Survey on the implementation status and reproductive outcomes of oocyte and ovarian tissue cryopreservation in Japan: Historical comparison with nationwide surveys.

PURPOSE: To clarify the reproductive outcomes of fertility preservation (FP) treatment. METHODS: We conducted a mailed-in questionnaire survey at institutions certified by the Japan Society of Obstetrics and Gynecology to investigate the number of oocyte cryopreservations (OC) and ovarian tissue cryopreservations (OTC) performed from December 2016 to the end of 2020. And, we conducted a detailed investigation of cases in which frozen specimens were used during the investigation period, and made historical comparisons with previous nationwide studies. RESULTS: Responses were received from 114 out of 150 facilities (response rate: 76.0%) for OC and 43 out of 51 for OTC (response rate: 84.3%). Breast cancer was the most common disease among patients whose FP specimens were used. During the study period, 1237 OCs and 198 OTCs were performed. In addition, 57 cycles of embryo transfer (ET) using cryopreserved oocytes and 12 cases of ovarian tissue transplantation (OTT) were performed. The mean age of patients who underwent ET using cryopreserved oocytes was 34.8 (±5.8) years, with a median age of 36 years. The pregnancy rate per ET using cryopreserved oocytes was 26.3% and the live birth rate (LBR) was 17.5%. Further, the LBR per patient was 43.3%, and the pregnancy rate following OTTs was 33.3%. Also, controlled ovarian stimulation using the random start method or the combination of aromatase inhibitors had no effect on pregnancy outcome. CONCLUSION: Implementation of both OCs and OTCs have markedly increased over time in Japan, with comparable reproductive outcomes as other reports.

The study of the efficiency of in vitro maturation of ovarian tissue oocytes in pediatric patients.

PURPOSE: Although recent in vitro maturation (IVM) studies in pediatric patients have demonstrated successful retrieval and maturation of oocytes, the studies included only a small number of premenarchal patients. In the present study, we examined the potential use of oocyte retrieval and maturation for pediatric patients who undergo ovarian tissue cryopreservation (OTC). METHODS: We retrospectively examined the clinical records of pediatric patients who underwent OTC at our institution between October 2015 and December 2022. Data on the age, primary disease, menstrual history, pre-procedure chemotherapy, anti-Müllerian hormone (AMH) level, number of oocytes collected ex vivo from ovarian tissue, and number of mature oocytes from IVM were examined. RESULTS: Data of 60 pediatric patients (aged 1 to 17 years) were included for analysis. Oocytes were retrieved from 36 patients; the oocytes of 18 of these patients could be cryopreserved. The IVM rate was significantly lower in the premenarchal patients than in the postmenarchal patients. The number of mature oocytes retrieved from IVM was higher in the no-chemotherapy group than in the chemotherapy group. A significant positive correlation was observed between the AMH level and the IVM outcomes. CONCLUSION: Oocyte retrieval and maturation in pediatric patients undergoing OTC is particularly useful in those not receiving chemotherapy. In patients receiving chemotherapy, the AMH level may be useful for predicting the IVM outcome. Activation of the oocyte maturation process in vivo in pediatric patients and better understanding of the major regulators of oocyte maturation are necessary to improve the utility of the IVM procedure.

Quantification of residual cryoprotectants and cytotoxicity in thawed bovine ovarian tissues after slow freezing or vitrification.

