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Endocrine Abstracts (2010) 22 S7.3

Tecnobios Procreazione, Bologna, Italy.

IVF treatment would gain considerable advantage from a safe and efficient oocyte cryopreservation method. Embryo freezing, which involves important legal and ethical drawbacks, would be no longer needed. Women facing a destiny of premature ovarian failure or requiring oocyte donation would also benefit from oocyte cryopreservation. Studies conducted in the last few years suggest that oocyte cryopreservation can be applied in a systematic and reproducible fashion, in some cases with success rates which appear to compete with those routinely achieved with embryo freezing. Vitrification techniques, recently introduced in the field of human IVF, have further enhanced the hope of developing oocyte storage as a viable assisted reproduction strategy. Nevertheless, doubts have been raised against oocyte freezing, especially with respect to its safety. Several investigations have been conducted to detect possible freezing-induced cellular alterations which might compromize oocyte viability and, indirectly, long term effects on the conceptus. From a clinical perspective, the options by which the stored material can significantly influence the clinical outcome of oocyte freezing, and make the comparison between different experience difficult. Since the introduction in Italy of an IVF law in 2004, the approach of thawing at each treatment cycle a small number of oocytes has been adopted in various studies (Borini et al. 2006, Levi Setti et al. 2006, De Santis et al. 2007, Parmegiani et al. 2008), in compliance with the legal restriction of not producing more than three embryos. In other contexts, oocyte thawing cycles has been performed without limitations to the number of embryos which may be generated and therefore the number of oocytes which may be thawed and used at each cycle (Chen et al. 2005, Boldt et al. 2006). The two approaches clearly are not directly comparable. The thawing of only a few oocytes per cycle involves a higher risk of premature treatment interruption as a consequence of increased incidence of failed fertilization or cleavage (Borini et al. 2004). Besides, the option of embryo selection is not practicable and in some cases the number of transferred embryos may be insufficient. Obviously, this scenario, which does not occur when an excess of oocytes are thawed in individual attempts, can significantly affect the clinical outcome in terms of pregnancy rate per thawing cycle and per transfer, as well as the implantation rate classically defined as the ratio between implantations and embryos transferred. For such a reason, it is difficult to compare the pregnancy rate of studies in which the mean number of embryos transferred was as different as 1.1 (Borini et al. 2004) and 4.6 (Lucena et al. 2006). An example of the inadequacy of the rate of pregnancy per transfer as an efficacy measure is offered by a study (De Santis et al. 2007) in which the conventional slow-freezing method was reported to produce a success rate of 16.7%. In reality, the rate of pregnancy per thawing cycle was much lower (7.7%), with more than 50% of cycles interrupted as a consequence of no oocyte survival after thawing or failed fertilization. Crucially, the validity of a criterion to assess the efficiency of oocyte cryopreservation depends on the inclusion in the calculation of all the events of attrition at pre- and post-storage stages. Only under such conditions do certain differences between alternative methods become apparent. For example, it is well known that a major improvement (from 35–40 to 70–75%) in the survival rate of oocytes frozen and then thawed via slow cooling may be obtained by increasing the sucrose concentration in the freezing solution from 0.1 to 0.3 mol/l (Fabbri et al. 2001). This change also improves the rate of fertilization (Borini et al. 2006, Levi Setti et al. 2006, De Santis et al. 2007). However, the different degree of attrition at the steps of survival after thawing and on fertilization in the 0.3 mol/l sucrose protocol is counterbalanced by a higher implantation rate of embryos generated by the protocol involving the lower sucrose concentration (Borini et al. 2004, De Santis et al. 2007). The overall outcome, considered as the proportion of implantations per thawed oocyte, ultimately makes the efficacy of the two methods very similar (2.4 vs 2.6%) and in any case insufficient for competing with embryo freezing. Other methods have suggested that implantation rates in excess of 5% per oocyte used could be obtained, approaching a success rate which normally is attained with frozen embryos. Using a freezing protocol involving the replacement of sodium with the less toxic cation cholin and the inclusion of Hepes as a pH buffer, Boldt et al. (2006) reported a remarkable implantation rate of 5.3% per oocyte used. It should be mentioned, however, that this result is derived from the treatment of only 23 patients and so far it has never been confirmed with a larger series of treatments. Our group reported a similar value (5.9%) of implantation rate per oocyte used, following the application of a protocol based on differential concentration of sucrose in the freezing (0.2 mol/l) and thawing (0.3 mol/l) solutions (Bianchi et al. 2007). A similar implantation rate per thawed oocyte has been more recently reported by another Italian group (Parmegiani et al. 2008).

