Post-thawing and culture comparison of three routine slow freezing methods for human ovarian tissue cryopreservation: Histological, molecular, and hormonal aspects

To find the gold standard out of three pre-established routine slow freezing (SF) methods, ovarian cortex tissues of nine transsexual individuals were cryopreserved and compared to each other, as well as the control (fresh) samples. Histological, genomic, and endocrinological effects of the SFs were assessed post-thawing and after a seven-day culture period. SF1 included 10% dimethyl-sulfoxide (Me2SO) in the base medium (BM), SF2 had 1.5 M/L ethylene-glycol (EG) and 0.1 M/L sucrose in the BM, and SF3 consisted of 6% Me2SO, 6% EG and 0.15 M/L sucrose in the BM. The cortical tissue strips went under a programmed cooling process and were stored in liquid nitrogen. Histological criteria (tissue damage and follicular quality), as well as gene expression levels, were assessed in the thawed and control tissues. Half of the thawed and control tissues were cultured for seven days and their histology, genetic profile, and hormonal status were examined as the reflection of the avascular tension effect. Post-thawing tissue damage was similar between all groups but significantly increased post-culture (P < 0.05). The percentages of high-quality follicles diminished in all SFs after thawing and culture (P < 0.05) except for the similarity of post-thawing SF3, compared to control. The genetic profile of the tissue after thawing and culture suggested quiescence/activation balance in SF1 and 2 and significant down-regulation in SF3, compared to the control specimens (P < 0.05). Post-thawing BAX:BCL2 was higher than control in SF1 and SF3 (P < 0.05), while this ratio in SF2 was similar to the control. However, after culture this ratio was similar to that of control in SF3 and diminished in SF1 and 2 (P < 0.05). The expression levels of gap-junction genes showed dramatic pre- and post-thawing fluctuations in all groups. After culture, estradiol in SF3 was significantly higher than SF1 and 2 (P < 0.05). In addition, progesterone in SF3 was similar to control but significantly lower in SF1 and 2 (P < 0.05). In conclusion, all SFs showed advantages and disadvantages, and the follicular quality and its function depend on the type of cryoprotectant and the speed of thawing. The effects of freezing/thawing continue to appear during the seven days of culture. According to the results of this study, SF3 seems to be more promising in keeping the follicles functional and safe from cell damage during culture.