Abstract Authors: Roksan Franko1,2 and Marcia AMM Ferraz1,2
1 Gene Center, Ludwig-Maximilians University of Munich, Munich, Germany
2 Clinic of Ruminants, Ludwig-Maximilians University of Munich, Oberschleißheim, Germany
Abstract Text: Assisted reproductive technologies (ART) like in vitro maturation (IVM), fertilization (IVF), and embryo production (IVP) are essential for reproductive practices and conservation. However, the manual handling required for each step, including removing gametes and embryos from the incubator and steps for exchanging media, can introduce stress and compromise their quality. To prevent it, we introduce OoTrap as a single device that allows oocyte capture, IVM and IVF, being a compact device with microwells in a dam channel, and operating in both static and perfusion-based functionality. For device fabrication, a two-piece mold was 3D printed using stereolithography, and devices were prepared using Polydimethylsiloxane. The two parts (perfusion channel and microwell dam) were assembled, and inlet reservoir and outlet tube were added. To evaluate the performance of OoTrap, we investigated fluid dynamics, cumulus-oocyte complexes (COCs) entrapment, IVM and IVF efficiency. For this, follicular fluid (FF) was added into the reservoir, COCs were allowed to settle into microwells and cell debris from FF were removed by washing the channel for IVM. Subsequently, fertilization was performed in device and presumptive zygotes were removed for denudation and culture. 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. A simulation study using COMSOL Multiphysics showed that hydrogen peroxide can be efficiently cleared, with 83% and 98% removal after 100s, under static and perfusion (20μl/h) conditions, respectively. These findings suggest that OoTrap could have effective diffusion and clearance capabilities, which promote optimal culture conditions. Evaluation of trapping efficiency showed 88% entrapment (n=6). Next, we analyzed IVM efficiency under static and perfusion (20μl/h) conditions, with conventional IVM as control. For this, FF from 5 ovaries were added per device and left for maturation in IVM media (10% FBS, 0.1UI/mL FSH, 25μg/ml gentamicin 0.2mM sodium pyruvate in TCM 199) for 22-24 hours. Nuclear staining for the assessment of maturation rate (Hoechst33342, n=5) showed a higher maturation rate in the dynamic OoTrap compared to conventional IVM (70% vs 62%, respectively, p=0.05), while no differences were observed for the static OoTrap (68%. p=0.83). Immunofluorescence for the analysis of spindle morphology and chromosomal alignment showed a tendency for decreased occurrence of abnormality in the perfusion group (7%) compared to both static (22%; p=0.09) and control (25%; p= 0.067). For IVF, following IVM, media was replaced by IVF medium (Stroebech) in the devices, frozen-thawed bull sperm was washed (Sperm Wash Medium, Stroebech), added in the reservoir of OoTrap and incubated for 18-22h under 20ul/h perfusion. Presumptive zygotes were then denuded and transferred to wells containing IVC medium (ECS50 as described in Dos Santos et al, 2021) and cultured until day 9. As control, conventional IVP was performed in wells. Although OoTrap group had a significantly lower cleavage rate compared to control (63 vs 73%, respectively, p=0.0046), there was no difference in blastocyst rate (15 vs 18%, respectively, p=0.524). Next, IVM, IVF and IVC in a single device was also tested, however due to the attachment of cumulus cells in the microwells, we did not see embryo development in these devices. Coating of the microwells to prevent such attachment is currently being investigated. OoTrap's streamlined workflow and elimination of cumbersome handling steps hold promise for improving efficiency, accuracy, and success rates in ARTs.
