The research, presented today (Monday 19 June) at the 22nd annual meeting of the European Society of Human Reproduction and Embryology, gives hope to hundreds of women who want to preserve their future fertility but who, for whatever reason, only have eggs, not embryos, available for freezing.
Dr Masashige Kuwayama told a news briefing that although sperm could be frozen, thawed and used for in vitro fertilisation with high levels of success, the freeze-thawing process could damage eggs and, until now, it had been very difficult to perform successful IVF using frozen-thawed eggs. Research by Professor Stefania Nottola and Dr Sandrine Chamayou, presented at the conference, gave examples of the current problems of conventional freeze-thawing and the need for more effective techniques. It is thought that worldwide less than 150 babies have been born using eggs that have been frozen.
Dr Kuwayama, scientific director of the Kato Ladies Clinic in Tokyo, Japan, has developed a new method of freezing eggs (oocytes) called the Cryotop method, which he first used for artificial insemination in sheep and cattle.
He said: "The Cryotop method is a highly efficient freezing procedure that opens a new way to resolve the various aspects of the problem of human oocyte cryopreservation. Using this method, we achieved a more than 90% survival rate for the freeze-thawed oocytes and a high pregnancy rate of nearly 42% after the oocytes had been fertilised and implanted in the women. This pregnancy rate is practically the same as the rate we can achieve in our clinic using fresh oocytes."
The Cryotop method involves very rapid freezing in a tiny amount (less than 0.1 microlitres) of a special vitrification solution, before storing in liquid nitrogen. This process prevents ice crystals forming, which do so much damage to the structure of the egg. Dr Kuwayama froze 111 eggs, of which 94.5% survived freeze-thawing, 90.5% were fertilised using ICSI (intra-cytoplasmic sperm injection), and 50% of the resulting zygotes were successfully developed for embryo transfer. Twelve pregnancies resulted from 29 embryo transfers (a pregnancy rate of 41.9%, compared with 42.5% using fresh eggs), with an average of 2.3 embryos transferred each time (compared with an average of 1.1 embryos formed from fresh eggs). Eleven healthy babies were born (nine singletons and one pair of twins), while two pregnancies miscarried. The women were aged between 25 and 37.
Dr Kuwayama said: "The conventional slow freeze-thawing method has been used successfully for human embryos in assisted reproduction for two decades, but has been far less successful for oocytes. The post-thaw survival rate for human embryos is about 85-90%. Now, using the Cryotop method, we can achieve post-thaw survival rates for oocytes of 90-95%, making oocyte cryopreservation a real option for women.
"This technology opens up new horizons for medically assisted reproduction in women, enabling them to have the option of having children at a later date by freezing eggs rather than embryos. Moreover, it will help to eliminate the existing time differences in fertility between men and women, whereby women's supplies of eggs decline at a faster rate than men's supplies of sperm."
Dr Chamayou, scientific director of the Unita di Medicina Della Riproduzione, Fondazione HERA, Catania, Italy, told the conference that Italy had banned embryo and zygote freezing in 2004, and any embryos created for IVF, up to a maximum of three, had to be implanted in one transfer. As a consequence of this new law, interest in egg freezing had grown. "Even although oocyte freezing is still considered experimental by the scientific community, nowadays in our centre it is routinely proposed to patients as an alternative to disposing of any surplus oocytes. Surplus oocytes become available in 78.5% of IVF cycles."
Her research into the potential of freeze-thawing eggs, using conventional methods of slow freezing and rapid thawing, revealed the consequences of freezing on fertilisation, subsequent initial cell division (cleavage) and embryo quality. Out of 337 thawed eggs, 263 survived (78%), 221 were fertilised using ICSI (67.9%), 116 underwent cleavage up to the second day (77.3%), 92 embryos were implanted in 37 embryo transfer procedures and two pregnancies resulted with two babies born (13.5%, although this figure was not significant due to the small numbers involved).
Dr Chamayou also found that the quality of embryos differed significantly depending on whether fresh or frozen eggs had been used; out of the total number of embryos, 36.7% were grade 1 embryos after IVF using fresh eggs, 19.5% were grade 1 after ICSI using fresh eggs (giving an overall percentage after IVF and ICSI of 24.7% grade 1 embryos), but only 12.1% were grade 1 using frozen-thawed eggs.
