Optimizing ovarian tissue preparation methods for vitrification: A two-part study on slicing methods and tissue size effects

Abstract

Ovarian tissue cryopreservation by vitrification is well-established, but the effects of procedural differences in the preparatory steps, such as dissection method and tissue size, remain underexplored. This study compared the thickness of cortical tissue pieces and the normality of primordial follicles prepared with either a Stadie Riggs tissue slicer or a conventional scalpel. Part two of the study assessed the viability of follicles vitrified within either 0.5–1 × 5 × 5 mm (thickness × length × width) or 0.5–1 × 10 × 5 mm pieces of cortical tissue. In part one, the proportion of morphologically normal follicles and section thickness did not differ by method ($p=0.309$ and $p=0.986$), although thickness varied by operator ($p=0.028$) without an interaction between method and operator. Upon visual H&E analysis, ovarian pieces prepared with the Stadie Riggs devices were more uniform in thickness and the sectioned edge less jagged than those prepared with a conventional scalpel. In part two, follicles were assessed on matched H&E, Ki67 and $\gamma$H2AX stained sections before and after four days of in vitro culture. Fresh tissue processed on day 0 yielded higher proportions of ‘good’ outcomes for morphology, proliferation, and DNA-damage compared to vitrified tissues ($p<0.001$, $p<0.001$ and $p=0.001$, respectively). By day 4, group differences were not significant. Direct comparisons between vitrified 0.5–1 × 5 $\times$ 5 mm and 0.5–1 × 10 $\times$ 5 mm vitrified pieces did not show significant differences at either time point (all $p>0.05$); pooled analyses yielded odds ratios $\sim$1 with no heterogeneity. In summary, both preparation methods provide comparable quantitative outcomes, with operator standardization appearing more influential than device choice. For vitrified ovarian cortex, both tissue sizes are interchangeable, and fresh tissue is superior at baseline. Considering the smoother cuts observed, we recommend Stadie Riggs preparation with either 0.5–1 × 5 $\times$ 5 or 0.5–1 × 10 $\times$ 5 mm fragments.