A new dynamic culture system for in vitro maturation of fresh and frozen ovarian tissues in mice

Abstract

Background: Reintroduction of malignant cells by ovarian tissue autotransplantation in cancer patients seeking fertility preservation is still a risk. Therefore, ovarian tissue cryopreservation followed by in vitro maturation could be a safer option. Objective: To establish a new dynamic culture system for in vitro maturation (IVM) of fresh & frozen ovarian tissue in mice. Materials and Methods: 72 ovaries were obtained from 36 female C57BL/6 mice 7–14 weeks old. 18 ovaries were frozen/thawed via slow freezing/rapid thawing protocol. 27 dynamic culture chambers (DCCs) were compared to 21 static culture chambers (SCCs), serving as a control. Each chamber contained 3 ovarian halves which were cultured in 2ml blank G-MOPS™ medium (Vitrolife, Sweden) for 6 days (1 estrus cycle) in a 38°C water bath. DCCs were continuously perifused via a peristaltic pump. Estradiol (E2) & Progesterone (P) were measured daily in all effluents to evaluate for folliculogenesis. After 6 days of culture, ovarian tissues were examined histologically for viability and follicular development, and oocytes were isolated for further IVM. Results: DCCs with fresh tissues (n=21) showed combined E2 & P peaks in 81% of chambers with E2 levels up to 120.2 pg/ml, P levels up to 28.1ng/ml. Histologically on day6: viability was 100% in 52.2% of chambers; Corpora Lutea (CL) were seen in all chambers producing E2. Oocyte isolation & further IVM were successful. DCCs with frozen/thawed tissues (n=6) showed combined E2 & P peaks in 50% of chambers with E2 levels up to 51.7 pg/ml, P levels up to 21ng/ml. Histologically on day6: viability was >60% in 50% of chambers; CL were seen in all chambers producing E2. Oocyte isolation was successful but no further IVM. SCCs with fresh tissues (n=15) showed combined E2 & P peaks in 80% of chambers with E2 levels up to 56.6 pg/ml, P levels up to 94.7ng/ml. Histologically on day6: viability was 100% in 26.6% of chambers; CL were seen in all chambers producing E2. Oocyte isolation & further IVM were successful. SCCs with frozen/thawed tissues (n=6) showed no E2 peaks. Histologically on day6: viability was >60% in 50% of chambers; CL were not seen. Oocyte isolation was successful but no further IVM. Conclusion: We preliminarily established a new dynamic culture system for IVM of fresh and frozen ovarian tissues in mice with superior results to the conventional static cultures regarding ovarian tissue viability, hormone production and oocyte outcome.