P-422 Ovarian tissue vitrification as a low-technology and cost-effective protocol for female fertility preservation: a bovine study
Authors: Deligiannis S P , Kask K , Modhukur V , Ivask M , Jaakma Ü , Damdimopoulou P , Tuuri T , Velthut-Meikas A , Salumets A 
Published: June 2023

Abstract

Study question
Which are the potential effects of vitrification on ovarian tissue, isolated follicles and short-term cultured follicles?

Summary answer
Vitrification-warming protocol using ethylene glycol as a cryoprotectant causes no severe damage to the ovarian tissue or the isolated follicles.

What is known already
Ovarian tissue cryopreservation (OTC) is used for fertility preservation (the only possible approach applicable for prepubertal girls). To date, more than 200 babies have been born from cryopreserved ovarian tissue. However, a slow-freezing protocol has been used most of the time, and only four babies have been born using the vitrification protocol. This suggests that current vitrification protocols may have an unknown effect on ovarian tissue and the developing follicles. Identifying the effects that vitrification may have on ovarian tissue will make vitrification for OTC more available to everyone while reducing high costs and new equipment requirements.

Study design, size, duration
We obtained 19 bovine ovaries from the local slaughterhouse and processed further by separating the medulla from the cortex. Hematoxylin and Eosin stain (H&E) was then used to calculate the follicle pool. Ovarian tissue pieces, where follicles were not identified, were excluded from the study.

Participants/materials, setting, methods
Ovarian cortical tissue pieces were fixed in Bouin’s solution and 4% paraformaldehyde and were used for morphological analysis using H&E, Ki67 immunofluorescence and TUNEL assay. Moreover, bulk RNA sequencing was used, and ovarian pieces with RIN&gt;7 were selected for library preparation. Finally, fresh and vitrified ovarian tissue pieces were used for follicle isolation. The isolated follicles were cultured for up to 6 days, followed by a viability test performed after every two days.

Main results and the role of chance
Vitrification and subsequent warming of the ovarian tissue showed no significant effect on the total population of follicles compared with the fresh tissue. However, from bulk RNA sequencing, we observed 165 differentially expressed genes (DEGs) in vitrified tissue (VT) compared with the fresh tissue (FT).
Vitrified cultured (VTC) pieces showed a significant decrease in monolayered and antral follicles. Moreover, a significant increase was observed in the population of atretic follicles in the fresh cultured (FTC) and VTC pieces compared with their non-cultured tissue pieces. Also, culturing fresh and vitrified tissue pieces for six days increased the proliferation of ovarian stromal cells and showed the presence of DNA damage in the stromal cells of both study groups. The RNA sequencing analysis yielded 1,042 and 1,191 DEGs in FTC and VTC pieces, respectively, compared with their uncultured control groups.
Furthermore, approximately 30% of the isolated follicles (152) from vitrified tissue were viable after isolation compared with the number of viable follicles isolated from the fresh tissue (525). The isolated follicles showed no changes in their viability rate for up to 4 days.
We used a Two-way ANOVA with multiple comparisons to analyse the collected data.

Limitations, reasons for caution
The study's main limitation was the lack of possibility to use the slow freezing protocol to compare the effect between the slow freezing and vitrification methods.

Wider implications of the findings
The vitrification and warming of bovine ovarian tissue did not show significant changes in ovarian tissue morphology. The follicle isolation process and subsequent culturing indicate that the vitrification method could be a way to preserve ovarian tissue and its follicles. However, culturing the tissue pieces affects their transcriptomic profile.

Trial registration number
N/A