G gamete

Microinjections were performed at ×400 magnification on a 37 °C heated stage inverted Nikon Eclipse Ti2 (Nikon, Tokyo, Japan). A Petri dish containing a microdroplet of ICSI ™ media in the center (ICSI ™, VitroLife, Sweden) under paraffin oil (OVOIL, VitroLife, Sweden) was used for sperms selection and immobilization. On the same dish, a microdroplet of G-Gamete ™ culture medium (G-Gamete ™, Vitrolife, Sweden) was used for placing the oocytes for microinjection. A single sperm was mechanically immobilized using the tip of the microinjection needle (Origio, USA) and then was aspirated inside the needle. The oocyte was held in place using a 35-degree angle holding micropipette (Origio, USA) with the polar body in the 6 or 12 o'clock position. Injection of a single spermatozoon within the oocyte cytoplasm was performed by using a micromanipulator (TransferMan® 4r, eppendorf, Germany). After ICSI, injected oocytes were cultured in G1-Plus ™ medium (G1-Plus ™, VitroLife, Sweden). Fertilization was confirmed by the presence of two pronuclei and the extrusion of the second polar body approximately 16–18 h after ICSI. The Fertilization Rate was calculated by dividing the number of obtained zygotes (2 PN) by the number of injected oocytes.

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.

The media used for the manipulation and culturing of mouse and human oocytes/zygotes were from Vitrolife Sweden AB. CB (C6762, Sigma, Saint Louis, MO, USA) was dissolved to a stock concentration of 10 mg/ml in dimethyl sulfoxide (DMSO, D2650, Sigma). Then the CB stock solution was diluted to gradient concentrations with G-GAMETE (10126, Vitrolife, Göteborg, Sweden). An additional amount of DMSO was added such that the final concentration of DMSO in each CB group was 0.1%. The 0 μg/ml CB group (DMSO group) represented zero CB + 0.1% DMSO, the blank group represented zero CB + 0% DMSO.