HOW I IMPROVED MY VITRIFICATION TECHNIQUE !

HOW I IMPROVED MY VITRIFICATION TECHNIQUE !

SAIPRASAD GUNDETI, SENIOR EMBRYOLOGIST, MALPANI INFERTILITY CLINIC PVT. LTD.

Vitrification involves freezing the embryo about 600 times faster than ever before. This ultrarapid process is so fast that it literally allows no time for intracellular ice to form. As a result, vitrification avoids trauma to the embryo.
In conventional (slow) freezing, 20-30% of embryos do not survive the freeze-thaw, and those that do survive have less than half the likelihood of generating a pregnancy as do fresh embryos. In contrast, vitrified embryos have a better than 95% freeze-thaw survival rate, and a pregnancy generating potential that is comparable to fresh embryos.
Vitrification is now regarded as potential alternative to conventional (slow)freezing.

Major advantages of Vitrifiction over Slow freezing are :

• Its prevents ice crystal formation within the cells, which can damage the embryo.
• It eliminates use of expensive freezing unit(required for slow freezing).
• High survival rate post thaw.
• High pregnancy rate following transfer of thawed vitrified embryos.

At Malpani infertility clinic, we have been using vitrification for a number of years to cryopreserve both embryos and eggs. When it comes to embryo cryopreservation, we freeze all stages of embryos i.e. Day 1, Day 2, Day 3, Day 5 and Day 6.

Steep Learning Curve :

To avoid embryo exposure to high concentrations of toxic cryoprotectants for extended periods, vitrification protocols generally require that embryos remain in the vitrification solution for a fixed time. This can be difficult, especially since vitrification technique have an extensive learning curve before the technique is mastered. Even with practice, one may not be able to quickly pick up embryos and vitrify within the time required.

Therefore it's critical that we determine the length of time the embryos can safely reside in vitrification solution, so that we don't overexpose embryos to toxic cryoprotectants, which can kill them or lead to very poor post thaw survival.

How difficult is it to master the technique?

Vitrification technique is a difficult art to master:

1. Due to high concentrations of cryoprotectants, vitrification medium is very viscous. High viscocity makes it very difficult to handle embryos, while they are in the medium. Needs lot of practice in order to get efficient.
2. One needs to be quick while handling embryos in vitrification medium, as long exposure of embryos to cryoprotectants can kill embryos or lead to poor survival rate post thaw.
3. Loading embryos onto cryolock is the most difficult part. It requires lot of practice. Improper loading can also affect the survival rate.
4. Thawing procedure (called Vitrification Warming) is as important as the Freezing. Improper thawing may lead to poor survival. So one needs to thaw embryos carefully in order to ensure good survival of embryos.

How do I vitrify your embryos and eggs?

VITRIFICATION FREEZING

We use Quinn's Vitrification Medium at our centre for freezing Embryos and Eggs.
Quinn's Vitrification Medium contains 2 media, "Equilibration Medium" and "Vitrification Medium"

Equilibration Medium contains Base Medium(Hepes buffered Medium) + 7.5 % ethylene glycol + 7.5 % DMSO (Dymethyle sulfoxide) + 20 % Protein supplement

Vitrification Medium contains Base Medium(Hepes buffered Medium) + 7.5 % ethylene glycol + 7.5 % DMSO (Dymethyle sulfoxide) + 0.5 M Sucrose + 20 % Protein supplement

Preparation of Vitrification Dish :

Place 1 drop of 0.1 ml of "Equilibration Medium" and 4 drops each of 0.05 ml of "Vitrification Medium" as shown in the figure.

The entire Freezing procedure is performed at room temperature.

We need to leave the Petri dish in the workstation for 15 min, for the medium to recover to room temperature.

Equilibration

• Place embryos or eggs to vitrify in equilibration medium. (This will be number of embryos or eggs that will be placed on 1 cryolock). We don't put more than 3 embryos or eggs on 1 cryolock.
• Once embryos or eggs are placed in Equilibration Medium, they spontaneously begin to shrink . This is when the intracellular water comes out.

Picture of shrunken embryo, after placing it in the equilibration drop.

• The shrunken embryo or egg will gradually recover back to its original size. This takes approximately 7 to 8 minutes. This may be less or more than 7 to 8 minutes in some embryos and eggs.

