A comparative study of biological sample preparation methods conventional cryopreservation of human tissue

Background/Aims: Data that compare the effectiveness of methodologies for cryopreservation of human tissue are very limited. Thus, two different biological sample preparation methods, the conventional cryopreservation of human ovarian tissue using either spontaneous or initiated (so-called “seeded”) ice formation, was carefully investigated and compared. Materials and Methods: Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy, and small pieces of experimental tissue (0.5 mm × 1 mm × 1–3 mm) were randomly distributed into three different groups: group 1 immediately after biopsy, experimental pieces after cryopreservation (thawing) with spontaneous ice formation (Group 2) and cryopreservation with initiated ice formation (Group 3). The pieces of tissue were cultured in vitro for 16 days, after which follicle viability and hormonal activity were evaluated. The level of statistical significance was set at P < 0.05. Results: The obtained results indicated that culture supernatants for Groups 1, 2, and 3 showed estradiol 17-ß concentrations of 476, 465, and 459 pg/mL, respectively. Whereas pProgesterone concentrations were 9.68, 5.77, and 5.61 ng/mL, respectively. In addition, the mean primordial follicle density per mm3 for Group 1 was 12.1 ± 3.9, 3.1 ± 1.4 for Group 2 and 6.0 ± 2.3 for Group 3. Moreover, it was recognized that 91%, 16%, and 87% follicles for Groups 1, 2, and 3, respectively, were normal (P2-1, 3 < 0.05; P1-3 > 0.1). Conclusions: The obtained results revealed that for the best results using cryopreservation of human ovarian tissue, the protocol of conventional cryopreservation must include a step of initiated ice formation. Moreover, an advanced analytical detection technique of high sensitivity, mass spectrometry, can further be used in the future for an accurate determination of hormonal levels and other related compounds and for screening of possible biomarkers using the best-obtained sample preparation method.