In recent years, the alternative cryopreservation approach of vitrification has raised several hopes. By adopting the cryotop vitrification method in cycles involving young donors (mean age 26.7 years), Cobo et al. (2008) achieved an implantation rate per embryo transferred of more than 40%. Nevertheless, when this study is analyzed considering the original number of oocytes used the implantation rate corresponds to a value (8.6%). Such a rate is not very dissimilar from the one (7.3%) resulting from a study conducted by a slow cooling method and including patients with a mean age of 33.7 years (Bianchi et al. 2007).

After February 2004, as an effect of the above mentioned IVF law, our program contemplated oocyte cryopreservation as an alternative to embryo freezing in order to maximize the potential of all oocytes retrieved from individual stimulation cycles. The cumulative pregnancy rate, including pregnancies from both fresh and frozen–thawed oocytes, was over 47%. Recently tested oocyte freezing methods which have shown to give rise to embryos with higher implantation ability (Bianchi et al. 2007) could further raise the hopes of achieving cumulative pregnancy rates well over 50%.

Evidence gained from approximately one thousand babies born from frozen oocytes, although still insufficient and incomplete, has not suggested so far that the process of freezing and thawing is associated with an increase in birth abnormalities. Although the number of published studies is relatively still modest, the number of oocyte cryopreservation treatments is dramatically increasing. This will have undoubtedly a significant impact on the practice of human IVF in the near future.

Bianchi V, Coticchio G, Distratis V, Di Giusto N, Flamigni C & Borini A. Differential sucrose concentration during dehydration (0.2 mol/l) and rehydration (0.3 mol/l) increases the implantation rate of frozen human oocytes. Reprod Biomed Online 2007 14 64–71.

Boldt J, Tidswell N, Sayers A, Kilani R & Cline D. Human oocyte cryopreservation: 5-year experience with a sodium-depleted slow freezing method. Reprod Biomed Online 2006 13 96–100.

Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M & Flamigni C. Pregnancies and births after oocyte cryopreservation. Fertil Steril 2004 82 601–605.

Borini A, Sciajno R, Bianchi V, Sereni E, Flamigni C & Coticchio G. Clinical outcome of oocyte cryopreservation after slow cooling with a protocol utilizing a high sucrose concentration. Hum Reprod 2006 21 512–517.

Chen SU, Lien YR, Chen HF, Chang LJ, Tsai YY & Yang YS. Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 2005 20 1975–1980.

Cobo A, Kuwayama M, Perez S, Ruiz A, Pellicer A & Remohi J. Comparison of concomitant outcome achieved with fresh and cryopreserved donor oocytes vitrified by the Cryotop method. Fertil Steril 2008 89 1657–1664.

De Santis L, Cino I, Rabellotti E, Papaleo E, Calzi F, Fusi FM, Brigante C & Ferrari A. Oocyte cryopreservation: clinical outcome of slow-cooling protocols differing in sucrose concentration. Reprod Biomed Online 2007 14 57–63.

Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S & Flamigni C. Human oocyte cryopreservation: new perspectives regarding oocyte survival. Hum Reprod 2001 16 411–416.

Levi Setti PE, Albani E, Novara PV, Cesana A & Morreale G. Cryopreservation of supernumerary oocytes in IVF/ICSI cycles. Hum Reprod 2006 21 370–375.

Lucena E, Bernal DP, Lucena C, Rojas A, Moran A & Lucena A. Successful ongoing pregnancies after vitrification of oocytes. Fertil Steril 2006 85 108–111.

Parmegiani L, Cognigni GE, Bernardi S, Ciampaglia W, Infante F, Pocognoli P, de Fatis CT, Troilo E & Filicori M. Freezing within 2 h from oocyte retrieval increases the efficiency of human oocyte cryopreservation when using a slow freezing/rapid thawing protocol with high sucrose concentration. Hum Reprod 2008 23 1771–1777.

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