Vitrification

• Once the embryo or Egg recovers back to its original Size, its ready to be vitrified. I generally vitrify the embryo or egg once it recovers to about 80% of its original size.
• Pick up the embryo or eggs from equilibration drop with a flexipet and place them in the 1st drop of vitrification medium.
• Once the embryo or egg is placed in the vitrification drop, it again starts shrinking. Keep the embryo or egg in the 1st vitrification drop for 10 sec. This is basically to get rid of Equilibration medium, which is carried along with the flexipet while transferring them from equilibration medium.
• While the embryo/egg is in the first drop of vitrification medium for 10 sec, rinse the flexipet by pipetting the vitrificaition medium to get rid of equilibration medium.
• After 10 sec, pick up the embryos/eggs from the 1st vitrification drop and transfer them to series of 3 drops of vitrification medium one after other.
• Quickly load the embryos onto cryolock with very little medium. Make sure to gently remove excess medium in case a bigger drop is made on the cryolock.
• immediately plunge the cryolock in Liquid Nitrogen. Now the embryos are vitrified.
• Slowly put the cap to the cryolock, while it is immersed in the liquid nitrogen.
• Put all the cryolocks in one visitube.
• Put the visitube into canister.

Vitrification Warming (thawing)

Quinn's vitrification warming medium contains 3 media, "Thawing Solution", "Dilution Solution" and "Washing Solution"

Thawing Solution (TS) contains Base media (Hepes buffered medium) + 1M Sucrose + 20% protein supplement.

Dilution Solution (DS) contains Base media (Hepes buffered medium) + 0.5M Sucrose + 20% protein supplement.

Dilution Solution (DS) contains Base media (Hepes buffered medium) + 20% protein supplement.

Preparation of dishes :

• Place a drop of 0.2 ml of Thawing solution (TS) in one dish as shown in the figure.
• Keep the dish for warming at 37 deg. C on a warming plate for about 15 min.
• Place 1 drop of 0.1 ml of Dilution Solution (DS) and 2 drops each of 0.2 ml of Washing solution in another dish as shown in the figure.
• Keep the dish at room temperature.

0.2 ml of Thawing Solution at 37 deg. C.

• Identify the cryolock (embryos we want to thaw) and put in the box containing Liquid Nitrogen.
• Remove the cap of cryolock, while it is immersed in Liquid nitrogen.
• Quickly submerge the cryolock tip into thawing solution under stereomicroscope view.
• The embryo will automatically dispel.
• Leave the dish at 37 deg. C for 1 minute.
• After 1 min, carefully collect the embryos from the thawing solution to Dilution Solution at room temperature making sure carrying forward minimal amount of thawing solution along with the embryos.
• After 3 min. pick up the embryos and transfer them to 1st washing solution. The embryos will recover back to its original size approx after 2 min.
• After 3 min. transfer the embryos to second washing solution.
• after 3 min transfer the embryos to culture dish.
• Incubate the embryos for 2 hours before transferring them to patient's uterus.

What mistakes can a beginner do while vitrifying, that can lead to a poor survival rate?

Preparation of vitrification dish

Mistake :

Placing only 1 drop of vitrification medium, trying to cut down on the amount of medium consumed.

Reasons :

• Using only 1 drop of Vitrification medium doesn't allow us to get rid of Equilibration medium before actually vitrifying the embryo/egg.
• The Equilibration Medium is not very effective when it comes to cryoprotecting , as compared to Vitrification Medium.

Solution :

• Use 4 drops of vitrification medium.
• Serially transfer embryos/eggs through the series of these 4 drops of vitrification medium. This allows us to get rid of the equilibration medium effectively.
• This will definitely ensure better survival rate.

Equilibration

Mistake :

• Keeping the embryos in Equilibration medium for a short period of period before transferring them to Vitrificaition medium.
• This happens when we rely on fixed time period for each medium.

Reasons :

• Once the embryo/egg is placed in Equilibration medium, it will shrink spontaneously and slowly recover back to its original size. The recovery period varies for different embryos.
• When we rely on fixed time period for e.g. 5 min in Equilibration medium, it may not be enough for the embryo/egg to recover to its original size.
• If we transfer the embryo/egg to vitrification medium before it recovers back to its original size, it doesn't vitrify properly, which leads to extremely poor survival rate post thaw.

Solution :

• Rather than relying on a fixed time, it's best to individualise the time, based on the shrinkage and recovery of each individual embryo/egg , to ensure better vitrification.
• The embryo/egg can take approximately 7 to 8 min. to recover back to its original size.
• In some embryos it can be less or more than 7 to 8 min.

Vitrification :

Mistake :

• Making a large drop of vitrification medium along with embryo/egg while loading it on cryolock.

Reasons :

• A large volume of vitrification medium on cryolock will not allow proper vitrification of the embryo/egg.
• If the embryo is not vitrified properly, it leads to poor survival.

Solution :

• Make a flat small droplet of vitrification medium.
• Even if the drop is large, get rid of the excess medium by aspirating it gently.
• An embryo placed in a minimal amount of vitrification medium on cryolock vitrifies perfectly.