"We concluded that oocyte preservation decreased the proportion of cleavage and grade 1 embryos, but did not influence fertilisation rates," she said. One reason for the high percentage of failure after fertilisation could be the damage done to the structure of the egg during freeze-thawing, in particular to a part called the meiotic spindle which is involved in cell division. The meiotic spindle is a bundle of microtubules, some of which become attached to chromosomes, providing the mechanism for chromosomal movement.
"Before freezing we observed meiotic spindles in 62.5% of oocytes, but in only 43.4% after thawing. This is statistically significant and I deduced that cryopreservation may induce irreversible damage to the bonding of the microtubules," she said.
Professor Nottola, an associate professor of anatomy in the Department of Anatomy, University La Sapienza, Rome, Italy, told the conference that a variety of abnormalities became apparent when frozen-thawed eggs were inspected more closely, even in those that appeared undamaged after thawing. In addition, the type and/or concentration of agent that was used to protect the egg during freezing (cryoprotectant agent or CPA) might affect the eggs as well.
Six eggs were frozen using a multi-step procedure that involved increasing concentrations of ethylene glycol (EG) as a CPA. Twelve were frozen using propane-1,2-diol (PrOH) and two different amounts of sucrose as CPAs. After thawing, the eggs were studied using light and transmission electron microscopy (LM and TEM).
The eggs appeared normal when observed with LM, although some eggs showed the development of vacuoles (small cavities that sometimes contain water, food or metabolic waste) in their cytoplasm. When TEM was used, more differences became apparent. These included:
- changes to the linkages between mitochondria (microscopic respiratory structures in the cytoplasm) and intracellular membranes amongst the EG-treated eggs
- changes to the amount and density of cortical granules (particles that harden the exterior of the egg once it has been fertilised to prevent other sperm penetrating it)
- changes to the structure of the egg exterior (the zona pellucida).
TEM also confirmed that some eggs, particularly those treated with EG and with the PrOH agent containing more sucrose, had vacuoles developing – a process known as vacuolisation.
Professor Nottola said: "These data suggest that frozen-thawed oocytes may look similar to fresh oocytes, but when they are examined more closely it becomes apparent that there are a number of very small but important alterations to specific parts of numerous oocytes, which presumably are responsible for their reduced developmental potential. The type and/or concentration of the CPA used may play a role, at least in part, in producing these alterations. In particular, EG appears less suitable than PrOH.
"In my opinion, the most important alteration found in frozen-thawed oocytes could be the presence of vacuolisation. This is a quite non-specific feature commonly found in cells that are responding to an injury and, even in absence of other alterations, might lead to an impairment of the developmental potential of the frozen-thawed oocytes.
"As far as the other possible alterations are concerned, the reduction in amount and density of the cortical granules and the consequent hardening of the zona pellucida in frozen-thawed oocytes may be an expected phenomenon that possibly could be by-passed by the application of ICSI technique at insemination; other zona pellucida damage may greatly affect oocyte survival but it depends on the type and amount of the damage; alterations in the mitochondrial organisation can be very subtle and deserve to be further investigated by transmission electron microscopy."
Professor Arne Sunde, former chairman of ESHRE, said: "Cryopreservation of human semen and embryos has been a routine procedure since the early 1980s, while cryopreservation of mature oocytes has proved to be very difficult. Current techniques for freezing oocytes have a very low success rates in terms of viable pregnancies per frozen oocyte. This is probably due to basic biological differences between oocytes and sperm cells and embryos.
"For decades men have had the opportunity to freeze sperm prior to treatment for malignant diseases, and thousand of babies have been born to couples where the male is an infertile survivor of cancer treatment. With the current techniques, women would need to freeze hundreds of oocytes in order to have a reasonable chance of obtaining a child, but by using the technique of Dr Kuwayama and his colleagues, it will be possible to achieve the same rates of success with 10-20 frozen-thawed oocytes as with fresh oocytes. This is a major improvement, and for the first time, cryopreservation of oocytes represent a realistic option for the preservation of fertility in women who are in need of aggressive treatment for malignant diseases."
Abstract nos: O-022, O-024, O-025, Monday 10.15-11.15 hrs CET (Meeting Hall 1, Level 1)