192 Microfluidics in assisted reproductive technologies: OoTrap for oocyte capture and in vitro maturation

Assisted reproductive technologies (ARTs) such as IVM and IVF play crucial roles in reproductive practice and conservation efforts. However, conducting IVM and IVF in field-based sample collection settings presents challenges, including limited access to equipment and the potential detrimental effects of transferring samples to equipped facilities on the survival and quality of cumulus–oocyte complexes (COCs). To overcome these challenges, we introduce OoTrap as an oocyte capturing and IVM system. OoTrap features a compact device with microwells (750 × 750 × 750 μm) in a dam channel, offering flexibility in operation with both static and perfusion-based functionality. The system allows efficient capture and maturation of COCs in the microwells while minimizing the need for laboratory equipment, such as a stereomicroscope and pipettes. Follicular fluid (FF) can be added into the device and the washing of the channel allows the removal of cell debris from the FF, while keeping COCs inside. To evaluate the performance of OoTrap, we investigated fluid dynamics, COC entrapment, and IVM efficiency. Data were analysed in R using a generalized linear mixed model, with ‘Treatment’ as fixed and ‘Replicate’ as random effect, and a Tukey post hoc test was performed for statistical analysis. For the fabrication of OoTrap, a two-piece mould was 3-D printed using stereolithography, and devices were prepared using polydimethylsiloxane. The two parts (perfusion channel and microwell dam) were sealed together, and an inlet reservoir and outlet tube were added. A simulation study using COMSOL Multiphysics showed that hydrogen peroxide in the OoTrap can be efficiently cleared, with 83% and 98% removal after 100 s, under static and perfusion (20 μL h⁻¹) conditions, respectively. These findings suggest that OoTrap could have effective diffusion and clearance capabilities that promote optimal culture conditions and COC survival. Trapping efficiency was evaluated by adding FF isolated from 5 bovine ovaries (~5 mL), allowing COC to settle in the microwells, washing the FF and cell debris out, and collecting the COCs by washing the chamber. On average, 93% of COCs were recovered (n = 3). Next, we analysed bovine IVM efficiency under static and perfusion (20 μL h⁻¹) conditions, with conventional IVM as control. For this, good quality COCs were selected and randomly assigned to the groups, IVM media (10% FBS, 0.1 IU mL⁻¹ FSH, 25 μg mL⁻¹ gentamicin, 0.2 mM sodium pyruvate in TCM-199) was used for 50 COCs per group and COCs cultured for 22–24 h. Nuclear staining was used to assess maturation rate (Hoechst 33342, n = 3). Our findings show a higher maturation rate in the dynamic OoTrap compared with conventional IVM (87% vs 78%, respectively, P = 0.007), while no differences were observed for the static OoTrap (86%). Analysis of spindle morphology and oxidative stress are currently being performed. OoTrap’s streamlined workflow, elimination of cumbersome handling steps, and potential for on-chip IVF and embryo culture capabilities hold promise for improving efficiency, accuracy, and success rates in ARTs.