Freezing human eggs - new findings point way to greater success

Freezing human eggs has proved one of the most difficult techniques to master in reproductive medicine and only about thirty babies have been born worldwide since the first attempts in 1986. But research published today*(Tuesday 27 February) in Europe's leading fertility journal, Human Reproduction, could lead to a significant improvement in success rates.

Italian fertility experts lead by Dr Rafaella Fabbri from the IVF Centre, Human Reproductive Medicine Unit at the University of Bologna, have identified crucial factors in the freeze-thaw process which contribute to the difference between success and failure. Putting their findings into practice in a laboratory study, they achieved a much higher than normal rate of egg survival and fertilised 57% by using ICSI (the injection of a single sperm into the egg). This is a figure comparable to fertilisation of fresh oocytes.

The key to their success was to increase the time the oocytes were exposed to the chemical protectants, propanediol plus sucrose, from around 10 minutes to 15 minutes and to double or even triple the normal amount of sugar (sucrose) in the freezing solution.

The main reason that human eggs disintegrate in the freezing process is inadequate dehydration. Longer exposure to the protectant and more help from the extra sucrose meant that the eggs were dehydrated gradually and more completely. This gentler freezing process minimised the formation of ice crystals, which can pierce the egg membrane and kill the cell.

The researchers further established that one factor thought to be important in successful cryopreservation - the presence of the protective nourishing cells surrounding the oocyte (cumulus oophorus) - did not actually have a bearing on success. There was no real difference in survival between eggs frozen with their cumulus oophorus partially removed or totally removed.

But, increasing the exposure time to the protectant and doubling the sucrose solution in the freezing process from 0.1mol/l to 0.2mol/l increased egg survival from 34% to 60%. If the solution was tripled, survival rose to 82%.

Dr Fabbri said: "Our next step will be to improve even further the oocyte survival rate after thawing. But our study has established that it is possible to cryopreserve human ooctyes and that ICSI could be an efficient method of achieving satisfactory fertilisation. Reassuringly, over 90 per cent of the fertilised oocytes reached cleavage stage on day two, which is an indication of the potential viability of the embryos, and three-quarters of the embryos were of very good or fairly good quality."

She said that ICSI would be the fertilisation method of choice for frozen oocytes because it could bypass potential problems arising from cryopreservation - possible hardening of the outside membrane of the egg or deficiencies in the cortical granules that normally prevent more than one sperm penetrating the egg.

Dr Fabbri hopes many centres worldwide will adopt the technique and that it will soon become routine practice. "It is very important that we improve the possibilities for women to become pregnant after having their eggs frozen. It will offer hope to women who lose their fertility through medical treatments or who may be at risk of premature menopause. It will also help to avoid freezing embryos and storing excess embryos."

*Human oocyte cryopreservation: new perspectives regarding ooctye survival. Human Reproduction. Vol 16. No 3. pp 411-416

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Two other papers in the current issue examine the problems and potential of freezing ovarian tissue. Scientists want to develop tissue freezing, as well as egg freezing, as another feasible method of helping women and girls whose fertility is threatened by treatment for illness, or who are at risk of a premature menopause.

First confirmation that ovarian tissue can develop normally after freezing Dr Debra Gook and colleagues from Melbourne have reported the first study** to confirm that human ovarian follicles have the potential to grow and develop after freezing.

Two women due to undergo cancer treatment donated ovarian tissue. This was preserved and frozen in sucrose and the chemical propanediol, and, after thawing, grafted under the kidney capsules of immunodeficient mice. Unfrozen tissue was also grafted as a control.

There was no evidence of ovarian follicle development in the tissue at the time of grafting. But, when the grafts were recovered from the mice around 20 weeks later, the researchers identified normal antral follicles in the majority of both fresh and frozen tissue. Antral follicle development is the crucial stage shortly before ovulation, when a central cavity is formed in the follicle.

The leader of the research team, Dr Debra Gook of the Reproductive Services Unit at the Royal Women's Hospital, said: "Our study is the first to confirm normal growth and development of human follicles to the antral stage following cryopreservation. Although this is preliminary work, development of these follicles after 20 weeks within almost all of the grafts, suggests that development following grafting is reproducible. The high rate of functional preservation of follicles following cryopreservation also suggests that cryopreservation of ovarian tissues has real potential for clinical application."

** Development of antral follicles in human cryopreserved ovarian tissue following xenografting.Human Reproduction.Vol 16 No 3 pp 417-422

Growing mouse ovarian follicles - future tips for freezing human tissue? A study*** by two Sydney-based researchers is the first to show that pre-antral mouse follicles isolated from frozen and thawed ovarian tissue can be grown in-vitro to the point of antral development. They can also release mature eggs after being stimulated with human chorionic gonodatrophin - a hormone used to stimulate the ovaries in IVF treatment.

Dr Helen Newton and Dr Peter Illingworth of the Department of Reproductive Medicine at Westmead Hospital, wanted to identify the best methods of preserving, freezing and thawing mouse follicles to find pointers towards the optimum method for dealing with human ovarian tissue.

They established that bigger pre-antral follicles grew rapidly and formed antral cavities but smaller follicles died rather than grew, that dimethyl sulphoxide or propylene glycol were better protectives than propylene glycol and that the optimum thawing temperature was 27 degrees C.

Dr Newton said: "The significance of these findings for the future of in-vitro human follicle culture is unclear. Nevertheless, since the number of patients having ovarian tissue stored is increasing it may be prudent at this early stage to optimise freezing protocols. This would ensure that if in-vitro growth of human follicles becomes a possibility, the success rate will not be jeopardised by poor initial freezing techniques."

*** In-vitro growth of murine pre-antral follicles after isolation from cyropreserved ovarian tissue. Human Reproduction. Vol 16. No 3. pp 423-429

Notes:

1. PDF version of this press release and full embargoed text of the papers with complete results and participating research teams, can be found from noon GMT Sunday 25 February at: http://www3.oup.co.uk/eshre/press-release/mar01.pdf
2. Human Reproduction is a monthly journal of the European Society of Human Reproduction and Embryology (ESHRE). Please acknowledge Human Reproduction as a source. ESHRE�s website is: http://www.eshre.com
3. Printed texts available on request from Dr Helen Beard, Managing Editor. Tel: 44-0-1954-212404 or email: beardh@humanreproduction.co.uk

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