Effect of ovarian tissue cryopreservation on gene expression and growth of human individual follicle in vitro

To investigate the effect of either slow freezing or vitrification for ovarian tissue cryopreservation on individual human early follicle gene expression and growth in three-dimensional (3D) culture system. Experimental study. Ovarian cortex tissue was collected from 11 patients (31.8±8.36 years of age) with obtaining written informed consent and cryopreserved using slow-rate freezing or vitrification. Tissues were fixed for histology analysis or used for early follicles isolation. Follicles were individually embedded in alginate(1% w/v) and cultured in vitro for 8 days. Follicle survival and growth were assessed by microscopy. Follicle viability was observed under confocal laser scanning microscope following Calcein-AM and Ethidium homodimer-I (Ca-AM/EthD-I) staining. Follicle gene expression was evaluated by single-cell mRNA analysis. After thawing, the percentage of damaged oocytes and granulosa cells was significantly higher in both slow freezing and vitrification group, as compared with fresh control (p<0.05). The growth of follicles in vitro was significantly delayed in vitrification group than fresh group (p<0.05), while the survival rate and viability appeared similar after both slow freezing and vitrification compared with the fresh control. Both of the slow freezing and vitrification down-regulated the gene expression of oct4, stella, zp3 and cyp11a (p<0.05), while there is no significant difference between the two groups. Although cryopreservation affect the morphology of human follicles and down-regulated gene expression of oct4, stella, zp3 and cyp11a, slow freezing offers similar conditions to fresh tissue for follicle growth, survival rate and viability than vitrification after in vitro culture. Compared to vitrification, slow freezing is considered to be the first choice for human ovarian cryopreservation.