Innovative multi-protectoral approach increases survival rate after vitrification of ovarian tissue and isolated follicles with improved results in comparison with conventional method

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.

Fertility Preservation in Women: Indications and Options for Therapy

“…In contrast, other studies concluded that slow cooling was superior to vitrification. [69][70][71][72][73] Still other data indicated both techniques to be similar in terms of the percentage of morphologically normal follicles and apoptotic cells. 74,75 Shi et al 76 recently conducted a metaanalysis that included 14 studies evaluating the efficacy of vitrification vs slow cooling of ovarian tissue.…”

The effect of high throughput vitrification of human ovarian cortex tissue on follicular viability. A promising alternative to conventional slow freezing ?

“…44 Partially, our handling approach ressembles the protocol of Nikiforov, using unlinked metal meshes for carrying ovarian tissue samples with 0.28 mm wire diameter and mesh aperture of 1.31 mm for vitri cation and insertion into cryovials after vitri cation. 45 Our approach included metal meshes with wire diameters of 0.25 mm and opening sizes of 0.38 mm. This ensures a large contact area of the cortex tissue on a metal carrier thin as possible, potentially in uencing on the thermal conductivity while the meshes were pre linked with the caps of the cryovials.…”

Equine Chorionic Gonadotropin as an Effective FSH Replacement for In Vitro Ovine Follicle and Oocyte Development

“…For a long time, eCG was used as an alternative FSH hormone for superovulation induction by improving the process of follicle recruitment in several mammal models [68]. Indeed, it was successfully used in small mammals, such as mice rats [69] rats alone [70], medium-large sized mammals such as sheep [71][72][73], goats [74], cows [75], mares [76], and pigs [77].…”