STUDY QUESTION: How much residual cryoprotectant remains in thawed/warmed ovarian tissues after slow freezing or vitrification? SUMMARY ANSWER: After thawing/warming, at least 60 min of diffusion washing in media was necessary to significantly reduce the residual cryoprotectants in ovarian tissues frozen by slow freezing or vitrification. WHAT IS KNOWN ALREADY: Ovarian tissue cryopreservation (OTC) by slow freezing has been the conventional method; while the vitrification method has gained popularity for its practicality. The main concern about vitrification is how much potentially toxic residual cryoprotectant remains in the warmed tissues at the time of transplantation. STUDY DESIGN, SIZE, DURATION: This was an animal study using the ovarian tissues from 20 bovine ovaries. The duration of this study was from 2018 to 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian cortex tissues were prepared from 20 bovine ovaries and assigned randomly to groups of fresh (non-frozen) control, slow freezing with 1.5 M dimethyl sulfoxide (DMSO), 1.5 M 1,2-propanediol (PROH) and vitrification with 35% ethylene glycol (EG). The residual cryoprotectant concentrations in thawed/warmed tissues were measured by gas chromatography at the following time points: frozen (before thawing/warming), 0 min (immediately after thawing/warming), 30, 60 and 120 min after diffusion washing in media. Next, the ultrastructural changes of primordial follicles, granulosa cells, organelles and stromal cells in the ovarian tissues (1 mm × 1 mm × 1 mm) were examined in fresh (non-frozen) control, slow freezing with DMSO or PROH and vitrification with EG groups. Real-time quantitative PCR was carried out to examine the expressions of poly (ADP-ribose) polymerase-1 (PARP1), a DNA damage sensor and caspase-3 (CASP3), an apoptosis precursor, in thawed/warmed ovarian tissues that were washed for either 0 or 120 min and subsequently in tissues that were ex vivo cultured for 24 or 48 h. The same set of tissues were also used to analyze the protein expressions of gamma H2A histone family member X (γH2AX) for DNA double-strand breaks and activated caspase-3 (AC3) for apoptosis by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: The residual cryoprotectant concentrations decreased with the extension of diffusion washing time. After 60 min washing, the differences of residual cryoprotectant between DMSO, PROH and EG were negligible (P > 0.05). This washing did not affect the tissue integrity or significantly elevate the percentage of AC3 and γH2AX positive cells, indicating that tissues are safe and of good quality for transplantation. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Since the study was performed with ovarian tissues from bovines, generalizability to humans may be limited. Potential changes in ovarian tissue beyond 120 min were not investigated. WIDER IMPLICATIONS OF THE FINDINGS: This study addresses concerns about the cytotoxicity of EG in warmed ovarian tissues and could provide insights when devising a standard vitrification protocol for OTC. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science to N.S.

Learning Curve of Surgeons Performing Laparoscopic Ovarian Tissue Transplantation in Women with Premature Ovarian Insufficiency: A Statistical Process Control Analysis.

STUDY OBJECTIVE: To analyze patient safety in laparoscopic ovarian tissue transplantation surgery by tracking the rate of postoperative complications and the learning curves of the surgeons by statistical process control analysis. DESIGN: A retrospective study. SETTING: A university-affiliated hospital. PATIENTS: A total of 100 patients with premature ovarian insufficiency who underwent ovarian tissue cryopreservation by vitrification and then autologous transplantation of frozen-thawed ovarian tissues with in vitro activation. INTERVENTIONS: Ovarian tissue cryopreservation, in vitro activation, and transplantation. MEASUREMENTS AND MAIN RESULTS: We assessed the surgery complications, differences in total surgery time, transplantation time, and transplantation time per ovarian sheet in operations performed by 3 experienced laparoscopic surgeons. Surgeon A performed 80 operations; surgeon B, 29 operations; and surgeon C, 20 operations. Complications occurred in 1.55% of the procedures. Although all 3 surgeons' performance never fell below the unacceptable failure limit, only surgeon A became competent after 66 cases. CONCLUSION: The laparoscopic ovarian tissue transplantation surgery was generally safe given that the postoperative complications were infrequent (1.55%). Although the performance of all 3 surgeons was acceptable, only surgeon A attained the level of competency after 66 cases. The transplantation method may not be the key factor for reducing surgery time in this surgery. An efficient ovarian tissue transplantation team is more important in reducing the surgery time than the surgeon's surgical technique alone.