Can human eggs and embryos be preserved indefinitely in a “frozen” state without losing their potential for normal development? The short answer is, probably; but the truth is that more experiments need to be done to answer the question of cellular resilience during the “suspended animation” that is the cryopreserved state. Here is why the matter is more complicated than was originally believed.
Liquid Nitrogen and Dewars
When freezing of mammalian embryos was first attempted in the 1960s, scientists were concerned about the long-term survival of the embryos. In turn, scientists decided to store the the embryos in one of the coldest environments known – liquid nitrogen. This was based on the premise that all biological processes would cease when samples were kept at such temperatures.
Liquid nitrogen is nitrogen gas in liquid form and it evaporates at the very low boiling point of −320 degrees Fahrenheit. In spite of that it is surprisingly easy to manufacture, cheap to buy, easy to store and relatively safe to transport. But liquid nitrogen is only as useful as the containers we keep it in: pressurized super-insulated vacuum containers called dewars, which allows us to store liquefied nitrogen over extended periods. Over the course of each day, only a small amount of the liquid is lost from these dewars due to evaporation. This minimal evaporation is countered by periodically topping off the dewars with more liquid nitrogen.
Early History With Slow Freezing
In the 1940s and 1950s, scientists had already frozen-stored human and other mammalian sperm in liquid nitrogen. It was relatively simple to freeze sperm, because the cells are small and have little liquid. Why does that matter? Because when a water-based liquid freezes, ice crystals are formed, which are known to be damaging to cells, even at low temperature. So, freezing larger cells, with lots of cytoplasm, was much more complicated. The challenge was most profound for oocytes (and embryos), which are larger than any other cell in the body. So how could this be accomplished without damaging the cells? Cryobiologists and embryologists were able to circumvent this by bathing the cells in cryoprotectants or “anti-freeze” before cooling them relatively slowly over one or more hours.
You might think adding “anti-freeze” to your eggs or embryos is a good way to kill them, right? Although high concentrations of cryoprotectants can be toxic to cells, the problem was largely circumvented by adding enough to allow cell survival both during storage in liquid nitrogen and short exposure at room temperature. As temperatures were slowly lowered in a cell-freezing machine and the fluid cooled, toxicity of the anti-freeze solutions diminished. This system of slow-cooling and low levels of cryoprotectant was used reasonably successfully for decades.
OK, so we proved we could freeze and then thaw mouse embryos. But now we needed to understand how long we could freeze them for. In experiments simulating 2,000 years of exposure, scientists showed that slow-frozen mouse embryos survived the most worrisome elements. But those are simulations, so what about actual clinical experience? Later data from mouse embryos showed that keeping embryos frozen for several years or even a decade seemed to have little effect on their survival after thaw.
The next obvious question is whether all of this experience with mice applies to humans? My lab was the first to study the effect of time on frozen-stored human embryos in 1988 and we found little, if any, effect of long-term storage on human embryos, although at that time human embryos had only been frozen for periods of up to three years. More than a decade later, babies were born following transfer of embryos that had been frozen-stored for years, some for longer than a decade. Several labs later confirmed those observations. A pregnancy was even reported in 2010 after embryo storage of 20 years by the IVF team in Norfolk Virginia. But more studies of long-term storage on cryopreserved eggs and embryos are still needed, particularly after vitrification. The table below shows results from a larger series obtained in a clinic in China by Dr. Liu and colleagues – again this was with slow-frozen embryos.
And here is where I should probably mention that we don't have enough data or experience to reveal if duration of egg / embryo storage has any impact on how the offspring ultimately develop.
Evolution To Vitrification
Despite its relative success, slow-freezing of human embryos had major drawbacks. The method was time-consuming, dependent on delicate equipment and not all eggs and embryos survived the thaw. These limitations were overcome with vitrification. Vitrification allows samples to be cryopreserved within one second (rather than 1 - 2 hours) without the risk of producing those damaging ice crystals I mentioned above. This method has revolutionized human embryo and egg cryopreservation and some laboratories now report 100% survival rate from eggs and embryos frozen .
With advances in technology comes progress in science and medicine, but also new worries and limitations. In the case of vitrification, samples can be kept apparently safe and held for a long time, years if not decades, but the samples must remain free of physical forces and completely submersed in liquid nitrogen at all times. Why is that a problem? Well, embryologists often must check and identify cryopreserved samples individually. At the very minimum, this must be done once a year during inventory checks or whenever samples are moved from one lab to the next. In order to do this, embryologists must lift the samples sufficiently out of the dewar to get a good read. This increases the risk of temperature elevation. A group of Argentinian scientists recently showed, in a theoretical model, that vitrified samples may lose their glass-like protective state even at fairly low threshold temperatures. They postulate that the vitrified state can be partially lost each time the cryo-devices are moved and exposed to the room even for brief periods or when there is not enough liquid nitrogen in the dewar tank. This problem will have solutions, but some engineering is needed before we can call the new methodology completely safe.
Are Eggs and Embryos Equally Sensitive?
It’s possible, yet unproven, that eggs are more sensitive than embryos if they are checked or handled carelessly. This may explain some adverse findings reported after egg and embryo transport from one location to another. In one experiment performed in 2010, a Chinese research team showed that vitrified mouse oocytes were more likely to survive, if the samples had only been stored in liquid nitrogen for a few weeks compared to a few months. No transportation had taken place. Those dramatic results only make sense if the older cryo-devices were exposed to adverse conditions and not fully immersed in liquid nitrogen at all times, although some researchers believe the effect was achieved by direct contact between the mouse egg and liquid nitrogen. This brings us back to the question posed earlier. Can human eggs and embryos be stored indefinitely? The answer, at this point, is still ‘probably’. Scientists and engineers need to develop ways for embryologists to store eggs and embryos safely, identify samples, perform inventory and transport samples without disrupting temperature. When this goal is achieved, spending an eternity in liquid nitrogen and coming out of it unscathed would not be a far-fetched idea.
References:
Cohen J, Inge KL, Wiker SR, Wright G, Fehilly CB, Turner TG Jr. Duration of storage of cryopreserved human embryos. J In Vitro Fert Embryo Transf. 1988, 5:301-303.
Dowling-Lacey D, Mayer JF, Jones E, Bocca S, Stadtmauer L, Oehninger S. Live birth from a frozen–thawed pronuclear stage embryo almost 20 years after its cryopreservation. Fertil Steril 2011; 95: 1120.
Liu Q, Lian Y, Huang J, Ren X, Li M, Lin S, Liu P, Qiao J. The safety of long-term cryopreservation on slow-frozen early cleavage human embryos. J Assist Reprod Genet. 2014 Apr;31:471-475.