Vitrification versus controlled-rate freezing in cryopreservation of human ovarian tissue.
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.
Department of Clinical Science, Technology and Intervention, Division of Obstetrics and Gynaecology, Karolinska Institutet, Karolinska University Hospital Huddinge, K 57, Stockholm SE 141 86, Sweden.
Victoria
V
Keros
Susanna
S
Xella
Kjell
K
Hultenby
Karin
K
Pettersson
Maryam
M
Sheikhi
Annibale
A
Volpe
Julius
J
Hreinsson
Outi
O
Hovatta
D000328:Adult; D001706:Biopsy; D002467:Cell Nucleus / Q000473:pathology / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005260:Female; D005615:Freezing; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods; D009685:Nuclear Envelope / Q000473:pathology / Q000648:ultrastructure; D006080:Ovarian Follicle / Q000473:pathology; D010053:Ovary / Q000473:pathology* / Q000648:ultrastructure
D003451:Cryoprotective Agents