Freezing and Fertility: What's it all About?

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 Introduction

The discovery that tissue kept at very low temperatures (typically in liquid nitrogen) almost completely halts normal metabolism and decay, led to the development of the science of cryobiology (the study of low temperature biology) and the concept of cryopreservation (freezing). Cryopreservation could allow storage – some have argued indefinitely – and use of the tissue at a later date. The successful freezing of sperm, embryos, and more latterly eggs and ovarian tissue has allowed cryopreservation to become an integral component of infertility treatment and its application continues to grow.

Successful cryopreservation in the field of reproductive biology owes its origins to a fortuitous discovery in 1948 by a research group in the United Kingdom led by Alan Parkes (1). They had been attempting to freeze fowl semen. However, most cells were damaged by the freezing and thawing process. Then, partly by error, a sample was frozen with the addition of glycerol. It proved to be an ideal protective agent against damage from the cryopreservation process. This impetus, through further research, led to a greater understanding of the biological effects of cryopreservation and successful techniques were established for many cell types, including sperm cells.

Cryopreservation technology initially had its most significant impact on veterinary science, especially in relation to the cattle breeding industry. The ability to successfully freeze bull semen allowed the improvement of cattle stocks worldwide and could be used to replenish stocks following outbreaks of various bovine related epidemics. Today, most cattle are sired using frozen semen.

The concept of human “sperm banking” dates back to the mid 1800’s. Although it had been shown that sperm could survive freezing and storage at temperatures as low as -l96°C, the ability of previously frozen sperm to function normally and result in pregnancies did not occur until the widespread use of glycerol and other cryoprotectants. Sherman was the first to demonstrate the functional capacity of previously frozen sperm to fertilize eggs in 1949 and the first successful human pregnancy conceived from previously frozen sperm was in 1953 (2). Following this, the freezing of human sperm was largely done on an ad hoc basis until the first dedicated sperm bank was opened in California in 1977. Nowadays, in Canada, frozen sperm is stored only in licensed sperm banks and its use and screening carefully monitored by Health Canada.

The birth of the first child with in vitro fertilisation (IVF) in 1978, heralded the onset of a global expansion in the use of infertility treatments. This renewed the interest in cryobiological technology in an effort to preserve surplus embryos for future use. The first child conceived from frozen embryos was born in 1985 (3). Cryobiology has continued to develop in order to keep pace with the rapid advances in reproductive technology and both gametes (sperm and eggs) and embryos at varying stages of maturity can now be cryopreserved with variable success rates being reported.

More recently, the successful re-transplantation of previously frozen ovarian tissue in 2003 (4) and the first reported live birth in 2004 (5) have led to renewed research and success in preserving female fertility – particularly for women who will undergo potentially sterilizing anti-cancer treatment.

Cryopreservation of human sperm is the most widely applied aspect of cryobiology in reproductive medicine. Insemination with frozen-thawed donor sperm is one of the most widely used treatments for male factor infertility. Frozen donor sperm is also often used as a back-up when surgical sperm retrieval procedures prove unsuccessful during IVF treatment.

Sperm freezing has also been used for many years to preserve fertility potential in men undergoing chemo- or radio-therapy which would lead to testicular failure and little or no sperm production. It has also been used to preserve fertility and sperm quality in men where there is evidence of deteriorating sperm quality (for example – where there is damage to vas deferens or following vasovasostomy surgery) as well as for those who choose to undergo vasectomy. 

Sperm freezing is also indicated for couples having fertility treatment where the male partner may experience difficulty in producing a sample at the appropriate time or where there are difficulties with normal sexual intercourse.

Most programs using frozen sperm for insemination report pregnancy rates varying from 10-30% per month. Higher pregnancy rates are achieved when the female partner undergoes mild ovarian stimulation. When frozen sperm has been used in women undergoing IVF, the pregnancy rates are the same as when using ‘fresh’ sperm – typically 30-50%.

Over 1,000,000 children have been born following conception with frozen sperm and data from countries which closely monitor the use of frozen donor sperm (such as the United Kingdom) have shown no increase in birth defects or childhood illnesses compared with the general population.

