• Journal of Embryo Transfer
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• v.14 no.3
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• pp.145-153
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• 1999

This study was conducted to investigate the effects of vitrification solution and developmental stage on the survival rate of vitrified-thawed human blastocyst embryos. Human blastocyst embryos were cryopreserved by vitrification using EFS and GE solution, and their survival rates were examined after thawing and further culture. EFS solution was consisted of 40% ethylene glycol, 18% Ficoll 70 and 0.3M sucrose. GE solution was consisted of 25% glycerol and 25% ethylene glycol. Embryos were exposed to EFS and GE solution by 2 steps and 3 steps, respectively, and plunged into liquid nitrogen after loading into 0.25ml plastic straws. Blastocysts were classified into 4 groups in accordance with their developmental stage: into 1) EEB, 2) MEB and 3) EdB, of blastocysts developed on day 5, and 4) 6d-Bla(the blastocysts which formed on day 6). The blastocysts at each stage were vitrified by GE solution and cryopreserved in LN2. After thawing them, we examined their survival rates, respectively. The resulted of this study were as follows: 1. The survival rate of blastocysts vitrified by GE solution was 64.4%, significantly higher than that (5.7%) vitrified by EFS solution (P<0.001). 2. When the blastocysts were vitrified by GE solution according to each developmental stage, the survival rates of EEB, MEB, EdB and 6d-Bla were 65.9%, 65.9%, 73.2% and 58.1%, respectively. In conclusion, the cryopreservation of human blastocysts by vitrification is likely to have a marked advantage in terms of cost, work and time as compared to the conventional slow freezing in IVF-ET programs.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.367-372
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• 2000

Objective: This study was performed to evaluate whether vitrification method could be used for the cryopreservation of human blastocysts derived from IVF program. Methods: Surplus embryos were obtained from consented IVF patients. Controlled ovarian hyperstimulation was done with midluteal GnRH agonist, gonadotropin and hCG. After oocyte retrieval and insemination, fresh embryo transfer was done at $4{\sim}8$ cell stage. The surplus embryos after ET were cultured in blastocyst medium up to 6 days after oocyte retrieval. Obtained blastocysts were cryopreserved with our vitrification method. Blastocysts were exposed to 1.5 Methylene glycol (EG) in phosphate buffered saline (PBS) for 2.5 minutes, followed by 5.5 M EG plus 1 M sucrose for 20 seconds. Then 1 to 3 blastocysts were mounted on electron microscope (EM) grid and the grid was plunged into liquid nitrogen for storage. For thawing, blastocyst-containing EM grids were sequentially transferred in 1.0 M, 0.5 M, 0.25 M, 0.125 M and 0 M sucrose solution at the intervals of2.5 minutes. And blastocysts were cultured for about 6 hours and only re-expanded blastocysts were transferred to uterus of the patients on 4 to 5 days after ovulation in natural cycle or on 18 to 19 day of artificial cycle. Results: From Oct. 1998 to Jul. 1999, 34 patients were agreed to participate in this study. The mean age and duration of infertility of the patients were 31.6 years and 4.1 years, respectively. Among 34 cycles. replacements could be done in 20 cycles (58.8%). A total 93 blastocysts were thawed and 48 (51.6%) of them survived. Thirty-eight blastocysts, mean 1.9 embryos per patient, were transferred, resulting in 5 clinical pregnancies which consisted of 1 triplet, 2 sets of twins and 2 singleton pregnancies. The pregnancy rate per transfer was 25% and implantation rate was 23.6%. Five patients delivered 7 healthy babies including 2 sets of twins at term. Conclusion: Successful pregnancies and deliveries were established after transfer of vitrified human blastocysts. Vitrification using ethylene glycol as cryoprotectant and electron microscope grid is a rapid and simple method that can be effectively applied for the cryopreservation of human blastocysts.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.177-186
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• 1998

While tubal pregnancy is frequently observed in human, it has been reported to rarely occur in other mammals. To investigate the reason of the absence of tubal pregnancy in other mammals, the ability of bovine tubal(oviductal) fluid to su, pp.rt the outgrowth of mouse embryos waw examined by using an in vitro model system wherein the trophoblast cells of hatched mouse blastocysts attach to and outgrow on tissue culture plates coated with FBS. When mouse blastocysts grwon in vitro from 2-cell embryos were cultrued in the dishes coated with FBS, human follicular fluid(hFF) and bovine follicular fluid(bFF), respectively, underwent outgrowth by spreading onto the plastic dishes during 48 hr. In contrast, none of the embryos cultured in the dishes coated with BSA or bovine obiductal fluid(bOF) did outgrow but remained as late blastocysts. Since addition of bOF at 5mg/ml or higher conc. to the culture medium resulted in degeneration of all embryos during 48 hr culture, 10mM conc. of glutathione(GSH) was added to the bOF-containing medium to circumvent the toxicity of bOF. In addition, bOF was heated $65^{\circ}C$ for 30 min(hbOF) to get rid of its precipitating properties and then added to the culture medium. When blastocysts were cultured in the presence of both hbOF and GSH 45.4% of embryos attached to the culture dishes. However, none of these embryos underwent outgrowth. Fially embryos were cultured in the presence of both hbOF and GSH but in the dishes coated with FBS. When they were examined after 48 hr, all of the blastocysts exhibited well-developed outgrwoth. Based upon these results, it is concluded that bovine oviductal fluid is capable of su, pp.rting the attchment of mouse blastocysts onto the culture plaste whereas it cannot promote the outgrwoth of mouse blastocysts in vitro, probably due to the lack of outgrwoth factor.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.105-111
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• 1994

