Viability of human ovarian tissue confirmed 5 years after freezing with spontaneous ice-formation by autografting and chorio-allantoic membrane culture

Abstract

To achieve optimal and uniform outcomes, slow cooling protocols for human ovarian tissues generally initiate ice formation at high sub-zero temperatures (−6 to −9°C). The aim of the study was to investigate the function of ovarian tissue that had unintentionally self seeded at −20°C during the freezing step, by examining its development following chicken embryonic chorioallantoic membrane (CAM) grafting and after transplantation back to the patient. Ovarian tissue was frozen in 6% (v/v) dimethyl sulfoxide, 6% (v/v) ethylene glycol and 0.15M sucrose which had self-seeded at −20°C. Five years after cryopreservation, 8 pieces were thawed and transplanted back to the patient. Two small (1×2×1mm) pieces of this thawed tissue were cultured in a CAM-system for 5days to assess the tissue viability. The autografted ovarian tissue re-established spontaneous menstrual bleeding within five months and raised serum 17-β Estradiol from 19 to 330pg/ml. Ultrasound revealed a dominant follicle at the site of the transplanted tissue in the follicular phase after the menstrual bleed. Analysis of the CAM cultured tissue established that 88% of the primordial follicles are degenerated and there was limited in growth of blood vessels. In conclusion, in spite of the damage caused by the cryopreservation with spontaneous ice-formation the viability could be confirmed by CAM culture and the restoration of ovarian function after auto-transplantation.