Is it safe to aliquot IVF media?

A question we often get from clinics is for how long time an opened bottle can be used. And if it is possible to aliquot and use the medium until expiry date. In this blog post I make an attempt to answer these and other related questions on safe usage of media.

What happens to the media if I use the same bottle for several weeks?

Evaporation is inevitable when working with media but there are ways to minimise the process.

Evaporation is temperature related but also occurs during storage in the refrigerator. Every time a bottle is opened and medium is removed, the atmosphere in the bottle is changed and the proportion between medium and air changes, resulting in increased evaporation. 

Repeated bottle openings in combination with storage at room temperature results in gradually increasing evaporation of the medium in the bottle. This evaporation affects osmolality and eventually osmolality will go out of specification. Consequently, it is advised to keep the bottle at room temperature as short a time as possible and to minimise the time it is open.

Evaporation affects amino acids and ammonium

It is not only an increase in osmolality that will occur if bottles are kept out of the refrigerator. Amino acids degeneration and increasing levels of ammonium will also occur faster and medium quality will be reduced.

Additionally, to avoid bacterial contamination it is crucial to use aseptic working technique when using the same bottle for several days.

How to minimise evaporation

Two easy ways to minimise evaporation are:

• Prepare dishes immediately after the bottle has been removed from the refrigerator
• Do not leave the bottle at room temperature to warm up before use 

For how long can I use an opened bottle with maintained quality?

Vitrolife has performed stability studies on our media products* after re-use of opened bottles. According to these studies it is safe to use a bottle (10-125 mL) of Vitrolife media for up to two weeks after the first opening - provided that you work according to what is described above.

Hence, work quickly when you prepare your dishes. Medium dishes should be prepared one by one and medium droplets covered with cover oil as soon as possible to avoid further evaporation. Minimise medium bottle exposure to room temperature and do not leave the bottle open. Return the bottle to the refrigerator as soon as your dishes are ready.

The number of openings during the two weeks must also be taken into consideration. How many times per day do you open a medium bottle? If possible, plan your work so that bottles are not removed from the refrigerator more than once a day.

I cannot use a bottle within two weeks and do not like to discard. Can I aliquot?

First it should be noted that aliquoting is performed on clinics' own responsibility and initiative. It can be done for several reasons, the above mentioned reason being but one of them. Another common reason is that many clinics aliquot media to save time during busy work days. To prepare test tubes with media for sperm preparation only once per week for example is rather common.

There are several risks to aliquoting. First, there is the obvious risk of contamination. Second, increased evaporation and osmolality when you pipette and if a small volume of medium is stored in a container containing a large volume of air. Make sure to fill the storage vessels to the top to reduce air inside and close the cap tightly.

The importance of using non-toxic disposables

However, the most important risk to consider before aliquoting is the vessel that you will use. It may be tempting to use flasks or test tubes that are already available in your lab but are these vessels mouse embryo tested for human IVF? Most flasks and tubes on the market are not manufactured and tested for IVF purposes and may contain embryo toxic molecules which can enter your embryo culture system and impair embryo development¹. This can be very treacherous. It may be easy to identify batches of lab materials with high levels of toxicity using, for example, a sperm survival assay^{2, 3}, but non-tested labware can contain low levels of toxic substances which may go undetected for a long period of time⁴. You may notice that the pregnancy rate is slightly reduced over time or that you cannot increase your results as expected when implementing new protocols or equipment. It will take a long time to find that the reason is a vessel used for aliquoting media.

Today there are products developed and tested specifically for use in IVF. Vitrolife Labware are examples of this, making them safe to use whenever aliquoting is employed.

Safe aliquoting is possible

All above considered, osmolality, non-toxic vessels for aliquoting and of course also aseptic work procedures, it is possible to safely aliquot media and use the full shelf-life, as long as you are aware of the risks and make sure to minimise them.

Every bottle of medium from Vitrolife comes with a package insert with instructions regarding how to handle Vitrolife media products. These instructions should be read before first opening of a bottle.

^{*Please check the bottle label before use of the product. If the medium can be re-used you will find this symbol on the label ②.} 

References

1. Nijs et al.Reprotoxicity of intrauterine insemination and in vitro fertilization-embryo transfer disposables and products: a 4-year survey.Fertil Steril; 2009. 92(2): 527-35.
2. Hossain et al; Human sperm bioassay for reprotoxicity testing in embryo culture media: some practical considerations in reducing the assay time. Adv Urol. 2010; doi: 10.1155/2010/136898.
3. Critchlow, J. D., et al. Quality control in an in-vitro fertilization laboratory: use of human sperm survival studies. Hum Reprod 1989. 4(5): 545-549.
4. Van den Bergh et al. Randomized autocontrolled comparison of the embryo culture performance of Nunc and Falcon petri dishes. J of Ass Reprod & Genet; 1999: 16(6): 306-9.