Abstract
BackgroundIn recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. The slow freezing is the most effective technique which resulted in greater live birth incidence so far. Despite that, interest to vitrification of the ovarian tissue is swiftly growing, thereby undermining the necessity for further improvements in the technique. In present study, we evaluated possibilities to increase follicle survival rates adopting innovative multi-protectoral vitrification protocols, applied to the slivers of ovarian cortex or isolated early-antral follicles, frozen individually. These experimental protocols have been compared with with validated vitrification and slow freezing ones, clinically used for female fertility preservation.ResultsThe results showed that third tested variation of experimental vitrification protocol, with four cryoprotectants in relatively low concentrations and applied to pieces of ovarian tissue at 0 °C during equilibration, increased survival rate of ovine ovarian tissue and improved results in comparison with conventional vitrification method. This variation of experimental protocol showed significant increase in percentage of follicles with good morphology (69,3%) in comparison with only commercially available vitrification protocol for ovarian tissue (62,1%). Morphology results were confirmed by TUNEL assay. Analysis of estradiol and progesterone production by cultured individual follicles after freezing/thawing revealed that steroids secretion remained significantly higher after multi-protectoral vitrification and slow freezing protocol, when follicles after standard vitrification protocol demonstrated decline in steroidogenic activity.ConclusionsThe multi-protectoral approach represents a workable solution to improve vitrification outcome on ovarian tissue and isolated follicles. The reduction of individual cryoprotectants concentrations, while maintaining their sufficient cumulative level in the final freezing solution, helps to increase efficiency of the procedure. Moreover, equilibration with lower temperatures helped to decrease even further the toxic effects of cryoprotectants and preserve original quality of ovarian tissue. Therefore, multi-protectoral vitrification can be suggested as an improved method for the clinical cryopreservation of ovarian tissue.
Highlights
In recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility
Slow freezing was the first method adopted for ovarian tissue cryopreservation back in 1996 and was successfully in use ever since [12, 13]
Progesterone secretion by follicles, cultured after cryopreservation In the same experimental set up as described for the estradiol secretion, the culture medium was analyzed on the presence of progesterone in three control points: day Discussion In the present study, we successfully developed a vitrification protocol for ovine ovarian cortical tissue and isolated follicles using a multi-protectoral approach
Summary
Autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. Vitrification has not been proved yet as an advantageous method for ovarian tissue cryopreservation, but shall continue to be considered a promising technic as it proved to be secure method for cryopreservation of oocytes and preimplantation embryos [14,15,16] Another prospective technique, which aims to restore fertility for patients who need gonadotoxic treatment, is an in-vitro approach, when isolated early stage (pre-antral and early antral) follicles are being cultured in laboratory conditions with further in-vitro maturation of oocyte. Such technique is still considering as an experimental and not resulted in a live-birth yet
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