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

Abstract

Background: In 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.

Results: The 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.

Conclusions: The 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.

Keywords: Cryopreservation; Fertility preservation; Isolated follicles; Ovarian tissue; Ovary; Slow freezing; Vitrification.

Fig. 1

Stainless steel devises for carrying ovarian tissue slivers (a) or isolated follicles (b). Devices used for the vitrification procedure, made of the stainless steel wire with the diameters of 0,28 mm and 0,04 mm respectively

Fig. 2

The scheme illustrating “in drop” system for in-vitro culture of isolated follicles. The scheme demonstrates V-shaped well with the 10 μL oil drop on the bottom with 70 μL drop of the culture medium, covered all together in approximately 80 μL of the oil. Additional small oil drop on the bottom ensured that follies were not attachment to the surface of the dish and were suspended in the drop of culture medium, which helped to maintain their 3D structure

Fig. 3

Percentage of good morphology follicles in ovarian tissue after freezing/thawing with different cryopreservation protocols. Results are % ± SE. Follicles were assessed in 5 μm sections after stating by hematoxylin and eosin. Asterisk represents significantly different results from matched protocols at p < 0,05

Fig. 4

Primordial follicles morphology after cryopreservation and further warming/thawing. Primordial follicles were categorized into two quality groups: (a) healthy-looking follicles, (b) - degenerated/damaged follicles. Follicle morphology was determined by analysis of hematoxylin and eosin stained sections of ovarian cortex tissue

Fig. 5

Analysis of DNA fragmentation in sections of sheep ovarian tissue using the TUNEL apoptosis detection kit. Positive control sample after the treatment with DNAse I (a); negative control sample with omitted terminal deoxynucleotidyl transferase enzyme (b); sample tissue with apoptotic follicles showed by arrows (c and d). TUNEL signal showed with green color, Propidium Iodine staining showed with red color

Fig. 6

Histogram of live and apoptotic follicles distribution in the sheep ovarian cortex after cryopreservation with different protocols. Samples were analyzed by the TUNEL apoptosis detection kit. Histogram illustrates that slow freezing method ensures the better preservation of primordial follicles in the tissue, while vitrification methods 1 and 2.3 showed best results amongst tested vitrification protocols

Fig. 7

Box-plot of estradiol concentrations in different control points of the culture period. Values of estradiol concentrations in control points on day 4, 6, 8 and 10 were compared to the value on day 2 (first controlled point during the culture period) in purpose to evaluate dynamic of estradiol production. Asterisk represent significantly different results at p < 0.05

Fig. 8

In-vitro growing follicle after 10 days of the culture. The follicle demonstrates signs of attachment to the bottom of the well (arrows) due to the external cells overgrowth, which however didn’t change normal 3D structure of the follicle

Fig. 9

Production of estradiol by growing ovine follicles. Data are mean + SEM representing medium for 20 follicles analysed in each experimental group measured in five control points - day 2, 4, 6, 8 and 10. Asterisk represents significantly different results between matched groups at p < 0,05

Fig. 10

Sheep oocytes after the live-dead assay. Oocyte were derived from the follicles which were subsequently mechanically isolated, frozen and warmed/thawed. Viability of oocytes were evaluated in staining by calcein-am and ethidium homodimer-1. Letter “a” shows alive oocyte after freezing/thawing showed, “b” - degenerated oocyte

Fig. 11

Production of progesterone by growing ovine follicles. Data are mean + SEM representing medium for 20 follicles analysed in each experimental group measured in three control points - day 2, 6 and 10. Asterisk represents significantly different results between matched groups at p < 0,05

Fig. 12

Box-plot of progesterone concentrations in different control points of the culture period. Values of progesterone concentrations in control points on day 6 and 10 were compared to the value on day 2 (first controlled point during the culture period) in purpose to evaluate dynamic of progesterone production. Asterisk represent significantly different results at p < 0.05