title: Optimization of cryoprotectant loading into murine and human oocytes.
abstract: Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me(2)SO) into mouse oocytes at 23°C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me(2)SO exposure time, revealing that neither shrinkage nor Me(2)SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me(2)SO addition appears to result from interactions between the effects of Me(2)SO toxicity and osmotic stress. We also investigated Me(2)SO loading into mouse oocytes at 30°C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me(2)SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me(2)SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.
authors.affiliation: Department of Mechanical Engineering, Villanova University, Villanova, PA 19085, USA.
authors.forename: Jens O M
authors.initials: JO
authors.lastname: Karlsson
authors.affiliation: Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Edyta A
authors.initials: EA
authors.lastname: Szurek
authors.affiliation: School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331, USA.
authors.forename: Adam Z
authors.initials: AZ
authors.lastname: Higgins
authors.affiliation: Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA.
authors.forename: Sang R
authors.initials: SR
authors.lastname: Lee
authors.affiliation: Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA; Department of Obstetrics and Gynecology, and Cancer Center, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, USA. Electronic address: aeroglu@gru.edu.
authors.forename: Ali
authors.initials: A
authors.lastname: Eroglu
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004622:Embryo, Mammalian; D047108:Embryonic Development; D019855:Ethylene Glycol / Q000494:pharmacology; D005163:Factor Analysis, Statistical; D005260:Female; D005306:Fertilization / Q000187:drug effects* / Q000502:physiology; D005307:Fertilization in Vitro; D006801:Humans; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D009994:Osmolar Concentration; D011247:Pregnancy; D019946:Propylene Glycol / Q000494:pharmacology; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D019946:Propylene Glycol; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide
keywords: Cryopreservation; Cryoprotectant; DMSO; Dimethyl sulfoxide; Freezing; Human; Oocyte; Propylene glycol; Simplex optimization; Vitrification