Treatment with trypLE before freezing improves thawing integrity and functionality of sheep ovarian tissue

Abstract

Objective: To study innovative approaches to ovarian tissue cryopreservation, a critical issue for fertility preservation in pediatric cancer patients. Despite historical attempts, recent advances in cancer treatment have underscored the urgent need for more effective and reliable ovarian tissue cryopreservation methods. Our research aims to evaluate if decreasing the rigidity of stroma before cryopreservation by investigating pre-treatments with enzymes can enhance the quality of ovarian tissue post-thawing.

Design: Our research evaluated the use of five commonly used enzymes to disaggregate tissue (trypLE, collagenase, dispase, accutase and hyaluronidase) before freezing ovarian tissue to decrease rigidity and facilitate cryopreservation. Sheep ovaries, with high similarity to human ovaries, were used as an animal model. Tissue structure, cell proliferation, apoptosis and viability were assessed before and after thawing.

Results: Our findings showed that enzymatic treatment with trypLE before freezing offered immediate benefits post-thawing with the highest viability values and percentage of intact follicles. A decrease in viability was observed after thawing and culturing the samples. The pretreatment with accutase damaged the tissue severely with also the lowest viability values. Ki67-positive follicles and stromal cells were observed in fresh samples, but only trypLE and hyaluronidase maintained Ki67-positive antral follicles after 2 days culture. Besides, only trypLE maintained all follicles negative to caspase-3 after thawing, and 7 days after culture primordial follicles were apoptotic in all treatments apart from trypLE.

Conclusion: our findings suggest that trypLE pretreatment could provide a beneficial approach for maintaining the functions and viability of cryopreserved ovaries after thawing. Further research is needed to fully understand their impact and optimize cryopreservation protocols in this important clinical context.

Keywords: Cryopreservation; Enzymes; Fertility; Follicles; Ovarian tissue; Prepuberal cancer.

Fig. 1

Viability preparations in of ovarian tissue (0.5 cm × 0.5 cm × 1 mm squares of tissue) in TIFF format for FIJI analysis at 0 h after thawing and 7 d after thawing. a TrypLE. b Collagenase. c Dispase. d Accutase. e Hyaluronidase. f Control

Fig. 2

Percentage of tissue viability measured as a percentage of the total treatments over time (Mean ± SEM)

Fig. 3

Percentage of follicular damage at 0 h, 2 d and 7 d post-thawing in TrypLE, collagenase, dispase, accutase, hyaluronidase and control. (Mean ± SEM). Different letters indicate significant differences among treatments

Fig. 4

H&E preparations of ovarian tissue at 0 h post-thawing. a TrypLE. b Collagenase. c Dispase. d Accutase. e Hyaluronidase. f Control. Blue arrows indicate healthy follicles. Yellow arrows indicate damaged follicles. Red arrows indicate areas of necrosis or pyknosis. Purple arrows indicate areas of decellularization

Fig. 5

H&E preparations of ovarian tissue at 2 days post-thawing. a TrypLE. b Collagenase. c Dispase. d Accutase. e Hyaluronidase. f Control. Blue arrows indicate healthy follicles. Yellow arrows indicate damaged follicles. Red arrows indicate areas of necrosis or pyknosis. Orange rectangles indicate damaged edges

Fig. 6

H&E preparations of ovarian tissue at 7 days post-thawing. a TrypLE. b Collagenase. c Dispase. d Accutase. e Hyaluronidase. f Control. Blue arrows indicate healthy follicles. Yellow arrows indicate damaged follicles. Red arrows indicate areas of necrosis or pyknosis. Purple arrows indicate areas of decellularization. Orange rectangles indicate damaged edges

Fig. 7

Percentage of positive Ki67 at fresh and 0 h and 2 d after thawing in TrypLE, collagenase, dispase, accutase, hyaluronidase and control.. Percentage of positive Caspase-3 at fresh and 0 h, 2 d and 7 days after thawing in TrypLE, collagenase, dispase, accutase, hyaluronidase and control. (Mean ± SEM). Different letters indicate significant differences among treatments

Fig. 8

Immunohistochemistry. a Follicle Ki67 positive in fresh tissue after dispase treatment. Blue arrows indicate cells in proliferation. b Follicle Ki67 negative in 0 h after thawing in accutase. c Follicle caspase-3 positive in 2 days culture after thawing in dispase treatment. Blue arrows indicate apoptotic cells. d Follicle caspase-3 negative in fresh sample after trypLE treatment