In Vitro Maturation

The oocytes were searched from the aspirated solution containing the COCs. Collected oocytes were graded according to IETS guidelines before processing for IVM. The grading was based on cumulus density and characteristics of ooplasm, viz., Grade 1 included COCs with more than three complete and compact layers of cumulus cells, Grade 2 had one or two compact cumulus cell layers, Grade 3 had less than one complete layer, and Grade 4 had expanded cumulus cell layers or signs of degeneration ( Fig. 1 ). The good-quality oocytes (Grades 1–3) were washed in IVM media and graded before placing them in preequilibrated IVM drop (media were preequilibrated overnight). Oocytes with maturation media were placed in an incubator with 5% CO ₂ in air (O ₂ concentration around 14–16%), 38.5 °C temperature, more than 90% relative humidity (RH), and incubated for around 22 hours for IVM.

The figure illustrates various grading patterns of cumulus–oocyte complexes as outlined in the IETS Manual, 5th Edition. Grade 1 : COCs have more than three complete and compact layers of cumulus cells covering the surface of the zona pellucida (ZP). The ooplasm is dense and has even granulation. Grade 2: COCs have one or two compact cumulus cell layers covering the surface of the zona pellucida. Grade 3: COCs have less than one complete layer of cumulus cells covering the surface of the ZP. COC, cumulus–oocyte complex; IETS, International Embryo Technology Society.