Purpose To evaluate the efficiency of an original slow freezing protocol on the quality and function of human ovarian cortex. Methods Human ovarian tissues were cryopreserved using a freezing medium supplemented with propanediol and raffinose as cryoprotectants and antioxidants (L-glutamine, taurine). Samples were then frozen using a faster cooling rate than the usual one. Viability and morphology of follicles, DNA fragmentation in follicles and stroma as well as histology of the vascular endothelium were analyzed before and after freezing/thawing. Moreover, a functional analysis was performed based on the evaluation of follicular growth and development in thawed ovarian tissues that were cultured in vitro. Results Our freezing/thawing protocol allows preservation of a high proportion of viable follicles and the preservation of the different follicle developmental stages (p>0.05 versus fresh control). 70.5±5.2 % of follicles retained an intact morphology after cryopreservation (p=0.04). Stroma cells but not follicles exhibited a slight increase of DNA fragmentation after thawing (p<0.05). Microvessel endothelium within thawed tissues appeared to be preserved. Granulosa cells showed signs of proliferation in follicles cultured for 12 days. Secretion of 17β-oestradiol significantly increased during in vitro culture. Conclusions This protocol leads to good preservation of ovarian integrity and functionality post-thawing and thus appears as a suitable technique of ovarian tissue cryopreservation in clinical settings. Further research could be extended to optimize conditions of in vitro culture.

Compared with CaAM/EthD-1, TB appears to be more reliable as a staining method for follicle viability evaluation. TB staining is a quick and useful method, complementary to histological analysis for quality control in ovarian tissue cryopreservation.

BackgroundCryopreservation of ovarian tissue can be used to preserve the fertility of patients who are about to receive treatment(s) that could compromise their future ovarian function. Here we evaluate the effectiveness of a vitrification protocol by carrying out a systematic comparison with a conventional slow-freezing method on human ovarian tissue.MethodsHuman ovarian samples (mean age 28.0 ± 1.1 years) were processed in parallel for each cryopreservation procedure: vitrification and slow-freezing. Following warming/thawing, histological observations and a TUNEL assay in ovarian follicles were performed and compared to unfrozen control.ResultsBoth cryopreservation protocols gave comparable histological outcomes. Percentage of intact follicles was 83.6 % following vitrification in a 1.5 M 1,2-propanediol (PrOH), 1.5 M ethylene glycol (EG) and 0.5 M raffinose solution, 80.7 % after slow-freezing in 1.5 M PrOH and 0.025 M raffinose, and 99.6 % in fresh tissue. Follicle density was unchanged by vitrification (0.6 follicles/mm2) or slow-freezing (0.5 follicles/mm2) compared to fresh tissue (0.7 follicles/mm2). Percentage of follicles with DNA fragmentation was not statistically different in vitrified (20.8 %) or slow-frozen (31.3 %) tissues compared to the unfrozen control (35.0 %). There was no difference in proportion of stroma cells with DNA fragmentation in vitrified (6.4 %) and slow-frozen (3.7 %) tissues compared to unfrozen tissue (4.2 %).ConclusionsThis vitrification protocol enables good preservation of ovarian quality post-warming. The evaluation of endocrine function after vitrification need to be perform in a higher cohort to evaluate if this protocol may offer a relevant alternative to conventional slow-freezing for the cryopreservation of human ovarian tissue.