Ocyte Cryopreservation
Printer-friendly versionA new opportunity for women
by Andrea Borini, MD
Spring 2006
Last May, the McGill University Health Centre (MUHC) announced the first successful birth in Canada resulting from frozen eggs. This was fantastic news for fertility health research and for women! Moreover, 15 McGill Reproductive Centre patients had embryo transfers produced from vitrified eggs, resulting in 7 pregnancies. A press release reported that the McGill Reproductive Centre was pioneering a revolutionary new freezing technique, which had dramatically increased the egg survival rate to approximately 90 percent.
My friend and colleague,Dr. Andrea Borini, Medical Director of the Tecnobios Procrazione Centre for Reproductive Health in Bologna, Italy, provides some history on oocyte cryopreservation and highlights what’s on the horizon for this exciting new technology. Dr. Borini also suggests how experts might continue to investigate innovative techniques for preserving female fertility. – Bev Hanck
Almost twenty years have gone by since the first pregnancy using frozen oocytes was announced.1
In the first ten years following that announcement very few babies were born, and the technique was abandoned. It was only after another ten years that the first pregnancy using thawed oocytes and ICSI was announced.2
This new insemination technique bypassed the difficulties the sperm had encountered in penetrating the zona pellucida – the protein shell surrounding the thawed oocytes. In this way great progress was made in understanding why frozen oocytes were giving such highly unsatisfactory results.
Since then, over 100 children have been born, especially in Italy.
In our country, even before Law 40 was passed, couples undergoing IVF expressed a keen interest in trying, for ethical-moral reasons, to avoid the generation of supernumerary embryos during an ART cycle. They would therefore ask to freeze the unimplanted oocytes in order to use them in subsequent ART cycles.
Since 2003, after the introduction of Law 40, which forbids the formation and hence the freezing of supernumerary embryos, patients have increasingly requested oocyte cryopreservation and results have steadily improved.
Furthermore, the oocytes cryopreservation gives women the same opportunities men have had for decades – the chance to preserve their fertility.
There are in fact at least two good reasons why a woman might want to freeze her oocytes for later use: the need, if she happens to develop cancer, to undergo radiation or chemotherapy treatments, which might cause her to become sterile; or quite simply the desire to become a mother later on.
Women are most fertile around age 20, after which fertility steadily declines.. If oocyte freezing were reliable and offered a high probability of success, many women might well decide to freeze oocytes at age 20 and use them when they are 35 or older, an age when many career women decide to try for children with obviously lower chances of a successful pregnancy.
The benefits of cryopreservation are even more obvious for women who could remain sterile after radiation or chemotherapy. Many studies are being carried out on this front, and reasons for hope come not only from the increasingly positive results obtained using frozen oocytes but also from the number of pregnancies achieved from the freezing of parts of the ovary, their thawing and their subsequent autotransplantation.
Up to now, the only chance these women had was to form embryos, freeze them and use them later. It is fairly obvious, however, that this was a realistic option only for women with a stable, permanent relationship.
Oocyte freezing means it is possible to bypass the legal, moral and ethical problems posed by embryo freezing. Indeed, the possibility of freezing female gametes and destroying them later, if not used, does not involve particular problems.
But what are the technical and biological problems that make the freezing of oocytes and their later use to achieve pregnancy so difficult? About ten years ago at the ESHRE conference we reported 15 pregnancies in 68 patients who had requested the freezing of supernumerary oocytes, obtained during an ART treatment, so as to avoid the creation of supernumerary embryos.
We recently reported in Fertility and Sterility the birth of 13 healthy children (one pregnancy was twins while three out of the 15 pregnancies miscarried).3
The problem addressed in these early treatment cases was the low rate of 37% oocyte survival after thawing. The fertilization rate of 45.4% was low compared to that obtained from fresh oocytes, though the embryo cleavage rate of 86.3% was perfectly normal.
Once formed, these embryos were able to implant with rates (about 16%) comparable to those derived from fresh oocytes. Overall, this initial experience showed that births from cryopreserved oocytes were not necessarily sporadic and that could be achieved in a reproducible fashion.
In order to make this treatment efficient, it was obvious the need to increase the rate of survived oocytes after freezing and thawing. Studies on the cryobiology of the human oocyte have been insufficient. It was not until 2001 that preliminary experiments conducted at the University of Bologna4 demonstrated that it was possible to obtain survival rates as high as 80% by increasing the concentration of the cryoprotectant (CPA) sucrose in the freezing solution. The mode of action of CPA is complex and not entirely understood, but in this specific case it is thought that a higher sucrose concentration causes an increase in cell dehydration. This effect, in turn, lowers the risk of ice crystals forming between cells and possibly causing of cell death through mechanical damage during ice formation. We recently tested this protocol5 in one of the largest studies published so far on this subject.
Our results confirmed that survival rates could be dramatically improved, while fertilization rates could be raised to figures as high as those of fresh oocytes (75% to 80%). The embryos derived from oocytes stored with this protocol showed normal morphology (in terms of blastomere shape, texture of the cytoplasm, presence of extracellular cytoplasmic fragments, etc.) in a proportion similar to embryos generated from fresh oocytes. Even so, the rate of implantation was very low – not higher than 6%. In final analysis, the number of implanted embryos, expressed as a fraction of thawed oocytes, remained unchanged compared to the previously tested protocol.
It is a somewhat paradoxical situation. Cryopreservation conditions, with their high sucrose concentration, seem to solve the problems of low survival and fertilization rates, which have so far limited the adoption of oocyte storage as a standard procedure. At the same time, however, they appear to compromise embryo viability. Whatever the cause of this failure, it seems to occur in a way that is not detectable through the routine assessments, mainly based on standard microscopic observation performed in the embryo lab.
Even after investigation with more sophisticated techniques, it remains unclear what causes partial loss of developmental ability in oocytes frozen with such an “improved” protocol, although we have recently obtained evidence on the effect of freezing on oocytes. Initially, we have observed the constitution of the meiotic spindle, the element of the cell skeleton that is crucial to cell viability and that is believed to be irreversibly disrupted after exposure to low temperatures. Surprisingly, this structure is in fact preserved unaltered in frozen-thawed oocytes, evidence that makes the problem of oocyte freezing rather intriguing.
In electron microscopy studies, however, we found that in oocytes frozen with the protocol involving a higher sucrose concentration, the intracellular organization appears to be damaged in some respects – a condition that could account for the observed loss of viability.
These findings show that the development of efficient oocyte cryopreservation protocols is an arduous task and that initial results that could induce to optimism should be considered with prudence.
We are continuing our commitment in the development of novel and more efficient oocyte cryopreservation methods. Recently tested alternative protocols have generated preliminary results that approach those normally achieved with embryo freezing in terms of overall efficiency. But more extensive investigations are needed before coming to a definite conclusion.
1. C. Chen, “Pregnancy after human oocyte cryopreservation,” Lancet1, no. 8486 (April 19, 1986).
2. E. Porcu et al., “Birth of a healthy female after intracytoplasmic sperm injection of cryopreserved human oocytes,” Fertility and Sterility 68, no. 4 (October 1997).
3. A. Borini, M. A. Bonu, G. Coticchio et al., “Pregnancies and births after oocyte cryopreservation,” Fertility and Sterility82, no. 3 (September 2004).
4. R. Fabbri, E. Porcu, T. Marsella et al., “Human oocyte cryopreservation: New perspectives regarding oocyte survival,” Human Reproduction 16, no. 3 (March 2001).
5. A. Borini, R. Sciajno, V. Bianchi et al. “Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration,” Human Reproduction AdvanceAccess, October 20, 2005.