OBJECTIVE: Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos. DESIGN: Experimental prospective controlled study. SETTING: Research center. PATIENT(S): Human Blastomeres were obtained after biopsy. INTERVENTION(S): Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing. MAIN OUTCOME MEASURE(S): Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres. RESULT(S): After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip. CONCLUSION(S): The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives.
OBJECTIVE: Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos. DESIGN: Experimental prospective controlled study. SETTING: Research center. PATIENT(S): Human Blastomeres were obtained after biopsy. INTERVENTION(S): Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing. MAIN OUTCOME MEASURE(S): Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres. RESULT(S): After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip. CONCLUSION(S): The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives.
Authors: Eun Kyung Ryu; Yong Soo Hur; Ji Young Ann; Ja Young Maeng; Miji Park; Jeong Hyun Park; Jung Yoon; San Hyun Yoon; Chang Young Hur; Won Don Lee; Jin Ho Lim Journal: Clin Exp Reprod Med Date: 2012-12-31