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                                                        Image result for vitrification freezing

We freeze your embryos at Malpani Infertility Clinic using the ultra-rapid freezing or vitrification technique. Here is how we vitrify your precious embryos !

Dr. Sai, Embryologist, Malpani Infertility Clinic Pvt. Ltd.

When do we freeze your embryos?

We freeze your embryos when :

  1. you have surplus ( supernumerary) embryos of good quality, after we have transferred the best embryos to your uterus. We freeze these embryos to preserve them , so that you can use these in the future to complete your family. Using frozen embryos after thawing them has a high success rate ; and is much less expensive ( and much easier) than starting a fresh cycle.
  2. You have a risk of developing OHSS ( ovarian hyperstimulation syndrome) . We freeze all your embryos and use them later, to prevent OHSS.
  3. When your uterine lining is thin, and is not receptive for embryo implantation. In the next cycle , we can prepare your endometrium with estrogens, and transfer the frozen embryos after thawing them, to maximise chances of successful implantation

  • Like sperms, the embryos are also stored in Cryocans.
  • Cryocans contain Liquid Nitrogen to keep the embryos at -196 C, where all metabolic activity is suspended .
  • Embryos can be frozen at any stage :
Day 1 Day 2 Day 3
2pn stage 2-Cell / 4-Cell Stage 6-Cell / 8-Cell Stage
Day 4 Day 5 Day 6
Morula stage Blastocyst Hatching Blastocyst

There are two techniques of freezing embryos :

  1. Slow Freezing :

    • Embryos are frozen by gradually decreasing their temperature.

    This technique requires a specialized programmable freezing unit called a Cryobath.

  2. Vitrification :

  • Embryos are frozen ultrarapidly, by vitrifying them. Vitrification is a simple technique, but has a steep learning curve. If not done properly, it can easily kill your embryos. The skill of the embryologist is crucial and it's important that the embryologist be experienced, to maximise success rates
  • Vitrification is the latest technique of freezing embryos.
  • It has a better survival rate as compared to the older conventional slow freezing of embryos.

Steps involved in Vitrification

  1. Assesment of Embryos to vitrify :

    All your embryos don't grow at the same rate and they won't all be of the same quality.
    After transferring the best embryos to your uterus, the remaining embryos are analysed; and the best ones are kept in a separate dish.

  2. Preparation for Freezing :

    Preparation of Medium :
    1. Vitrification of embryos require special medium, containing cryoprotectants.
    2. Vitrification medium contains high concentration of cryoprotectants, compared to medium used for slow freezing.
    3. The Vitrification medium that we use in our IVF lab, is commercially manufactured, and is sold as a kit containing 2 solutions, Equilibration medium and Vitrification medium.

    4. The medium is removed from the refrigerator, and small droplets of medium are deposited in a petri dish , as shown in the figure.
    5. The medium is allowed to come to room temperature, before we actually start the vitrification process.

    Preparation of Cryolocks :
    1. Cryolocks are devices on which we load your embryos to store them.
    2. Cryolock come in different colors to help in identification. Different colored cryolocks are used for different patients.
    3. We write the following details on each cryolock :
      1. Patient's Full Name - your full name will be written in capital letters.
      2. Code - This code will be a unique serial number.
      3. Number of embryos -The number of embryos we load in that particular cryolock.
      4. Date - The date on which we freeze your embryos.

  3. Vitrifying your Embryos:

    1. The embryos to be vitrified are transferred to Equilibration medium.
    2. After 5 min, the embryos are transferred from equilibration medium to Vitrification medium.
    3. In less than 1 minute, the embryos are picked up from the vitrification medium and loaded onto a cryolock which has been pre-labeled with your name. Excessive exposure to this medium at room temperature can kill your embryos, which is why we have to work with great speed !

    4. The embryos are picked up from Vitrification medium using a fine pipette, under a stereozoom microscope, and then loaded on to your cryolock. The embryos need to be transferred in a very small volume of medium ( as little as one microliter, which means I have to be very careful when doing this )

Picture of embryos being loaded on a cryolock

  • The cryolock along with your embryos is plunged in Liquid nitrogen to vitrify your embryos.

  • The embryos are vitrified and we store all your cryolocks in cryocans.

    Your Embryo Your Vitrified Embryo

  • Note that a vitrified embryo looks shrunken because the water in its cells has been frozen and solidified
  • The cryolocks are kept in a visitube which come in different colours.

  • The visitube with your cryolocks is stored in cryocans.

    How we Thaw your embryos :

    We thaw your embryos a day before your Embryo Transfer. This allows us to check how well they have survived the thaw; and allows us to select the embryos which continue to divide well in the incubator.

    1. Identification of your embryos :

      Your Embryos are identified by-
      1. Canister Number
      2. Colour of Visitube
      3. Colour of Cryolock
      4. Code and your name on the cryolock.
    2. Preparation for Thawing :

      Preparation of Medium :
      1. The Media required to thaw (warm) your vitrified embryos is removed from the refrigerator.
      2. Thawing medium is a kit of 3 solutions.

      3. Small droplets of medium are prepared on a dish as shown :
    3. Thawing your Embryos :

      Post - Thaw Embryo
      • Once your cryolock with embryos is identified, it is removed from the cryocan.
      • The cryolock with your embryos is dipped into Solution 1. The embryos will get expelled in the medium

      • The embryos will remain in Solution 1 at 37 C for 1 min.
      • After 1 min, your embryos are transferred to Solution 2. The embryos will remain in solution 2 at room temperature for 3 min.
      • After 3 min, the embryos are transferred to Solution 3 for 5 min. They are properly washed in this solution.
      • The embryos are now thawed and we transfer them to culture dishes which are prepared in advance, labeled with your name.

      • Note that this looks as good as a fresh embryo, and no one can make out it was frozen for many months
      • Not all embryos will survive the thaw. In a good clinic, about 80% of embryos survive the thaw.