Hum. Reprod. Advance Access published online on April 9, 2009
Human Reproduction, doi:10.1093/humrep/dep079
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Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue
1 Department of Clinical Science, Technology and Intervention, Division of Obstetrics and Gynaecology, Karolinska Institutet, Karolinska University Hospital Huddinge, K 57, SE 141 86 Stockholm, Sweden 2 Fertility Unit, Mother-Infant Department, Institute of Obstetrics and Gynaecology, University of Modena and Reggio Emilia, 41100 Modena, Italy 3 Department of Laboratory Medicine, Karolinska University Hospital Huddinge, SE 141 86 Stockholm, Sweden 4 Department of Women's and Children's Health, Uppsala University, SE 751 85 Uppsala, Sweden
5 Correspondence address. Tel: +46-8-58583858; Fax: +46-8-5858-575, E-mail: Outi.Hovatta{at}ki.se
BACKGROUND: Controlled-rate freezing of ovarian cortical tissue for preservation of fertility among young women facing chemo- or radio-therapy is a widely accepted procedure. To improve the method for cryopreservation of ovarian tissue, particularly the stroma, we carried out a systematic comparison of vitrification versus slow programmed freezing.
METHODS: Ovarian tissue from 20 women, donated during Caesarean section, was used for parallel comparison of survival and detailed light and electron microscopic (EM) morphology of oocytes, granulosa cells and ovarian stroma after freezing (slow freezing and vitrification), thawing and 24-h culture. Using tissue obtained from the same patient, we compared four cryopreservation protocols and fresh tissue. The cryoprotectants used in slow freezing were 1,2-propanediol (PrOH)-sucrose and ethylene glycol (EG)-sucrose. For vitrification, tissues were incubated for 5 or 10 min in three solutions containing a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP).
RESULTS: Cryopreservation using controlled-rate freezing and vitrification preserved the morphological characteristics of ovarian tissue generally well. As revealed by morphological analysis, particularly EM, the ovarian stroma was significantly better preserved after vitrification than after slow freezing (P < 0.001). The follicles were similarly preserved after all freezing methods.
CONCLUSIONS: Vitrification using a combination of PrOH, EG, DMSO and PVP was comparable to slow freezing in terms of preserving follicles in human ovarian tissue. Ovarian stroma had significantly better morphological integrity after vitrification than after controlled-rate freezing.
Key words: cryopreservation/human ovarian tissue/ultrastructure/vitrification
Contributed equally to this article. Submitted on November 25, 2008; resubmitted on March 5, 2009; accepted on March 12, 2009.
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