The effects of human or mouse leukemia inhibitory factor(hLIF or mLIF) were examined as a means of increasing the development of in vitro matured(IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted using Hochest dye staining. Two-to 8-cell embryos derived from bovine IVM/IVF oocytes were cultured 5 to 6 days in CRI aa with or without mLIF or hLIF. All culture media were contained 3mg/ml bovine serum albumin. In experiment 1, the proportion of embryos developed to morulae and blastocysts in CRI aa containing 5,000U/ml mLIF(37.8%) was slightly higher than those of CRIaa containing 1,000U/ml mLIF(34.6%) and 0 U/ml mLIF(27.4%; P>0.05). In experiment 2, 0, 1,000 and 5,000U/ml of hLIF added to CR1aa media yielded 27.6%, 43.0% and 35.5% morulae and blastocysts, respectively(p>0.05). These were no significant increases in cell number among treatments(p>0.05). These results were indicating that mLIF or hLF can increase the proportion of embryos that develop into morulae and blastocysts without and increase in the cell number.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.3
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• pp.241-248
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• 2003

Objective: The purpose of this study was to evaluate the survival rate of vitrified blastocyst according to the freezing vessels, equilibration time in cryoprotectant and artificial dehydration method. Methods: Human blastocysts were vitrified after loading onto the plastic straw, open-pulled straw (OPS), electron microscopy grid (EM grid) for 1.5 min or 3 min. They also were directly plunged into LN2 within 30sec. For artificial shrinkage of blastocysts, 36 gauge fine needle was pushed at the cellular junction of the trophectoderm into the blstocoele cavity until it shrank without damage of inner cell mass. Results: The survival rate of vitrified blastocysts on plastic straw, OPS, EM grid as freezing vessels were 26.7, 13.0 and 60.5%, respectively. The survival rate of EM grid was significantly higher than that of plastic straw and OPS (p<0.05). For 1.5 min equilibrium, the survival rates of early blastocyst (EB), middle blastocyst (MB) and late blastocyst (LB) were 64.4, 81.0, and 20.0% respectively. For 3 min equilibrium, the survival rates of EB, MB, and LB were 69.9, 50.0 and 57.5% respectively. The survival rates of EB and MB were significantly higher than that of LB in 1.5 min equilibrium group (p<0.05), however, the significance was not observed in 3 min equilibrium groups. In cytoplasmic shrinkage before vitrification, the survival rates of EB, MB and LB were 92.9, 100 and 75.9% respectively. The survival rate of MB was significantly higher than that of LB (p<0.05). The survival rates of vitrified blastocysts by artificial dehydration and slow-frozen blastocysts were not significantly different as 88.9 and 66.7%, respectively. Conclusion: This study showed that the vitrification of human blastocysts using EM grid and artificial dehydration is an effective method. Therefore, these methods would be an useful techniques for blastocyst cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.67-74
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• 2000

Objective: This study was conducted to investigate the effect of vitrification on the implantation and the pregnancy of human blastocysts. Method: The transfer of the frozen-thawed blastocysts by the slow freezing or vitrification was performed between January 1998 and July 1999. The zygotes derives from IVF were cocultured with cumulus cells in YS medium containing 20% hFF for 5days. Two or three of the best balstocysts produced on day 5 were transferred into the uterus, and then supernumerary blastocysts were randomly divided into two groups. One was frozen by slow freezing and the other was frozen by vitrification method. The slow freezing procedure was performed in two steps (5% glycerol and 9% glycerol + 0.2 M sucrose for 10 min, respectively) using programmed freezer ($-2^{\circ}C$/min to $-7^{\circ}C$, manual seeding at $-7^{\circ}C$, $-0.3^{\circ}C$/min to $-38^{\circ}C$ and plunged into $LN_{2}$). The blastocysts frozen by slow freezing were thawed at $36^{\circ}C$ then removed glycerol in 7 steps. The vitrification procedure was performed in three steps (10% glycerol for 5 min, 10% glycerol + 20% ethylene glycol for 5 min, 25% glycerol + 25% ethylene glycol and directly $LN_{2}$ within 1 min). The blastocysts frozen by vitrification were thawed at $20^{\circ}C$ water then removed cryoprotectant in 3 steps. In each group, thawed blastocysts were cocultured with cumulus cells in YS medium containing 20% hFF for 18h and transferred into the uterus. The implantation rate was evaluated per transferred blastocysts and the pregnancy rate was evaluated per transfers. Results: The survival rate of vitrified group (74.5%) was higher than slow freezing group (68.0%), but not significant. When 98 thawed blastocysts of vitrification were transferred in 40 cycles, 19 pregnancies (clinical pregnancy rate; 47.5%) were established. One miscarriage occurred in the eighth week of pregnancy (ongoing pregnancy rate; 45.0%). 7 pregnancies were ongoing, 11 pregnancies went to term, and 16 healthy infants were born. The Implantation rate was 31.6%. These results were higher than those obtained by the slow freezing (clinical pregnancy rate; 40.3%, ongoing pregnancy rate; 32.5% and implantation rate; 25.3%), but not significant. Conclusion: Vitrification is a simple, quick and economical method when compared to slow freezing. It will be chosen as a good method of human embryo freezing in IVF-ET programs.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.33-40
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• 2001

