be done even better. Even if such a concept is proven or not, if your passionate about embryology and do not consider it as a job, please read and understand the concept of slow freezing as well.

To begin with, vitrification is a technique and freezing and thawing a process and hence it is important to know, thawing is also called vitrification by the process of thawing. Those of you, who use any software for storing your data, please notice that the system will show vitrification or slow freezing as the options while you enter data for both freezing and thawing. The reason to stretch this point is because, it is important to know what you are doing. Methods developed for vitrification in embryology use a combination of these two possibilities. There are multiple vitrification kits available in the market, proven to give the same results. Each laboratory adopts the use of any one of them due to their experience and trust on that kit. Some of the commonly used kits include, Kitazato, Irvine Scientific, Origio, Life Global to name the most common. Even in vitrification, though it is a newer concept, it could be either an open system vitrification or closed system. The main difference between the two being; after loading the embryo, in an open system liquid nitrogen comes in direct contact with the embryo. Whereas, in a closed system, you have to cover the loaded embryo with a cap and then immerse it in liquid nitrogen. Both these systems are widely accepted and there has been no consensus on which is the best vitrification kit. Whichever be the system being used to vitrify, it is important to follow the kit’s protocol consciously. The secret ingredient to a good vitrification is 3P’s – Practise, perseverance and patience.

Essentially any vitrification cooling kit contains 3 solutions; 

  1. basic solution (BS),
  2. equilibration solution (ES) and
  3. vitrification solution (VS)

Similarly, any vitrification thawing kit will contain; 

  1. thawing solution (TS),
  2. dilution solution (DS) and
  3. washing solution (WS)

The key to a good vitrification freezing program is understanding the principle, proper adherence to protocol and lot of practice. Vitrification thawing is a relatively easier process and is the reflection of a good freezing process. If you do not freeze well, the survival will also be low, on the other hand, if you freeze well, the survival will be good.

Principle

Vitrification can be defined as an extreme elevation in viscosity, i.e. solidification of solutions without ice crystal formation at low temperature. This phenomenon can be induced by either applying an extreme cooling rate or by using high concentrations of cryoprotectant solutions. Although human embryo and oocyte vitrification was slow to evolve, it has become an invaluable technology in the field of reproductive medicine.

Vitrification uses a higher concentration of cryoprotectants, avoids ice crystal formation, and places embryos in a glasslike state by reducing the chilling and osmotic injury seen in slow-cooling methods. The elimination of this type of injury may increase their chances for survival. The high toxicity produced by increased concentrations of cryoprotectants is avoided by quickly loading embryos into special straws, cryo-loops, or droplets and directly plunging them into liquid nitrogen. Additionally, the time required for equilibration and cooling is considerably reduced.

Tip no 1 – The ratio of freezing

If we have not yet noticed, it is important to notice the difference in size as the egg becomes and embryo and an embryo becomes a blastocyst. As the transition takes place the size keeps increasing and thus we use different sized denupets to handle each of these. However, at the same time, it is important to remember that the size of the device (cryotop/cryolock or cryo-loop) does not change. Thus, this means, as the size goes up we need to reduce the number we freeze on 1 device. As per good practice, it is advised to freeze in the following order:

Oocyte/Embryo # on each device
Oocyte Up to maximum 4
Day 3 embryo Up to maximum 3
Day 5/6 blastocyst Up to maximum 2

We have been part of practices, where 1 vitrification freezing device had 7 oocytes or 5 Day 3 embryos or 4 Day 5 blastocysts. Such practice is conducted most commonly when ;

  1. The clinic is trying to cut costs since each of these devices is an expensive consumable item
  2. Clinics which have free-lancer embryologist and thus are time bound due to other commitments
  3. Clinic which conduct batch IVF cycles, thus making time management difficult leading to such short cuts

You may be able to save some money and time, but there will surely be an impact on the frozen gametes or embryos. The post thaw survival benchmarks are set as per the best practices shared above in the table. Changes in the above scheme, will have an impact on the percentage of survival and we already know the reason for the same.

Tip 2 – Media left on the device

The number of oocytes/embryos loaded on the device is also associated with addition of more media. The technique of vitrification demands leaving as less amount of media as possible surrounding the oocytes/embryos. More media left on the device can cause the either sticking of the oocytes/embryos or raising the chances of ice crystal formation. Ice crystal formation can  In either case could result in non-survival of the frozen material. Thus it is important to drain off as much media from the device as possible. Another way to reduce the media would be not to use a larger bore sized denupet than required for loading. So for Oocyte to use 135 micrometer, cleavage stage 175 micrometer and blastocyst 275 micrometer.

