Intracytoplasmic Sperm Injection after Vitrification of Immature Oocytes in Follicular Fluid Increases Bovine Embryo Production

Background: Despite the low efficiency caused by its harmful effects, vitrification is the technique of choice for oocyte cryopeservation, especially at the germinal vesicle (GV) stage. This enables the banking of female gametes without linkage to the male genotype. Follicular fluid (FF), in vivo, is known to provide an adequate environment to the immature oocyte. The intra-cytoplasmic sperm injection (ICSI), by the other hand, can be used to bypass any sperm penetration disorder, including the ones caused by cryopreservation. This study aimed to evaluate oocyte vitrification in FF based solution, and to asses ICSI efficiency in the fertilization of vitrified/warmed bovine GV oocytes.Material, Methods & Results: Follicles of 2-8 mm in diameter were aspirated from bovine ovaries obtained from a slaughterhouse, selected and maintained into FF from aspiration, until their allocation in the experimental groups. The FF used to prepare the vitrification solution was centrifuged, heat inactivated, filtered through a 0.22 mm pore and stored at -20°C. Oocyte vitrification was done into one of these three solutions: The standard solution TCM-Hepes (TH-Vitri) was compared to a totally FF based solution (FF-Vitri), and to a 50:50 (v/v) mix of both solutions (TH:FF-Vitri). Oocytes were submitted to in vitro embryo production in order to assess embryo production efficiency. A second set of experiments using the FF-Vitri solution compared IVF versus ICSI. With basis on cleaved structures, the morula + blastocyst rate obtained in the Fresh Control (43.9%) was similar to FF-Vitri (31.1%). Conversely, the TH-Vitri (15.7%) and the TH:FF-Vitri (20.4%) rates were significantly lower than the Fresh Control. ICSI showed a positive effect in comparison with IVF. The embryo development rate of Vitri-IVF (18.8%) was the lowest, whereas Vitri-ICSI (37.3%) was similar to the Fresh-IVF (43.9%), but lower than the Fresh-ICSI (57.8%).Discussion: Oocytes cryopreserved in TH based solution are known to show certain rigidity in the zona pellucida, being this event a possible cause to spermatozoa penetration disruption. Our results agree with that, since the fertilization rate for TH-Vitri was significantly lower than for the FF-Vitri. In contrast, GV oocytes vitrified in total versus partial FF based solution showed similar maturation and fertilization rates as the Fresh Control, evidencing the beneficial effect of FF during the course of vitrification. It is possible that FF helped to adjust oocyte maturation, allowing a better nuclear-cytoplasmic synchrony. Also, it might have provided some protection due to its antioxidant properties. The releasing of cortical granules induced by freezing, lead to a zona pellucida hardening and failure in sperm penetration. Factors present in the FF might block this premature releasing of cortical granules, thus ensuring that the egg retains its ability to be fertilized after maturation. The blastocysts produced from the FF-Vitri oocytes were the only ones that had the average ICM similar to the Fresh Control, evidencing that besides the similarity in morula + blastocyst rates, the embryos derived from oocytes vitrified in FF solution have also yielded best quality. When vitrified warmed oocytes were submitted to ICSI, there was an increase in the blastocyst production. This increment of embryo production with ICSI evidences a pathway to overcome the zona pellucida biological barrier. In conclusion, the use of FF as base for vitrification solution improves further embryo development; ICSI increases the embryo production of vitrified/warmed bovine GV stage oocytes.