Molecular and Cellular Endocrinology 169 (2000) 33 – 37 Clinical experience and applications of oocyte cryopreservation E. Porcu *, R. Fabbri, G. Damiano, S. Giunchi, R. Fratto, PM. Ciotti, S. Venturoli, C. Flamigni Uniersita di Bologna, Clinica Ostetrica e Ginecologica, Via massarenti 13, 40138 Bologna, Italy Abstract Oocyte cryopreservation is a viable solution for the ethical problems related to embryo storage, and the only available technique for preservation of fertility in women who have to undergo chemo- or radiotherapy. The main problems with oocyte cryopreservation are concerned with the survival rate and the fertilization rate. Recently the introduction of the intracytoplasmic sperm injection (ICSI) led to an increase in the fertilization rate. The success achieved with the first case treated encouraged us to set up a clinical trial on human oocyte cryopreservation. In the first stage of the study, 23 women with tubal infertility were enrolled. Superovulation was induced and 375 oocytes were retrieved; of these 338 oocytes were frozen. The survival rate was 59.5% and was independant of the duration of cryopreservation or the presence of cumulus. The normal fertilization rate was 64.4%, and only 7.5% of fertilizations were abnormal. A total of 90.8% of fertilized oocytes cleaved. A mean of 3.1 1.3 embryos per patient were transferred. Three pregnancies were achieved. In the second stage of our investigation, more patients were enrolled and similar results were observed. Sixteen pregnancies were achieved. A further stage of the investigation involved the fertilization of frozen oocytes with frozen sperm and even these resulted in a pregnancy. Our study demonstrated that pregnancies can also be achieved when frozen eggs are fertilized by testicular and epididymal sperm. As a consequence of the success of our investigations, a program of oocyte cryopreservation for oncological patients has been initiated in our centre. In our opinion, oocyte cryopreservation is, at present, a safe and efficient technique as documented by the birth of several healthy children. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Oocyte cryopreservation; intracytoplasmic sperm injection; In vitro fertilization www.elsevier.com/locate/mce 1. Introduction The cryopreservation of female gametes is the solu- tion of several problems in assisted reproductive tech- niques. It is a successful alternative, for storing the excess oocytes and avoiding the development of more embryos than those required for a single transfer in human in itro fertilization (IVF). Besides, this could be a useful method for all the patients at risk of losing their ovarian function because of pelvic diseases, surgery or radio/chemotherapy and consequent ovarian damage. The main problems of oocyte cryopreservation con- cern the survival rate and the fertilization rate. Varying percentages have been reported but in most experiences they are very low. The main reasons accounting for this can be found in oocyte damage, such as cracks in the zona pellucida, disruption of the meiotic spindle, spon- taneous discharge of the cortical granules and plasma membrane and cytoplasmic organelle damage. These alterations may lead to the increased incidence of parthenogenesis, reduced fertilization rate or the in- creased incidence of polyspermic fertilization. Recently intracytoplasmic sperm injection (ICSI) has been proposed as a solution to cryopreservation dam- age involving the zona pellucida and cortical granules. ICSI should increase the fertilization rate and avoid polispermic fertilization. Kazem et al. (1995) recently demonstrated a higher fertilization rate in frozen oocytes inseminated with ICSI compared to IVF while Gook et al. (1995) showed a higher number of embryos cleaving up until the seventh day. Our team decided to undertake a study to ascertain the efficacy of ICSI in fertilizing frozen thawed oocytes and to translate into clinical results the basic knowledge deriving from previ- ous investigations of other authors. The data of Gook et al. (1994) documenting normal kariotypes and the absence of stray chromosomes were reassuring. In addi- * Corresponding author. Fax: +39-051-305806. 0303-7207/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0303-7207(00)00348-8