A new in vitro model for pre-transplantation diagnosis of frozen/thawed human ovarian tissue

Introduction: Ovarian tissue freezing and autotransplantation is a promising option for preserving fertility of female cancer patients. There are several in vivo, but no established in vitro models to evaluate frozen/thawed human ovarian tissue prior to autotransplantation. Objective: To establish a new in vitro model for endocrine assessment of frozen/thawed human ovarian tissue prior to autotransplantation. Materials & Methods: Ovarian tissues were obtained by laparoscopy from 5 patients 19 to 34 years old recently diagnosed with cancer. 200mg cortical tissue from each patient was used and further divided into two groups: 100mg tissue as fresh (control group) and 100mg tissue as frozen/thawed (study group) after using slow freezing/rapid thawing protocol. Either fresh or frozen/thawed tissue from each patient was separately cultured in blank G-MOPS medium (Vitrolife, Sweden) for 6 days, at 37°C in an incubator without CO2. Estradiol (E2), Progesterone (P) and Anti Müllerian Hormone (AMH) were measured every 2 days in all supernatants to evaluate the endocrine function. After culture, all cortical tissues were examined histologically for viability and follicle development assessment. Mean values±SD were calculated. Results: Frozen/thawed tissues (study groups) showed slower recovery of endocrine function. On days 2, 4 and 6 of culture, E2 was 0.5±1.3, 4.4±9.8 & 14±13.3 pg/ml, P was 0.2±0, 0.2±0 & 0.3±0.1ng/ml, and AMH was below the detection limit. Viability after culture was 72±8.36%. Fresh tissues (control groups) showed faster recovery of endocrine function. On days 2, 4 and 6 of culture, E2 was 37.5±33.8, 62±44.7 & 104.2±58.9 pg/ml, P was 0.4±0.3, 0.6±0.4 & 1±0.5ng/ml, and AMH was under the detection limit. Viability after culture was 94±5.47%. Conclusion: For each patient, reasonable endocrine and histological data can be obtained by culturing only 100mg of fresh or frozen/thawed cortical tissue in G-MOPS medium for just 6 days. This new in vitro model can be used for each patient prior to autotransplantation to assess the endocrine function of the frozen/thawed ovarian tissue. These data will be complimented by follicle density assessment to confirm the endocrine results. In addition, oocyte isolation and in vitro maturation will be performed to assess the reproductive function.