L26/P-394 Alleviating cryopreservation-induced damage with a mitochondria-targeted therapy to improve ovarian tissue survival and transplantation outcomes

Abstract

Study question To investigate the key injuries during ovarian cryopreservation, and assess if M1 (mitochondria targeted compound) can mitigate cryoinjury and improve follicular survival after transplantation. Summary answer Mitochondrial dysfunction is a major cause of ovarian cryoinjury. M1 may enhance mitochondrial function, stromal integrity and cellular proliferation, thereby improving follicular survival after transplantation. What is known already Ovarian tissue cryopreservation and transplantation (OTCTP) offers a promising fertility preservation technique for prepubescent patients or those requiring urgent oncological treatment. However, the cryopreservation process causes cryoinjury through multiple mechanisms, including oxidative stress and cold stress, and eventually results in significant follicular loss after transplantation. Various strategies have been explored to mitigate cryopreservation-induced injury. M1, a sulfur-containing histidine derivative, acts as a natural mitochondrial-targeted product with antioxidant and anti-inflammatory properties. It can be transported into mitochondria to reduce ROS production. However, further investigation is needed to demonstrate the role of M1 during OTCTP. Study design, size, duration High-resolution spatial transcriptomics (Stereo-Seq) was used in 3 samples of human ovarian tissue (obtained from patients undergoing gynecological surgery, with informed consent and ethical approval), comparing fresh and slow-freezing samples. Ovarian tissue from 3-week-old female C57 mice was used. After in vitro culture for 2 days, mitochondrial function, stromal integrity and cellular proliferation were assessed in each group (M1 or control, n = 3). In vivo, the follicle survival was evaluated one week after transplantation. Participants/materials, setting, methods Stereo-Seq was employed to profile the molecular impacts of slow-freezing on human ovarian tissue. In mouse models, ovarian tissue from 3-week-old female C57 mice was slow-frozen with either 50 μM M1 or control added to cryoprotectant, thawing, and culture media. In vitro, tissues were analyzed for mitochondrial function (COX8A), stromal integrity (ACTA2) and proliferation (PCNA) with immunofluorescence (IF). In vivo, tissues were transplanted under the renal capsule and collected one week later for follicular analysis. Main results and the role of chance Spatial transcriptomics revealed significant downregulation of mitochondrial respiratory chain complex assembly genes (NDUFB4, NDUFS4) and cellular respiration markers (COX4I1, COX8A, COX7C) in slow-thawed human ovarian tissue. The trend was also confirmed by IF staining and quantitative reverse transcription polymerase chain reaction (qRT-PCR). In mouse ovarian tissue, M1 treatment significantly elevated the expression of markers associated with mitochondrial function (COX8A, ∼2.3-fold, P < 0.05), stromal integrity (ACTA2, ∼4.4-fold, P < 0.001) and proliferation (PCNA, ∼2.4-fold, P < 0.05) compared to controls in vitro (n = 3). In vivo, ovarian tissue treated with M1 exhibited a significant increase in primordial follicle count (1,222 ± 43 vs. 608 ± 110, p < 0.01) and total follicle count (1,476 ± 103 vs. 874 ± 185, p < 0.05) one week post-transplantation (n = 3). Limitations, reasons for caution The study is limited by the small sample size in each group for both human and animal experiments. Further research is required to elucidate the underlying molecular mechanisms of M1 on ovarian tissue cryopreservation. Wider implications of the findings Our findings demonstrate that slow-freezing significantly impairs mitochondrial function in ovarian tissue. M1 may promote mitochondrial function, stromal integrity and proliferation during slow-freezing of ovarian tissue and could contribute to improved follicular survival following transplantation. Trial registration number No