Gamete and Embryo Storage

Jacques Cohen, PhD

Procedures developed for freezing embryos of rodents and farm animals were applied with minimal adaptation to the needs of human assisted reproduction a few years following the birth of Louise Brown in 1978. The clinical purpose was obvious, since the earliest outcome data suggested that clinical IVF after fresh embryo transfer had a high failure rate and extra embryos were often discarded, compounding the general lack of success. Cryopreservation was the most important process introduced in the fledgling field of assisted reproductive technology (ART), allowing multiple embryo transfer attempts after a single retrieval procedure. Although cryopreservation increased the efficiency of ART, cumulative outcomes remained modest, due to sub-optimal post-freeze survival rates and an early embryo culture system which often limited embryo growth. Although many thousands of thawed embryos resulted in apparently normal live births, overall, the efficiency after a single retrieval procedure increased by just a few percent. A turning point occurred 20 years later with new and improved embryo culture methods, and the routine application of egg freezing and vitrification. The latter significantly improved post-thaw survival of stored eggs and embryos. Although the dynamic aspects of cryopreservation progressed over the years, leading to an exponential increase in successful cryopreservation of eggs and embryos, their actual storage technology and the legal context remained surprisingly archaic.

Added to the sharp climb in numbers of cryopreserved samples were new applications such as freeze-all approaches, to avoid transfer of fresh embryos in a potentially sub-optimal uterine environment; freezing of eggs and embryos in fertile women undergoing gonadotoxic treatments; and young fertile women who opted to defer family building. Increased cryo-storage inventory and its potential risks has been criticized in the mainstream media, particularly after the publicity from adverse events, such as those reported in early 2018 in two IVF clinics in the USA, and the failure of two large-capacity cryo-storage devices. Such failures may not always be preventable. Potential breakdown of systems must be monitored, and possible losses anticipated or circumvented through improved engineering, continuous oversight and quality management. In this issue of SRM, expert authors in 6 articles review technological, ethical, clinical, legal and quality control aspects of egg and embryo cryo-storage.

The past, present and future of technological aspects of cryopreservation of embryos as well as gametes are extensively reviewed by Rienzi and coworkers. Four articles are dedicated to the technology, quality management aspects, implications and physiological consequences of cryo-stasis and cryo-storage. Alikani and Parmegiani discuss the exponential usage of cryopreservation and possible implications of increasing inventories such as gamete and embryo abandonment, health consequences and risks associated with handling and transport. They present a breadth of demographic data detailing the extensive rise in cryo-usage and its global status, suggesting that for every ART-baby born there are approximately five eggs or embryos in cryo-storage. The authors review obstetric and neonatal outcomes and suggest that while cryopreservation appeared to offer reduced risks, recent studies show that the incidence of ‘large for gestational age’ and macrosomia is higher among neonates following cryopreservation than these events arising from fresh transfers and natural conception. They suggest that the association with cryopreservation awaits confirmation through large prospective investigations. The authors offer further insights into embryo abandonment. This is a a complicated condition where intended recipients (owners) remain unreachable, deceased or disabled with no clear instructions available regarding their embryos. Some jurisdictions have adopted guidelines to limit storage, but most countries do not have such rules and clinics remain in an uncertain environment, either storing embryos in perpetuity or discarding them without a clear regulatory framework. The authors also discuss the duration of cryo-storage. Their position is that prolonged storage is generally safe if storage conditions are stable and monitored, but that the recent change in technology, from slow freezing in large volume devices to fast vitrification in very small volumes, may warrant additional safeguards. In this context they are particularly concerned about adverse transport conditions because small volume devices may be at risk of accidental thawing at relatively low but slightly increased temperatures. Specimen loss and the potential for contamination are also discussed.

Malter's review discusses the nature and consequences of suspended cellular life or cryo-stasis. This author explains that freezing of single cells or uniform tissue itself can be detrimental, but that contemporary antifreeze-mediated cryopreservation systems are mostly benign and that ‘existence’ at ultra-low temperatures can be a true form of viable stasis. Malter attests to the complete lack of knowledge regarding prolonged cell cryo-stasis, and subsequently discusses what is known or not known about arrested cellular activity. He describes cryo-stasis as a “rather mysterious state” and that it could provide “an obscure window of understanding into the definition of life itself.”

Pomeroy and Marcon review the quality control processes involved with cryo-storage as well as the use and management of inventory software. They discuss adverse events such as rapid accidental loss after tank failure and the causes of such major malfunctions. They ponder over flaws in the storage tanks, design of monitoring procedures as well as failures related to quality control protocols and diligence in following the protocols. They point out that catastrophic failures usually engender litigation which causes an absence of details that normally would be shared with colleagues. Solutions regarding adverse events are offered but the authors are careful in pointing out that certain solutions may not be in the interest of patients and their frozen material. The advantages and drawbacks of on-site or off-site storage and tank location are discussed as well as the use or preference of small or large-capacity tanks. The authors emphasize that ongoing quality control must include checking tanks visually and analyzing evaporation rates. We may never eliminate all accidents with stored reproductive tissues, but we can reduce the probability of future accidents. The authors further discuss how to manage the risks inherent in cryo-storage, where stored tissue should be kept, and how to reduce risks through quality control.

In his review, Tomlinson extends this and argues that there is no time for complacency. He suggests that increasing levels of regulation, and the requirement to adhere to total quality management practices, may avoid most catastrophic incidents such as the death of an employee, the risk of contamination or the loss of a freezer full of patients' eggs, embryos or spermatozoa. He highlights the need to make practices considerably safer for patients as well as for staff. Essential monitoring of oxygen and appropriate ventilation are still under-valued aspects of cryo-storage risks. Such measures can easily be retrofitted. Tomlinson argues that in the light of recent events, clinics and storage outfits should place risk management of their cryo-facilities high on their agenda.

In the final review by legal experts Crockin and Gottschalk, it is skillfully discussed how the rapidly emerging medical practice of cryo-storage has sometimes outpaced the official context. While this should not be surprising, it is evident that national and international legal bodies in association with clinical organizations and health providers must design thoughtful frameworks to deal with ethical concepts of cryo-storage. Many IVF clinics with embryos that have been stored for prolonged periods wrestle with issues of disposition, such as embryos which may be discarded pursuant to prior instructions or considered “abandoned” and potentially discarded. The legal characterization or definition of what cryopreserved embryos and eggs are (or are not) seems astoundingly complex given the lack of practical context in many countries. The lack of legal frameworks confounds the many complicated issues, that may arise from the practice of cryo-storage.