Objective: This study was to establish the human embryonic stem (ES) cells derived from frozen-thawed blastocyst stage embryo that were destined to be discarded after five years in routine human IVF-ET program. Methods: Frozen-thawed and survived human blastocysts were treated by immunosurgery, and recovered ICM cells were cultured onto STO feeder cell layer and ICM colony was subcultured by mechanical dissociation into clumps. To identify ES cell, alkaline phosphatase staining and expression of Oct4 in replated ICM colonies were examined. Also, to examine the possibility of ES cell differentiation, retinoic acid (RA), basic fibroblast growth factor (b-FGF), nerve growth factor (NGF) were added in culture medium. In addition, to classify the specific cell type, differentiated cells were stained by indirect immunocytochemistry. Results: One ICM colony recovered from frozen-thawed six blastocysts was subcultured, continuously replated during 40 passage culture duration without differentiation. Subcultured colonies were strong positively stained by alkaline phophatase. When the expression of Oct4 in cultured ES colony was examined, Oct4b type is more clearly indicated than Oct4a one although there was not detected in embryoid body or differentiated cells. In differentiated cardiomyocytes from ES colony, cells were beaten regularly (60 times/min). In differentiated neural cells from ES colony, neurofilament (NF) 200 kDa protein, microtubule associated protein (MAP) 2 and ${\beta}$-tubulin of specific marker in neurons, glial fibrillary acidic protein (GFAP) of specific marker in astrocytes and galactocelebrocide (GalC) of specific marker in oligodendrocytes were confirmed by indirect immunocytochemistry. Also, muscle cells were detected by indirect immunocytochemistry. In addition, ES colonies can be successfully cryopreserved. Conclusion: This study suggested that establishment of human ES cells can be successfully derived from frozen-thawed blastocysts that were destined to be discarded, and obtained specific cell types (cardiomyocytes, neurons and muscle cells) through the in vitro differentiation procedures of ES cells.

• Clinical and Experimental Reproductive Medicine
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• v.42 no.3
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• pp.94-100
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• 2015

Objective: The goal of this study was to ascertain optimal assisted hatching (AH) method in frozen embryo transfer. We compared the effect of depending on whether mechanical or laser-AH was performed before or after the vitrification of embryo development rate and blastocyst cell numbers. Methods: In order to induce superovulation, pregnant mare's serum gonadotropin followed by human chorionic gonadotropin were injected into 4- to 5-week-old female mice. 2-cell embryos were then collected by flushing out the oviducts. The Expanded blastocysts were recovered after the collected embryos were incubated for 48 hours, and were then subjected to artificial shrinkage (AS) and cross-mechanical AH (cMAH) or quarter-laser zona thinning-AH (qLZT-AH) were carried out using the expanded blastocysts before or after vitrification. After 48 hours of incubation, followed by vitrification and thawing (V-T), and blastocysts were fluorescence stained and observed. Results: The rate of formation of hatched blastocysts after 24 and 72 hours of incubation was significantly higher in the AS/qLZT-AH/V-T group than in the other groups (p<0.05). The cell number of the inner cell mass was higher in AS/V-T/non-AH and AS/V-T/cMAH groups than those of others (p<0.05). In the control group, the number of trophectoderm and the total cell number were higher than in the AS-AH group (p<0.05). Conclusion: The above results suggest that AS and AH in vitrification of expanded blastocysts lead to the more efficient formation of hatched blastocysts in mice.

• 대한생식의학회:학술대회논문집
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• 2001.03a
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• pp.171-179
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• 2001

• Development and Reproduction
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• v.20 no.3
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• pp.219-225
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• 2016

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ${\geq}38$ years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen ($LN_2$) and then directly immersed into the first WS for 1 min at $37^{\circ}C$ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrified-warmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.