Cryopreservation and fertility preservation

Cryopreservation means storing living cells or tissues at very low temperatures, usually in liquid nitrogen at −196 °C, so that they can be used years later. In reproduction it underpins semen banks for livestock, the storage of surplus embryos after IVF, egg freezing, and attempts to protect the fertility of children and adults whose medical treatment may damage their ovaries or testes. It is also used to conserve the genetic material of rare and endangered animals, a theme well represented in the journals once published on this domain.

An accidental beginning

The modern field dates from 1949, when Christopher Polge, Audrey Smith and Alan Parkes reported in Nature that spermatozoa could be revived after exposure to very low temperatures when glycerol was present. The protective effect of glycerol, first shown with fowl sperm, introduced the idea of cryoprotectants: substances that help cells survive freezing.

Embryos followed more than two decades later. In 1972 researchers reported that mouse embryos survived freezing to −196 °C, and some to −269 °C, provided they were cooled slowly (0.3 to 2 °C per minute) and warmed slowly. Depending on the rates used, 50 to 70% of more than 2 500 frozen and thawed embryos developed into blastocysts in culture, and when survivors were transferred to foster mothers, more than 40% of the embryos in the resulting pregnancies gave rise to normal, living young. Sixty-five per cent of the foster mothers that received surviving embryos became pregnant.

Why freezing harms cells

Once safely frozen, cells can in principle endure storage at −196 °C for centuries. The danger lies in the journey down and back up. Classic work on the physics of freezing identified two opposing risks:

• Cooling too fast: not enough water leaves the cell by osmosis, and ice forms inside it, which is usually lethal.
• Cooling too slowly: as ice forms outside the cell, the cells are trapped in shrinking channels of increasingly concentrated, unfrozen solution, which also causes damage.

Slow-freezing protocols try to find the best compromise for each cell type. An alternative approach, vitrification, uses high concentrations of cryoprotectant and very rapid cooling so that the solution solidifies into a glass-like state without ice crystals forming. According to ESHRE, vitrification has improved the outcome of both embryo and egg freezing, and the UK Human Fertilisation and Embryology Authority (HFEA) notes that it is more successful than slow cooling for eggs.

Eggs, embryos and ovarian tissue

The development of egg vitrification has been one of the main drivers of the growth of fertility preservation over the past two decades: ESHRE’s 2020 guideline on female fertility preservation states that oocyte cryopreservation should be offered as an established option. The HFEA cautions, however, that success rates with frozen eggs tend to be quite low and that eggs frozen at a younger age are of higher quality.

For women who cannot undergo hormone stimulation before cancer treatment, an alternative is to freeze pieces of the outer layer of the ovary, the cortex, which contains immature follicles, and later graft them back. In 2004 a Belgian team reported a live birth after this procedure: the tissue had been frozen in 1997 before chemotherapy for Hodgkin’s lymphoma and reimplanted in 2003, after which ovulatory cycles returned. ESHRE recommends offering ovarian tissue cryopreservation to patients at moderate or high risk of losing fertility when freezing eggs or embryos is not feasible, considers it an innovative method in adult women, and recommends slow freezing as the standard for tissue, with vitrification of tissue offered only within research programmes. Before transplantation, the tissue should be checked for residual cancer cells.

The guideline also shows how broad the population seeking fertility preservation has become. It covers:

• people with cancer facing treatments that can damage the ovaries;
• people with non-cancerous diseases that require such treatments;
• people with genetic conditions that predispose to premature ovarian insufficiency;
• transgender men (assigned female at birth);
• women who choose to freeze eggs because of age-related fertility decline.

Storage, law and practice

Legal frameworks govern how long gametes and embryos may be kept. In the United Kingdom, eggs, sperm and embryos can be stored for up to 55 years, provided consent is renewed every 10 years. If consent is not renewed, or the clinic cannot reach the person concerned, the stored material can be disposed of. Other countries apply different limits and rules. The HFEA describes the main risk of egg freezing as the possibility that treatment will not work, and points out that complications of pregnancy increase with the age at which the eggs are eventually used.

Not every proposed protective measure is equally established. For example, ESHRE advises that drugs called GnRH agonists may be offered to protect ovarian function in premenopausal women receiving chemotherapy for breast cancer, but notes that evidence for a protective effect on the ovarian reserve is limited and that they should not replace cryopreservation. In vitro maturation of eggs collected without full stimulation is likewise regarded as an innovative procedure.

What remains uncertain

The basic physics of cell freezing and the effectiveness of sperm, embryo and egg vitrification are well established. Open questions concern the long-term outcomes of ovarian tissue transplantation, methods for preserving the fertility of prepubertal boys, the reasons for differences in freezing tolerance between species and breeds, and how best to adapt protocols to wild species, where samples are scarce and each animal’s biology may differ. ESHRE’s guideline itself stresses that more evidence is needed on pregnancy outcomes after these techniques. The clinical context is described on the page on IVF and assisted reproduction.