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.

Is oxygen availability a limiting factor for in vitro folliculogenesis?

Transplantation of ovarian tissue for the preservation of fertility in oncological patients is becoming an accepted clinical practice. However, the risk of re-introducing tumour cells at transplantation has stirred an increased interest for complete in vitro folliculogenesis. This has not yet been achieved in humans possibly for the lack of knowledge on the environmental milieu that orchestrates folliculogenesis in vivo. The main aim of this study was to investigate the effect of oxygen availability on follicle health and growth during in vitro culture of ovarian tissue strips. To this end, a model was developed to predict the dissolved oxygen concentration in tissue under varying culture conditions. Ovarian cortical strips of bovine, adopted as an animal model, and human tissue were cultured in conventional (CD) and gas permeable (PD) dishes under different media column heights and gaseous oxygen tensions for 3, 6 and 9 days. Follicle quality, activation of primordial follicles to the primary stage, and progression to the secondary stage were analysed through histology. Follicle viability was assessed through a live-dead assay at the confocal scanning laser microscope. Findings showed a higher follicle quality and viability after culture of bovine ovarian strips in PD in adequate medium height and oxygen tensions. The best culture conditions found in the bovine were adopted for human ovarian strip culture and promoted a higher follicle quality, viability and progression. Overall, data demonstrated that modulation of oxygen availability in tissue plays a key role in maintaining follicles' health and their ability to survive and progress to the secondary stage during ovarian tissue in vitro culture. Such culture conditions could increase the yield of healthy secondary follicles for subsequent dissection and individual culture to obtain competent oocytes.

Successful slush nitrogen vitrification of human ovarian tissue.

OBJECTIVE: To study whether slush nitrogen vitrification improves the preservation of human ovarian tissue. DESIGN: Control vs. treatment study. SETTING: University research laboratory. PATIENT(S): Ovarian biopsies collected from nine women (aged 14-35 years) during laparoscopic surgery for benign gynecologic conditions. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian cortical strips of 2 × 5 × 1 mm were vitrified with liquid or slush nitrogen. Fresh and vitrified cortical strips were analyzed for cryodamage and viability under light, confocal, and transmission electron microscopy. RESULT(S): Compared with liquid nitrogen, vitrification with slush nitrogen preserves [1] follicle quality (grade 1 follicles: fresh control, 50%; liquid nitrogen, 27%; slush nitrogen, 48%); [2] granulosa cell ultrastructure (intact cells: fresh control, 92%; liquid nitrogen, 45%; slush nitrogen, 73%), stromal cell ultrastructure (intact cells: fresh control, 59.8%; liquid nitrogen, 24%; slush nitrogen, 48.7%), and DNA integrity (TUNEL-positive cells: fresh control, 0.5%; liquid nitrogen, 2.3%; slush nitrogen, 0.4%); and [3] oocyte, granulosa, and stromal cell viability (oocyte: fresh control, 90%; liquid nitrogen, 63%; slush nitrogen, 87%; granulosa cells: fresh control, 93%; liquid nitrogen, 53%; slush nitrogen, 81%; stromal cells: fresh control, 63%; liquid nitrogen, 30%; slush nitrogen, 52%). CONCLUSION(S): The histology, ultrastructure, and viability of follicles and stromal cells are better preserved after vitrification with slush nitrogen compared with liquid nitrogen.