JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):15
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263536

P-03. Advancing Embryo Production: Development and Evaluation of the Nano.Follicle 3D In Vitro Maturation System

Bruno Carrino Suave¹, Sarah Gomes Nunes^2,3, Caroline Schiavão Fernandes¹, Enzo Dare Rissi¹, Thaisy Tino Dellaqua³, Ana Caroline Silva Soares³, Alan Brunholi Giroto¹, Amanda Talys Sampaio¹, Rafael Martins Tonzar¹, Raquel Zaneti Puelker², Guilherme Colla⁴, Janice Koepp⁴, Anthony Cesar de Souza Castilho^1,2

¹Universidade do Oeste Paulista - UNOESTE - Presidente Prudente - SP - Brasil
²BlastoCell Biotechnologies - Presidente Prudente - SP - Brasil
³Universidade Estadual Paulista “Júlio de Mesquita Filho” Botucatu - SP - Brasil
⁴BioCelltis Biotecnologia - Florianópolis - SC - Brasil

Objective: In vitro production (IVP) of bovine embryos is widely applied in both commercial and research settings. Conventional IVP systems typically employ two-dimensional (2D) monolayer cultures, in which oocytes remain in direct contact with plastic surfaces. This artificial environment poorly reproduces in vivo conditions and may contribute to abnormalities in oocyte development. Recent advances have focused on three-dimensional (3D) culture systems to enhance oocyte viability, morphology, cellular development, and intercellular communication. Spherical 3D environments improve nutrient and oxygen diffusion, thereby supporting a more physiologically relevant maturation process. This study aimed to develop and evaluate a closed 3D in vitro maturation (IVM) system, termed Nano.Follicle, and to assess its impact on oocyte quality and subsequent embryonic development.
Methods: The biomaterial used in Nano.Follicle was characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FTIR), and cytotoxicity testing following ISO 10993-5 standards, while cell viability and migration were evaluated via scratch assay. For IVP, bovine ovaries were collected from a slaughterhouse, and antral follicles (3–8 mm) were aspirated. Only Grade I and II cumulus–oocyte complexes (COCs) were selected and allocated to either a conventional 2D culture or the Nano.Follicle 3D system. Following IVM, COCs were fertilized in vitro and cultured for 7 days. Meiotic progression was assessed using Hoechst staining, apoptosis via TUNEL assay, and embryo development by cleavage rate, blastocyst formation, and total cell counts.
Results: Meiotic progression showed no significant differences between systems: metaphase II rates were 84.43±8.22 (Nano.Follicle) vs. 76.89±8.97 (2D), p=0.5102; metaphase I rates were 8.43±3.70 vs. 18.24±8.21, p=0.2222; and degenerated oocytes were 7.12±5.43 vs. 4.86±4.35, p=0.6813. Apoptotic oocytes were 10.22±3.72 (Nano.Follicle) vs. 2.42±1.24 (2D), p=0.1037. Cleavage rates were 75.41±5.14 vs. 83.42±2.93, p=0.2275, and blastocyst production was 28.58%±2.32 vs. 26.98%±2.64, p=0.6634. No significant differences were found in total cell counts of blastocysts (BL): 83±5.13 vs. 98.8±7.60, p=0.1521; or hatched blastocysts (BE): 117.42±9.95 vs. 137.75±19.66, p=0.3943. However, expanded blastocysts (BX) exhibited a significantly lower total cell number in the Nano.Follicle group (107.63±5.36) compared to 2D (130.6±6.35), p=0.0068.
Conclusion: In conclusion, the Nano.Follicle 3D IVM system displayed favorable physical, thermal, and cytocompatibility profiles, supporting its potential for application in IVP. While embryo development outcomes were comparable to the conventional 2D system, Nano.Follicle effectively reproduced key aspects of the in vivo microenvironment and represents a promising alternative for both research and commercial embryo production.