Hyase 10xtm

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.

The COCs retrieved at ovum pick-up were maintained in an incubator for 2 h (37 °C, 5% CO₂ and 5% O₂). The procedure of mechanical removal of CCs was facilitated by the exposure of COCs to hyaluronidase (HYASE-10XTM, Vitrolife, Goteborg, Sweden). At this purpose, each COC was moved into the hyaluronidase droplet (200 µL, HYASE-10X™, Vitrolife, Sweden) where it was gently blown for several times, but for no more than 30 s. At the end of this step, the oocyte in the hyaluronidase droplet was transferred into the operation droplets (50 µL) with oocyte medium (G-GAMETE, Vitrolife, Sweden), where they were gently and repeatedly aspirated with a denudation pipette. The oocytes categorized as mature (nuclear metaphase II stage) were fertilized in vitro using intracytoplasmic sperm injection (ICSI) procedure. The relative CCs were removed and washed in three droplets of medium (G-GAMETE, Vitrolife, Sweden) and were used for the aim of the present study. CCs deriving from MII oocytes were pooled and collected separately for individual patients and stored at −80 °C until the use for the genetic analysis.