**7. The thawing process**

**6. Trends in embryo storage**

150 Cryopreservation in Eukaryotes

embryos are termed "abandoned".

proposed.

lacking.

population.

pre-natal testing may be of benefit.

by adverse events or long-term culture [136].

Worldwide, 1.5 million ART cycles are performed each year and this number continues to rise [133]. Up to a third of patients who undergo IVF have supernumerary embryos that are cryogenically stored. A case is made for the perpetually increasing reserve of embryos and more importantly those of patients lost to follow up. For reasons undescribed here, these

At least in the USA, estimates provide that there are up to 1.4 million abandoned embryos [134] and though unknown it is safe to assume this number dwarfs the combined worldwide total. Even with permitting signed patient consents, clinics are hesitant to act and discard unclaimed embryos, largely because of the lack of regulatory guidance [135], leaving clinics vulnerable to unanticipated legal ramifications. Recently, several solutions have been

Consideration of imposing strict time limits on storage, outlining relevant responsibilities of fertility clinics and patients, and clarifying absolute guidelines related to unrestricted utility of embryos in terms of donation for third party, teaching or research use must be defined within a strict legal framework. Above all adequate long-term storage facilities are

A somewhat limited solution supporting non-fertile couples in conception and familial continuity is embryo donation. It is noteworthy to mention that cryopreservation has indirectly found a place in a larger market in terms of the transport and exchange of all types of biological samples. Initial concern over the sensitivity of microscopic volumes employed in vitrification to potentially shifting conditions during shipment has largely been overcome by advanced

Specific to embryo donation, ethical and genetic consequences of donation to related and unrelated parties, including offspring, must be carefully considered. In addition, given lack of restrictions on storage time limits, decades old donated embryos, thus far, may provide relatively antiquated genetics which ultimately interfere with the natural evolution of the

Similarly, in the case of multiple embryos donated to different parties, even a marginal risk of unsuspecting, related siblings, procreating by chance may have devastating consequences for the developing fetus. This is even more likely if embryos are donated to a clinic within the same geographical region. As such, advanced screening methods prior to procreation and/or

Contrary to this line of thought, in a separate context, in animal and cell line research laboratory settings, recessive gene expression and cell line mutations confer an advantage for study purposes and cryopreservation provides cell line stability against undesired changes induced

vapor shipping dewars, temperature monitoring, and precautionary handling.

Given the worldwide exchange of reproductive cells, which is practically commonplace, IVF labs generally house a repository of embryos cryopreserved with various methodologies and formulations. The core responsibility of clinics is to ensure safety and best practice outcomes, and this requires staff training and laboratory access to a myriad of cryopreservation formulations and techniques.

In actual practice, IVF laboratories may permit revitalization of embryos using readily available thaw solutions/protocols. This is in contrast to purchasing the specific formulations matched to the cryopreservation solutions that the cells were frozen in. The impact of such mix and match freeze/thaw practices on embryos/oocytes is largely unexplored and may be quite significant given the unique actions of CPAs and unpredictability of carrier solutions as previously described. Observed success may be due to the robustness of certain reproductive cells and this may provide some artificial confidence in this regard. Prior to such validation cross-use of combinations of cryoprotectant solutions should be approached with caution.

The development of cryopreservation techniques has had immense impact across many disciplines, most notably reproductive medicine. While significant advances have been made, further advances are needed in the changing landscape of fertility. Cryopreservation has allowed IVF to evolve into a medical procedure that is efficient, safe, readily accessible, and relatively affordable.