Cryopreservation of human embryos has now become an integral component of assisted reproduction. The first human pregnancy was achieved in 1983 (6), but unfortunately resulted in a miscarriage. The first reported live birth was in 1985 (3), and since then 100,000s babies originating from cryopreserved embryos have been born worldwide.

Ovarian stimulation protocols used in IVF treatment will generate an average of 10 to 12 eggs being collected per treatment cycle in young women. Techniques for the cryopreservation of surplus embryos developed rapidly as a means of not only avoiding wastage but also maximizing the fertility potential of any given cycle of treatment.

Embryo cryopreservation has also been used for other reasons related to assisted reproduction. In patients who are believed to be at high risk for developing ovarian hyper stimulation syndrome (OHSS), all available embryos can be cryopreserved and then used in a subsequent cycle thereby avoiding the risk of severe illness. Also, in some cases, where patients require donor eggs, the availability of cryopreservation avoids the absolute necessity to synchronize the treatment cycles of the donor and the recipient as well as minimizing the small risk of transmission of an infective agent. Although, in both these cases overall successful pregnancy and live birth rates are typically about 20-30% lower than in ‘fresh’ embryo transfer cycles. 

In situations where a woman is about to commence potentially sterilizing chemo- or radio-therapy for many types of cancer, and where time and her condition permit, embryos can be created following an IVF cycle and then frozen for future use (once she has recovered from the initial cancer). Obviously, in such cases the women need to be in a committed relationship with a male partner in order to allow creation of such embryos.

Embryos may be frozen at various stages of development and the selection criteria for cryopreservation of embryos may vary from unit, although in general it will be determined by the total number of embryos available, the number of embryos required for ‘fresh’ transfer and the quality of the embryos remaining.

Overall there are a number of factors that will influence the viability and implantation potential of frozen-thawed embryos. Of prime importance is the quality of the embryo prior to freezing. Implantation rates for embryos frozen early following fertilization are superior to that of later stage embryos, possibly as a result of the lower risk of cryoinjury in the former (7). Embryos that have been biopsied for the purpose of pre-implantation genetic diagnosis (PGD) also have a lower survival rate following thawing (8).

Frozen-thawed embryos may be replaced into the uterine cavity in either a natural menstrual cycle or in a controlled hormone replacement cycle for women with irregular periods. There is no significant difference in pregnancy rates between these two embryo replacement protocols (9). 

Pregnancy rates from frozen-thawed embryos are lower than those for fresh embryos but in most centres will achieve pregnancy rates of between 20% to 25% per cycle. There is some evidence that assisted hatching may also improve implantation for froze thawed embryos – although further data is awaited. In a technique that was developed in the 1980s, it is reassuring that, so far, long-term follow-up studies have not indicated any increase in birth defects or any harmful effects on the children conceived as a result of embryo freezing (10).

The cryopreservation of human oocytes is an area of reproductive technology that has gained a lot of interest in recent years. It is an attractive alternative to embryo cryopreservation as it avoids some of the ethical issues surrounding embryo cryopreservation and storage. It would also offer a valuable option for fertility preservation in women facing chemo- or radio-therapy or other conditions that may have a deleterious effect on fertility.

The first births as a result of frozen eggs were reported back in 1986 (11). However the initial scientific work into freezing of mature eggs was dogged by both poor survival rates and low subsequent fertilization rates. Live birth rates per frozen-thawed egg varied from 1% to 10%. In fact results from the largest long-term study resulted in 9 births from over 1,500 thawed and subsequently fertilized eggs, giving a pregnancy rate of only 0.7% (12).

It appears that the mature egg is particularly susceptible to cryo-injury. The main biophysical factor that affects egg survival following cryopreservation is ice crystal formation within the egg leading to its demise. The newer freezing technique of vitrification – where the tissue is cooled ultra rapidly, thereby avoiding ice-crystal formation, has recently been used for these sensitive human eggs. A recent study comparing traditional slow freezing to vitrification in human eggs demonstrated improved survival and fertilization rates (13). Currently, preliminary data from the McGill University egg freezing research program has shown pregnancy rates between 30-40%.

