Supplementing in-vitro maturation media with human follicular fluid improves both the maturation rate of mouse immature oocytes and the subsequent embryonic development

Abstract

Patients of an advanced maternal age who have sometimes shown unsynchronized follicle growth often receive ovarian stimulation. In those cases, immature oocytes are collected from smaller follicles. Even immature oocytes are valuable for those patients, but it is necessary for these immature oocytes to be allowed to effectively mature in vitro. The aim of the present study was to evaluate whether supplementing culture media with human follicular fluid (HFF) could improve the maturation and subsequent embryonic development of immature mouse oocytes. This was an experimental study. Mouse germinal vesicle (GV) oocytes derived from 8-10 week-old female B6D2F1 mice were divided into three groups according to their concentration of HFF supplementation: The first group used 100% HFF without culture media (all-FF group), the second group used culture media with 50% HFF (v/v) (half-FF group), and the third group used only culture media (non-FF group). After obtaining informed consent, HFF obtained from the first puncture of a follicle during oocyte retrieval without blood contamination was used in this study. The culture media for in-vitro maturation (IVM) involved conventional media (Universal IVF media®) for human IVF treatment in addition to recombinant FSH (0.075 IU/ml) and hCG (0.1 IU/ml). The maturation rates of GV oocytes and the blastocyst formation rates were evaluated among the three groups. Maturation was defined as confirmation of MII stage chromosomes via staining Hoechst solution under fluorescence microscopy. The mature oocytes after IVM were inseminated and fertilized oocytes were additionally extended to the blastocyst stage (up to 124 hours). The maturation rate following IVM in the half-FF group was 100%, which was significantly higher than that of the non-FF group (67.0%, p<0.05), but was similar to the all-FF group (92.0%). The fertilization rates of the all-FF, half-FF and non-FF groups were 78.1, 56.2 and 21.9%, respectively, which showed a significant increase in accordance with the inclusion of HFF. The blastocyst formation rates of the all-FF and half-FF groups were 61.1 and 63.3%, respectively, whereas that in the non-FF group (16.7%) was significantly lower (p<0.05). Supplementing the culture media with HFF during IVM significantly improves the maturation rate of immature mouse oocytes, and the mature oocytes derived from IVM media with HFF possessed higher developmental potential for blastocyst formation. Supplementing IVM culture media with HFF could be useful option for the IVM of human immature oocytes.