Tip 3 – Start with Day 3, Day5 and then oocyte

The principle of vitrification demands, removing maximum water content from the oocytes/embryos. The failure to do so, could put the oocyte or embryo in an osmotic shock and can result in lysis of the same. We may not be able to see this lysis until we thaw, and hence it is necessary to observe the same while vitrifying. Thus it is advised, if you are beginning to vitrify, it is important you start practicing and performing Day 3 embryos first followed by Day 5 blastocyst and then oocytes. The prime reason for this is the water content in each of these stages. Oocytes have the highest water content followed by blastocyst and then Day 3.

Tip 4 – Immediate immersion in Liquid nitrogen

As suggested earlier, it is important to adhere to the protocol stringently. Thus as per each protocol, you will get approximately 60 seconds to load the material on the device. Once this step is done, you have to ensure device either open or close has to be immediately dipped in liquid nitrogen. Quick immersion will ensure we skip the prolonged exposure to vapour and avoid any change in temperature, thus enhancing the technique and resulting in better survival rate.

Tip 5 – Document your observations

Along with following the timeline and technique of vitrifying, it is extremely important to document your observations of the process. For e.g.; if you took more than usual time to load the oocytes/embryos or the shrinkage did not happen and the reason you think could cause it. These may not be important at the time of vitrifying, storing but extremely important to analyse if the oocyte/embryo does not survive.

Thus following the above mentioned steps along with the protocol mentioned in your kit, should help you upgrade your process of vitrification. If you are beginning your training in vitrification, then all the above mentioned points are applicable. If you have been performing vitrification for a long time, this could serve as a checklist for you to see if you have not missed any of the above. We are sure adopting the above mentioned tips will bring a positive change in your technique and its outcome.

 

FREQUENTLY ASKED QUESTIONS (FAQ):

Q. I have been vitrifying for sometime, but sometimes it takes me more than one minute to immerse the cryotop. Is that okay ?

The ideal time is 1 minute, However if you need to make a decision between time and draining the media on the cryotop always drain the media even if it takes a little more time. In my experience I have seen embryos in which I took a little more time to load and drain the media survived better than the ones in which I immersed in nitrogen on time but more media.

Q. How do I know if the media is adequately drained from around the embryos?

This is very subjective as their is no calculated quantity but you know the media is drained well if the embryos are together and there is no trail or pool of media around them.

Q. We frozen two embryos on a cryodevice, the patient now is adamant to transfer only one embryo as they do not want twins. what should we do?

Firstly, you need to check whether they are cleavage or bastocyst grade embryos and their quality. If they are good grade blastocyst embryos you can re freeze one after explaining the pros and cons to the patient (Possibility of embryo not surviving). However, if the two embryos are of cleavage stage two things can be done 1. Counselling the patient that the probability of twins with transferring cleavage stage embryos is much lower. 2. Transfer one cleavage stage embryo and take the other to blastocyst stage. If the embryo reaches blastocyst stage it should be vitrified at that stage.

Q. At what stage should the embryos be frozen?

You should ideally freeze the embryo on the stage that you hope to transfer at. For instance, if you plan a Day 5 FET for a patient, do not freeze the embryo as Day 3 as you will have to thaw and take the embryos to Day 5 for a possible transfer. This will have the following disadvantages 1. You froze all embryos on Day 3 and ended up using more media and cryodevice and increasing the cost to the centre significantly. 2. The patient is ready for a transfer but you do not know if and how many embryos will reach blastocyst stage.

What details should be mentioned on the cryodevice?

Labelling on the cryodevice is very important, Each cryodevice and the lid should have the full name of the patient and her partner, Date of freezing, Unique identity number of the centre and the quantity and stage of embryos loaded on the the cryodevice. Trust me, there is enough space to mention all the information.

Q. I have three A grade embryos , 2 B and 1 C (All blastocyst stage) How should I vitrify them?

Whatever the stage always remember, you must put the best embryos in the first cryo device, second best in the second cryodevice and so on. It is important to understand that each transfer that you give to the patient has to be her best. There is no point in transferring average embryos in the first FET and saying the best for the last.

Q. How many embryos should be put per cryodevice ?

As mentioned above, the cut off of oocyte, cleavage and blastocyst is different. However, the number put per cryodevice should be discussed with the clinician and patient before freezing. For instance you have two blastocyst and according to the cut off you can freeze upto two blastocyst on one cryodevice, but if the patient is sure that they do not want twins and want only one embryo transferred per cycle then you should be freezing one blastocyst per cryodevice making it a total of two cryodevice.

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