Gadjah Mada (Gama) Sleeved Cryoloop as a Novel Device of Vitrification to Preserve Embryo Viability Assessed by Embryo Development and Mitochondria Activity
Ita Fauzia Hanoum, dr.,MCE., PRof.Dr. Djaswadi Dasuki, MPH, Ph.D., SpOG(K)
2013 | Disertasi | S3 Kedokteran UmumBackground: Supernumerary good grade embryos resulted of improved culture system, avoiding multiple gestation, increased need to transfer embryo on natural cycle, as well as selecting best embryos for a better pregnancy rate all has raised the need to develop an optimal cryopreservation program in the ART laboratory. Vitrification is a method to preserve embryos, avoids intracellular ice formation (IIF) by rapidly changing the intracellular content from a liquid to a solid `glass like` state. Develop 0.1 μl Gama sleeved cryoloop as a device is proposing a vitrification procedure in a small volume of cryoprotectant (CPA) to support a very fast cooling and warming rate whilst reducing the toxicity of the cryoprotectant to the embryos. Methods: Swiss Albino 8 cells, morula and blastocyst was vitrified using loops: Gama sleeved cryoloop (0.1-3μl CPA), Cryologic(1μl CPA) and Hampton Research(0.2-0,3μ CPA). Following 24 hours storage embryos were warmed and observed for embryo development and hatching or hatched. Mitochondria activity was investigated by MitoTrackerRed 580 FM M22425 (Invitrogen, Eugene, Oregon, USA). Calculation of CTCF (Corrected Total Cell Fluorescence) were intensity and area of the fluorescence in the embryos. Results: Over all 215 stimulated female Swiss Albino mice aged 6-8 weeks, 147 mated which were then sacrified and from ampula of ovarian tubes dissected 836 cells were retieved. 594 good morphology 2 cells embryos were included in the study, the rest 242 cells either beyond 2 cells, empty zona, atretic and abnormal cell were excluded. The good morphology 2 cells embryo were then allocated in to groups of various devices: 0.1 μl Gama sleeved cryoloop, 1μl Gama sleeved cryoloop, 3 μl Gama sleeved cryoloop, Cryologic loop and Hampton Research loop. The protocol of embryo transfer in the clinical human ART laboratory is transfering the selected embryos at day three (D3) of culture that is 8 cells stage. In this mouse model study, vitrify warmed 8 cells stage resulted in retrieval, survival and good embryos the highest in percentage when used 0.1 μl Gama sleeved cryoloop (p=0.0095) while vitrify warmed morula (p=0.927) or blatocyst (p=0.976) resulted in percentage of no differ between devices. Further more, vitrify warmed 8 cells stage used 0.1 μl Gama sleeved cryoloop resulted fewest non hatching embryos comparing Hazard Ratio of 1 μl, 3μl Gama sleeved cryoloop, Cryologic loop and Hampton Research loop (2.8-3.6 times in resulting non good embryos compare to 0.1 μl Gama sleeved cryoloop). In this present study, vitrify warmed and cultured 8 cells stage embryos used 0.1 μl Gama sleeved cryoloop resulted in highest good embryo (p=0.047), and generated fewest non good ( non hatching ) embryos as compare to morula (2.7 times; p=0.040) and blastocyst (2.2 times; p=0.172) that generated more non hatching embryos. Grouped as pre-compaction (8 cells stage) and post-compation (morula and blastocyst) vitrify warmed embryos in present study resulted in comparable retrieval rate, survival rate and generated good embryos (hatching or hatched) (p=0.666). However, morula stage generated more non hatching embryos when vitrify warmed and cultured. Multivariate analysis was done by considering devices used (0.1 μl, 1 μl, 3μl Gama sleeved cryoloop, Cryologic loop and Hampton Research loop), stages of embryos (8 cells stage, morula, and blastocyst) and adjusted with embryo of better intrinsic factor of embryos to the non good embryos resulted in no statistically significant different (p=0.218). However, Hazard Ratio of group with better intrinsic factor of embryo resulted in less generated non good embryos (0.7 X) (p>0.05), while Hampton Research loop generated highest non hatching (non good embryos) 1.8 times comparing 0.1 μl Gama sleeved cryoloop (p=0.020), 3 μl Gama sleeved cryoloop 1.6 times (p=0.044), 1 μl Gama sleeved cryoloop 1.5 times (p=0.095) and Crylogic loop 1.4 times (p=0.153). The data of mitochondrial activity chaptured with red fluorescence in this present study for all devices used showed that there was no significant difference for 8 cells (p=0.101) and morula (p=0.105) vitrified warmed, while in the group of blastocyst differences in intensity of red fluorescence chaptured might probably influeced by varies of blastocyst size (p=0.029). Conclusions: 0.1 μl Gama sleeved cryoloop when used as device for vitrification procedures of 8 cells stage mouse embryos resulted in highest percentage of good embryos (hatching or hatching), no atretic embryos observed, and mitochondrial activity observed by Mitotracker was no difference between all cryoloop used in the study when 8 cells stage mouse embryo vitrify warmed.
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Kata Kunci : vitrification, cryoloop, volume of CPA, hatching and hatched rate, Gama sleeved cryoloop
