It’s getting cold out there, so let’s talk about freezing! Reproductive cryopreservation is what we are talking about today, not frostbite of your tender bits.
The process was pioneered in the 1930’s with rabbit sperm, then with human sperm in 1945. It was not until the early 1980’s that human embryos were frozen, and 1986 for human oocytes (although initially survival rates were dismal). It really has been less than decade since the modern techniques were refined.
Think about this scientific miracle: we can indefinitely place gametes or embryos in a state of suspended animation, then re-animate with a process that can lead to creation of human life! (Fun fact: The longest reported duration of embryo cryopreservation that eventually led to a live birth was 20 years!**) Mind-blowing, and yet completely routine these days. That said, it’s not as simple as throwing a steak in the freezer.
Let’s review the two different freezing techniques that have been used over the years.
- Slow freezing is the traditional technique that involves the phase change from liquid to solid. Cryoprotective agents (aka CPAs, or “antifreeze”) are used to dehydrate the cell. A process known as “seeding” induces an ice crystal-forming chain reaction. Using programmable freezing units, the temperature will decrease at a rate of –0.3°C to –0.5°C until it reaches -30°C, at which time the cells can be plunged into liquid nitrogen (-196 °C). While slow freezing worked well for sperm and was reasonable (but certainly not optimal) for embryos, egg freezing just didn’t work very well. Until, that is, vitrification came along!
- Vitrification is technically not freezing at all, but instead ultra-rapid cooling which results in the transformation from the solid phase to a glass-like state. Cooling occurs at an effective rate of -1300°C - -23,000°C per minute! This requires high concentrations of CPAs but does not require seeding or a programmed freezing unit. Most importantly, ice formation does not occur. Permeating CPAs enter the cell and push the water out from the inside. Nonpermeating CPAs don’t enter the cell but create an osmotic gradient to pull the water out across the cell membrane. Vitrification is much faster than slow freezing (duh), but it requires more skill and attention to detail.
Thawing is much easier: 30 seconds in the microwave and you’re good to go... Kidding! It is also a highly technical and precise process. Just as vitrification is not actually freezing, the reverse process is technically not thawing, but instead, "warming”. In order to do this, the cell is surrounded by high concentrations of sucrose (talking high carb here!) in a decreasing, stepwise process called elution. Each step draws out a little bit of CPA and allows a little bit of water to enter in a controlled fashion.
Compared to the slow freezing technique, vitrification significantly improved survival rates of blastocysts, but especially revolutionized the ability to cryopreserve eggs. Why the difference? The size of the mature oocyte and the blastocyst are essentially the same, but the ratio of water content to cell membrane is much higher in oocytes, which means more potential for ice crystals that disrupt the meiotic spindle, and preventing the orderly alignment and separation of chromosomes. Eggs get destroyed with slow freezing.
Vitrification avoids the ice crystal problem, so the cellular machinery in the oocyte remains intact. As of 2013, egg freezing was no longer considered experimental. Much like the advent of oral contraceptives in the 1960’s, vitrification has also created the opportunity for people with ovaries to take greater control of their reproductive choices.
How do we know that we are good at vitrification? First of all, we use the state-of-the-art techniques and the best materials and reagents available. Secondly, our survival rates exceed the published standards. And finally, we have the awards to prove it: Olive Fertility Centre just won the 2020 LOVE (Leaders in Vitrification Excellence) Awards, recognizing our technical expertise and the highest success rates with vitrified oocytes in the country! If I were freezing my eggs or embryos, I would want these talented scientists to be in charge.
