Cumulus Cells Isolation from COCs

The procedure used for the CCs isolation from COCs is the same as previously described in our study [10 (link)]. Briefly, the removal of CCs from COCs was aided with the exposure of COCs to hyaluronidase solution (HYASE-10XTM, Vitrolife, Goteborg, Sweden). A subsequent mechanical repeatedly aspiration of the COCs in denudation pipettes completed the procedure of CCs isolation. At the end of the practice, the oocytes categorized as mature (nuclear metaphase II stage) were used for fertilization in vitro (ICSI procedure) and the relative CCs were stored at −80 °C until the use for the purpose of the present study. Of note that CCs deriving from metaphase II (MII) oocytes were pooled and collected separately for individual patients.