Research Projects

Rationale: Reproductive ageing caused by diminished ovarian function leads to infertility in women and reduced livestock productivity. Adipose-derived mesenchymal stem cells (ADSCs) show promise in reversing ovarian ageing, but the efficacy of cryopreserved ADSCs remain unclear. Conventional cryoprotectants like DMSO are cytotoxic. This study explores ProGly-DES (deep eutectic solvents) as a safer alternative for ADSC cryopreservation and ovarian function restoration, including transgenerational safety. Hypothesis: Replacing DMSO with a biocompatible DES-based cryoprotectant will improve the post-thaw viability, genomic integrity, and functionality of ADSCs. These cells may restore ovarian function and oocyte quality in aged ovaries without transgenerational effects, confirming the safety of the DMSO-free cryopreservation strategy. Objectives: 1. To evaluate the cryoprotective efficacy of ProGly-DES in preserving the viability and functional characteristics of ADSCs compared to conventional cryoprotectants 2. To assess the regenerative potential of ProGly-cryopreserved ADSCs in restoring ovarian function and improving oocyte and embryo quality in a murine model of reproductive ageing 3. To investigate the transgenerational safety and developmental competence of offspring derived from ProGly-ADSC-treated aged ovaries Methodology: The ovarian regeneration potential of ADSCs cryopreserved using 10% DMSO and 20% ProGly will be compared in aged C57BL/6 mice. Post-thaw cells will be evaluated for viability, apoptosis, oxidative stress, and proliferation. Following intraovarian transplantation, ovarian recovery will be estimated by histology, hormonal profiling, follicle count, and marker expression (proliferative and angiogenic). Additionally, Oestrous cyclicity, oocyte quality, IVF outcomes and blastocyst DNA integrity will be analysed. To demonstrate transgenerational safety, F1 offspring from treated mice will be evaluated for growth, survival, sex ratio, organ weights, hormone levels, genomic and epigenetic stability. Significance and impact: If the proposed objectives are achieved, this will be the first study to evaluate the ovarian regeneration potential of cryopreserved ADSCs. Additionally, the study will provide insights into the comparative efficacy of DMSO and ProGly in cryopreserving ADSCs with limited cellular damage. The in vivo assessment of functional revival of aged ovaries and reproductive competence following ADSC transplantation will demonstrate the role of cryopreservation efficiency in stem cell-based fertility restoration. The observations made from this study can be translated to reverse ovarian ageing in advanced maternal age females and livestock, using readily available cryopreserved ADSCs without additional delay. Furthermore, if ProGly is proven to have better cryoprotective efficiency, it could serve as an alternative to DMSO for cryopreserving various cell types, due to its ease of availability and biocompatibility.