Early human follicle ultrastructure comparison after slow cryopreservation in two different cryoprotectants

[摘要] BACKGROUND: The cryopreservation and transplantation of ovarian tissue havebeen shown to restore ovarian function temporarily and may also preserve the fertilityof young female cancer patients until after their sterilizing cancer treatment. Sincetissue samples are large and morphologically complex, the cryopreservationmethodology is difficult to optimize and standardise. Ovarian tissue cryopreservationis therefore, still in its experimental stages and is not a routine option offered tocancer patients.OBJECTIVES: Our main aim was to initiate, develop and implement a practicalovarian tissue cryopreservation and re-transplantation protocol which would restoreovarian function, and possibly fertility, in young female cancer patients undergoingsterilizing cancer therapies in South Africa. The objective of this study was toimprove the slow cryopreservation protocol for human ovarian tissue. Theultrastructural effects after cryopreservation with two well-known cryoprotectants,dimethyl sulfoxide (DMSO) and 1,2-propylene glycol (PROH), on early humanfollicles were investigated and compared to identify and the better cryoprotectant.MATERIALS AND METHODS: A single group experimental study design was used.The participants consisted cancer patients of the Gynaecological Oncology Unit ofTygerberg Hospital who entered on a basis of voluntary informed consent. Ovariantissue was obtained by laparoscopic oophorectomy. After dissection of theovary(ies), some fresh cortical tissue was sent for metastatic analysis and a fewstrips taken as fresh control. Remaining dissected ovarian cortical tissue sections ofeach patient were equally divided into the two cryoprotectant groups. Five resultinggroups could be compared: i) fresh tissue (control group); tissue equilibrated in ii)DMSO; or iii) PROH and tissue equilibrated and cryopreserved in iv) DMSO or v)PROH. Five tissue samples per patient were therefore fixed for standard histologicalhaematoxylin and eosin (HE) staining and transmission electron microscopy (TEM).Tissue samples showing early follicles on HE slides were sent for TEM processing.Ultrastructural studies on micrographs of primordial and primary follicles wereassessed according to a scoring system which gave an indication of follicular health.Appropriate statistical tests were applied to analyse the mean scores where P≤0.05was considered as statistically significant. RESULTS: No significant overall cryopreservation treatment effect was evident inany of the follicular ultrastructures evaluated. This result indicated that thecryopreservation protocol used did not induce significant damage to the cortex tissuecompared to the fresh control group. Comparison of the effect of PROH and DMSOon the follicular ultrastructures showed that PROH tend to induce more extensivedamage, especially after cryopreservation. Correlation studies showed significantpositive relationships between the majority of the evaluated ultrastructures, especiallybetween the oocyte and granulosa cell layer and basal membrane. The stromal cellsand extracellular matrix did not correlate well with other structures. Correlationsindicated that the granulosa cells, oocyte and basal lamina are affected similarly andthat the damage in one of these structures may be representative of the damage inthe other structures.CONCLUSION: The main aim of the study was achieved since results showed thatno significant damage was induced by the cryopreservation protocols. Ovarian tissuecryopreserved in this study has shown to restore endocrine function temporarily afterheterotopic autotransplantation in menopausal patients. From the electronmicroscopy evaluations, DMSO showed better cryopreservation results. The DMSOcryopreservation protocol was also more time efficient and has shown the mostsuccessful outcomes in the literature. The stromal tissue seemed to be affecteddifferently by cryopreservation compared to the primordial follicle ultrastructures.Younger patients are needed for future studies since a larger initial follicular reservemay allow for larger follicle sample sizes.

[发布机构] Stellenbosch University