Saturday, June 18, 2011

How to improve the vitrification of blastocysts

This is a guest post by our embryologist, Sai Gundeti.

Vitrification is the newer alternative to the traditional ‘slow freezing’ technique of cryopreservation of embryos for storage and future use. It is far more efficient and effective than the older technique, which is why we now use only vitrification in our clinic for cryopreservation of eggs and embryos.
It is the duty of the embryology team to learn this new technique, so they can offer the best possible care to their patients.

Here’s how we vitrify embryos in our lab.

It’s usually the supernumerary embryos which are vitrified, which is why embryo vitrification is generally carried out after the best ( top ) embryos have been transferred in the fresh IVF cycle . However, in some cases we may electively freeze all embryos , and not do an embryo transfer(ET) at all. These include: (i) when a patient is unable to have ET for medical reasons; (ii) when there is a high risk of Ovarian Hyperstimulation Syndrome (OHSS) or (iii) oocyte donation cycles, in which the recipient’s endometrial receptivity is not optimal for implantation.

At Malpani Infertility Clinic, discussions regarding embryo cryopreservation are made in consultation with the patient and are documented in the patient’s notes.
Survival rates after freezing and thawing are better than 95 %. However, sometimes survival could be partial i.e. an embryo may lose 1 or more cells , but it is still considered to be viable. An embryo is considered non-viable if it loses more than 50% of its cell mass.

We use SAGE/QUINNS vitrification medium to vitrify embryos. It contains Sucrose, DMSO and Ethylene glycol.

DMSO/Ethylene glycol are used as the permeating cryoprotectants, while sucrose as the non-permeating cryoprotectant. The optimal concentration of these cryoprotectants is selected to permeate cells and transform them to a solid state without formation of ice crystals. Protocols have evolved to minimize damage due to ice crystal formation, cryoprotectant toxicity and osmotic shock by using ultra-rapid freezing and thereby short exposure to toxic reagents.

Prior to Vitrification :
• Embryos are stored in Cryolocks with a coloured identification.
• Cryolocks are labeled as :
- Full name of the patient
- Patient unique freezing identification number
- Number of frozen per cryolock
- Date of freeze.
• All of this identifying information is recorded on a freeze event register that also includes details of embryos that have been vitrified and storage location.

Artificial shrinkage of Blastocysts before vitrifying them :
Increasing blastocoelic fluid in expanding blastocysts has been associated with poorer survival rates. This is why we routinely reduce the blastocoelic cavity prior to vitrification.

Artificial shrinkage is carried out by laser pulse using Saturn laser.
Ablation is done between the trophectoderm cells

Procedure :
• Blastocysts suitable for vitrification are identified. Those which have a large cavity ( expanded blastocysts) are selected.
• The dish is placed on the heated stage of workstation.
• The dish is positioned in order to give a clear view of the Blastocyst.
• 1.2 ms LASER duration for desired pulse length is selected and a single ablation opposite the Inner Cell Mass (ICM) and between the trophectoderm cells is done.
• This causes the blastocyst to start shrinking very quickly !
• The blastocysts are vitrified immediately to avoid re-expansion.

Vitrification procedure :
• “Equilibration Medium” and “Vitrification Medium” are equilibrated until they are at room temperature upto to 30 minutes prior to use.
• The Blastocyst is transferred to the Equilibration medium and allowed to equilibrate for 8-10 minutes. The blastocyst initially shrinks and then returns to its former shape.
• The blastocyst is now transferred to the Vitrification Medium and quickly loaded onto cryolock labeled with patient’s name.
• The cryolock is quickly plunged into liquid nitrogen vertically and gently stirred in liquid nitrogen for approximately 5 seconds so as to avoid formation of an isolating air bubble layer around the cryolock.
• The procedure is repeated for subsequent Blastocyst. We usually freeze only one blastocyst per cryolock, because the survival rates are so high !
• When all embryos have been vitrified they are stored in an allocated dewar location.
• Full records of all details concerning the vitrificaiton, as well as details of storage are documented.
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