Despite these encouraging results, egg freezing for preservation of fertility should still be regarded as an experimental treatment. Only 100-200 babies have been born worldwide following vitrification and egg freezing – and although there appears to be no increase in problems during pregnancy, childbirth or infancy – this is still too early to draw definite conclusions. Therefore, at present, fertility preservation with egg freezing should only be considered as part of a research study and ideally only for women who are likely to lose their fertility as a result of anti-cancer or other potentially sterilizing treatments.

The concept of ovarian tissue freezing was developed in the 1990s because of the poor success and difficulties with egg freezing at this time, primarily for patients wishing to retain future fertility prior to sterilizing cancer treatments or in those at risk of premature ovarian failure.

It is hoped that frozen ovarian tissue can be cultured in the laboratory and ultimately mature eggs developed which could then be fertilized and any resulting embryos used for treatment. At present, although there have been encouraging developments, the complete maturation of the egg and its successful fertilization has not been possible.

The other alternative use for frozen ovarian tissue is to transplant some tissue back to the women (after full recovery from her cancer). Following the initial case in New York in 2000, there have been several other successful cases where a return to normal menstruation has been reported (4, 14). In 2004, the first birth following ovarian tissue re-transplantation using previously frozen ovarian tissue was reported in Belgium in a woman 5 years after completing successful cancer treatment for Hodgkin’s disease (5). This remains the only livebirth worldwide so far.

There are many attractions for ovarian tissue freezing, including the non-fertility related benefits of restoring ovarian function in women who suffer a premature menopause as a result of their chemo- or radio-therapy. Not unsurprisingly, therefore, research programs performing ovarian tissue freezing are currently on-going in many university centres all over the world – at present the Canadian centre is McGill University.

However, many concerns remain. Firstly the transplanted tissue function appears often to be short-lived (this may be a function of the freezing-thawing process or the graft transplant) and secondly, there continues to be a risk of re-activating or re-seeding cancer in women who have survived their original cancer. Finally, with only one pregnancy and birth reported so far, there is no data regarding the safety during pregnancy or to any children conceived following ovarian tissue freezing and subsequent transplantation.

There is little doubt that the advances in cryobiology have contributed immensely to the treatment options available for couples experiencing infertility. However, along with these advances a number of specific ethical problems have arisen.

Age and death have become less significant factors with respect to reproduction. Concepts such as posthumous insemination with sperm from a dead partner, twins being born years apart, donation of embryos, the treatment of single women or lesbian couples, and the possible preservation of fertility for purely social reasons are now realities.

The storage of excess embryos has also proved to be a complex issue. Difficulties can arise when couples separate or disagree over the fate of any stored embryos. When one partner dies the fate of the remaining embryos is a matter of considerable debate.

In some countries the subsequent use of the embryos by the remaining partner is illegal.

Many more embryos are cryopreserved and stored than are ever used in frozen-thawed embryo transfer cycles. The ultimate fate of these stored embryos is a problem that authorities have tried to deal with in many countries. In 1996 in the United Kingdom over 3,000 cryopreserved embryos were destroyed as embryos could not legally be stored beyond 5 years without additional consent (15). The maximum storage period varies from county. In the United Kingdom, France and Canada there is a maximum 5 year storage period, whereas in Australia it is 10 years. In other countries, such as the United States there is no legal time limit.

As a science, cryobiology has a long history. The widespread introduction of assisted reproductive infertility treatments over the past twenty years has also been associated with a concomitant dramatic increase in the development and use of cryopreservation technology.

Cryopreservation is now considered an essential adjunct to modern reproductive treatments. It is now possible to cryopreserve eggs, sperm, embryos and ovarian tissue at a variety of different stages of their development to offer couples a significant range of options to suit their particular needs.

Developments in both the use of differing cryoprotectants and the freezing process itself have led to major improvements in survival and development following thawing.

As with assisted reproduction in general, cryopreservation technology has also led to the development of a number of moral and ethical issues surrounding its use. A number of these issues are unresolved and will continue to generate considerable debate. 

ABOUT THE AUTHOR: Dr. William Buckett is   Attending Physician and Sub Specialist in Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynaecology at the Royal Victoria Hospital, McGill University Health Centre in Montreal, Quebec, Canada. He is also Assistant Professor, Department of Obstetrics and Gynaecology, McGill University, Montreal, Quebec, Canada 

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