Alginate engineered cumulus oocyte enclosing microbeads for modelling 3D oocyte maturation

Microencapsulation using hydrogels can provide a suitable structure mimicking the in vivo microenvironment. We recently produced ovarian follicle structures in the sheep model by encapsulation cumulus-oocyte complexes (COCs) in tailored alginate microbeads through the bio-printing technology [1, 2]. The aim of this study was to test the suitability of these 3D structures for In Vitro Maturation (IVM) by comparing their maturation rate versus those of COCs cultured in conventional 2D IVM. Two experiments were conducted in two different laboratories with oocytes from adult (Experiment 1; UniPisa) and prepubertal sheep (Experiment 2; UniBari). COCs recovered from the ovaries of slaughtered adult (>1 year) and prepubertal sheep (<6 months) were either included in alginate engineered microbeads [3D-IVM; 2] or directly placed in multi-well Petri dishes and cultured for IVM [2D-IVM; 3]. After IVM, oocytes were fixed overnight at 4°C in 3.8% formaldehyde solution in PBS, stained with Hoechst 33258 and observed under epifluorescent microscopy to assess their meiotic stage (Chi-square test: significance at P<0.05). In Experiment 1, 349 oocytes of adult sheep were analysed (n.5 replicates). Significantly increased oocyte maturation rates were observed in 3D-IVM compared to 2D (133/196, 68% vs 87/153, 57% for 3D and 2D, respectively; P<0.05) with a corresponding reduction in the rates of oocytes showing abnormal chromatin configurations (10/196, 5% vs 19/153, 12% for 3D and 2D, respectively; P<0.05). In Experiment 2, 221 oocytes of prepubertal sheep were analysed (n.4 replicates). Significantly higher rates of MII oocytes were obtained in 3D-IVM versus 2D-IVM (83/129, 64% vs 46/92, 50% for 3D and 2D, respectively; P<0.05). In conclusions: 3D-IVM allows to reach higher oocyte nuclear maturation rates than conventional 2D-IVM. The inter-laboratory (Pisa and Bari) analysis performed in two models, adult and pre-pubertal sheep, further validated our results. Studies are ongoing to evaluate the effects of 3D-IVM on oocyte developmental competence. These data may have potential clinical and toxicological applications. [1] Tirella et al. Biofabrication 2014;6:025009, [2] Mastrorocco et al. LXXII congresso SISVet., [3] Martino et al. Reprod Toxicol2016;65:204-21