Early human follicle ultrastructure comparison after slow cryopreservation in two different cryoprotectants
Els, Cecilia Lydia
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BACKGROUND: The cryopreservation and transplantation of ovarian tissue have been shown to restore ovarian function temporarily and may also preserve the fertility of young female cancer patients until after their sterilizing cancer treatment. Since tissue samples are large and morphologically complex, the cryopreservation methodology is difficult to optimize and standardise. Ovarian tissue cryopreservation is therefore, still in its experimental stages and is not a routine option offered to cancer patients. OBJECTIVES: Our main aim was to initiate, develop and implement a practical ovarian tissue cryopreservation and re-transplantation protocol which would restore ovarian function, and possibly fertility, in young female cancer patients undergoing sterilizing cancer therapies in South Africa. The objective of this study was to improve the slow cryopreservation protocol for human ovarian tissue. The ultrastructural effects after cryopreservation with two well-known cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propylene glycol (PROH), on early human follicles 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 of Tygerberg Hospital who entered on a basis of voluntary informed consent. Ovarian tissue was obtained by laparoscopic oophorectomy. After dissection of the ovary(ies), some fresh cortical tissue was sent for metastatic analysis and a few strips taken as fresh control. Remaining dissected ovarian cortical tissue sections of each patient were equally divided into the two cryoprotectant groups. Five resulting groups 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 histological haematoxylin 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 were assessed according to a scoring system which gave an indication of follicular health. Appropriate statistical tests were applied to analyse the mean scores where P≤0.05 was considered as statistically significant. RESULTS: No significant overall cryopreservation treatment effect was evident in any of the follicular ultrastructures evaluated. This result indicated that the cryopreservation protocol used did not induce significant damage to the cortex tissue compared to the fresh control group. Comparison of the effect of PROH and DMSO on the follicular ultrastructures showed that PROH tend to induce more extensive damage, especially after cryopreservation. Correlation studies showed significant positive relationships between the majority of the evaluated ultrastructures, especially between the oocyte and granulosa cell layer and basal membrane. The stromal cells and extracellular matrix did not correlate well with other structures. Correlations indicated that the granulosa cells, oocyte and basal lamina are affected similarly and that the damage in one of these structures may be representative of the damage in the other structures. CONCLUSION: The main aim of the study was achieved since results showed that no significant damage was induced by the cryopreservation protocols. Ovarian tissue cryopreserved in this study has shown to restore endocrine function temporarily after heterotopic autotransplantation in menopausal patients. From the electron microscopy evaluations, DMSO showed better cryopreservation results. The DMSO cryopreservation protocol was also more time efficient and has shown the most successful outcomes in the literature. The stromal tissue seemed to be affected differently by cryopreservation compared to the primordial follicle ultrastructures. Younger patients are needed for future studies since a larger initial follicular reserve may allow for larger follicle sample sizes.