Viability and function of the cryopreserved whole rat ovary: comparison between slow-freezing and vitrification

Initial findings suggest that a moderate concentration of the cryoprotectant dimethyl sulfoxide (DMSO) is superior to a high DMSO concentration for both vitrification and slow freezing.

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Milan Milenkovic, M.D., Ph.D., César Diaz-Garcia, M.D., Ann Wallin, B.Sc., Mats Brännström, M.D., Ph.D.

Vol 97, Issue 5, Pages 1176-1182



To investigate four different protocols for cryopreservation of the whole rat ovary with intact vasculature to evaluate whether differences exist in post-thawing viability of the ovary after either vitrification or slow freezing.


Experimental study.


Obstetrics and gynecology department.


Immature Sprague-Dawley female rats.


Ovaries were isolated with the vascular tree intact up to the bifurcation of the abdominal aorta and were subsequently cannulated. The ovaries were flushed with increasing concentrations of the cryoprotectant dimethyl sulfoxide (DMSO) to either 1.5 or 7 M. The ovaries underwent cryopreservation by vitrification or passive slow freezing.

Main Outcome Measure(s):

After thawing, the ovaries were subjected to neutral red viability staining to assess the density of viable small follicles and for long-term (48 hours) incubation evaluation of steroid secretion, histology, and apoptosis assay.


The follicular viability was decreased in both vitrification groups and in the slow-freezing group with the high concentration of DMSO, as compared with fresh controls. Estradiol levels in the incubation medium followed the same pattern. Light microscopy revealed well-preserved morphology in all groups after 48 hours' incubation. Apoptosis was increased in both vitrified and cryopreserved ovaries.


We have developed a new method that can be used in basic studies to improve cryopreservation protocols. Our initial findings suggest that a moderate concentration of the cryoprotectant DMSO is superior to a high DMSO concentration for both vitrification and slow freezing.

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Fertility and Sterility

Editorial Office, American Society for Reproductive Medicine

Fertility and Sterility® is an international journal for obstetricians, gynecologists, reproductive endocrinologists, urologists, basic scientists and others who treat and investigate problems of infertility and human reproductive disorders. The journal publishes juried original scientific articles in clinical and laboratory research relevant to reproductive endocrinology, urology, andrology, physiology, immunology, genetics, contraception, and menopause. Fertility and Sterility® encourages and supports meaningful basic and clinical research, and facilitates and promotes excellence in professional education, in the field of reproductive medicine.