Hyperosmotic HEPES-CZB Enhances Post-ICSI Survival of C57BL/6N Mouse Oocytes.

Purpose: This study aimed to determine whether performing intracytoplasmic sperm injection (ICSI) under transiently increased osmolality in HEPES-CZB can improve post-ICSI survival as well as embryo and offspring production in C57BL/6N mice. Methods: HEPES-CZB was supplemented with 0-200 mM NaCl or KCl, or 0-400 mM D(+)-glucose or D(-)-mannitol. Oocytes were transferred to each hyperosmotic medium and then subjected to ICSI. Post-ICSI survival, normal fertilization, and blastocyst development were evaluated as rates per injected oocytes. Blastocyst quality was examined by immunostaining to quantify cell types and numbers. Two-cell embryos generated under the best conditions were transferred to evaluate full-term development. Results: Across supplements, increased osmolality consistently reduced oocyte diameter. Following ICSI, the survival rate of these oocytes was significantly increased, and development to the blastocyst stage was maximized under conditions of 100 mM NaCl, 100 mM KCl, 150 mM glucose, and 200 mM mannitol. Under these conditions, the full-term development per injected oocyte remained comparable to the control. Conclusion: Hyperosmotic ICSI significantly improves post-ICSI survival but may compromise developmental competence at higher concentrations, resulting in no net increase in offspring production. Further optimization may increase the number of embryos produced using this approach and reduce workload on the embryologist.