Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture

Objective: To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended in vitro culture. Design: Randomized experimental study. Setting: University research laboratory. Patient(s): Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. Intervention(s): None. Main Outcome Measure(s): Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9 days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time–PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. Result(s): The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription–PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10–35) and a markedly lower up-regulation of only 5 genes (range, 3.6–7.8) in SN samples. Long-term in vitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. Conclusion(s): Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.