BIOENGINEERING OVARIES: A LEAP IN VETERINARY FERTILITY

Utilizing cutting-edge tactics to improve domestic animal reproductive performance or protect biodiversity by halting the extinction of mammalian endangered species is still one of the challenges of the assisted reproductive technologies/ART. In vitro recapitulation of early phases of folliculogenesis/ivF is one of the most challenging ART perspectives for attracting immature and low competent female gametes for use in reproduction. Although early-stage ovarian follicle cultures were originally developed, their limited translation to in vivo systems has constrained their broader use. However, current advances in reproductive tissue engineering/REPROTEN [1] have opened the potentiality of mimic the ovarian stroma environment on which engraft multiple isolated ovarian early follicles by generating transplantable artificial ovary. The purpose of this study was to assess the ovarian biomimicry of the Poly(epsilon-caprolactone)(PCL) electrospun patterned scaffolds in reproducing a suitable long-term (18 days) microenvironment supporting the transition from preantral (PA) to early antral (EA) follicle stage by recruiting incompetent oocyte towards the final step of specialization (fully grown dimension, ability to resume meiosis and embryo development). Ovine preantral (PA) follicles transition into early-antral (EA) stage was assessed through a stepwise refinement of the current ivF. Firstly, PCL scaffolds were introduced to provide microarchitectural 3D guidance to PA for single-follicle ivF (SF-PCL). Then, a multi-follicle culture system with PCL scaffolds (MF-PCL) was tested with the aim to better mimic the native ovarian microenvironment. Morphological and functional endpoints were evaluated, including follicle growth, antrum differentiation, steroidogenic switch off, chromatin configuration, oocyte maturation, and parthenogenetic activation rate. The synchronous growth of follicles and oocytes was supported by both SF- and MF-PCL systems. However, the MF-PCL exhibited a slower follicular growth rate, a delayed antrum differentiation but a prolonged resilience in long-term ivF. Indeed, MF-PCL showed consistently lower degeneration rates (5% vs. 28% of SF-PCL; p<0.001) and a higher percentage of antrum cavity differentiation (95% vs. 72% of SF-PCL; p<0.001). Furthermore, the follicles transited from PA to EA on MF-PCL group displayed a significant upregulation of the aromatase enzyme (3-fold change PA vs. EA; p<0.0001), which was similar to that recorded in EA follicles developed physiologically (EA in vivo). Notably, MF-PCL 3D long-term culture also improved the developmental quality of oocytes that displayed a more advanced chromatin configuration (53% Chromatin Surrounding-Nucleolus; 27% Surrounding-Nuclear-Envelope vs. 100% Non-Surrounding-Nucleolus of SF-PCL; p<0.001). The more advanced specialization of MF-PCL incubated oocyte was finally confirmed for their greater ability to resume meiosis after hormonal stimulation (Metaphase II stage oocyte: 62% vs. 50% for SF-PCL; p<0.05), and to be activated by parthenogenesis (> 8 cells embryo: 82% vs. 28% for SF-PCL; p<0.001). Of note, MF-PCL was effective in bypassing the embryo blockage (8 blastomeres stage). PCL-scaffolds have demonstrated their capability to effectively mimic the natural ovarian environment, fostering the growth and development of multiple follicles. This innovation, coupling the use of biomaterials and the ovary reconstruction, could lead to substantial advancements in the fields of animal reproduction and wildlife conservation, offering new strategies to enhance fertility and to preserve and use diverse genome materials.