• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.103-103
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• 2002

This study was conducted to investigate cryopreservation of pearl oyster, Pinctada fucata martensii larvae. Four cooling rates (-0.25, -0.5, -0.75 and -1.0$^{\circ}C$/min.) were used to examine a proper cooling rate during cryopreservation of trochophores before seeding temperature (-12$^{\circ}C$). Seven developmental stages (early and late trochophores, early and late D-shaped larvae and early, middle and late umbo stage larvae) and different sugars (fructose, glucose and sucrose) were used to investigate optimal larval stage and effective sugar in cryopreservation of larvae. The survival rates of frozen-thawed trochophores increased at cooling rate of -1.0$^{\circ}C$/min. As larval developing, survival rate of frozen-thawed larvae increased, except umbo stage larvae, and especially late D-shaped larvae highly survived as 91%. Addition of sugar revealed positive effect on cryopreservation in this experiment and 0.2 M glucose and sucrose mixed with 2.0 M dimethyl sulfoxide significantly enhanced survival rate of larvae (P<0.05). The results of our study indicate that desirable cooling rate, developmental stages of larvae and effective sugar far cryopreservation of pearl oyster, P. fucata martensii larvae are -1$^{\circ}C$/min, late D-shaped larvae and 0.2 M glucose and sucrose, respectively.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.13-19
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• 1992

This study were carried out to investigate the effective concentration of cryoprotective agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen porcine embryos. The porcine embryos foflowing dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined by FDA test. The results are sunnnarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen4hawing in the freezing medium with a various concentration of glycerol, DMSO and propanediol added 0.25M sucrose were higher survival rate than those of sucrose concentration of 0.50M. 2. The survival rates of porcine embryos after ultrarapid ftozen4hawing in the freezing medium added 0.25M and 0.SOM sucrose were higher survival rate than those of sucrose concentration of 0.75M and 1.00M. 3. The temperature thawed at 2$0^{\circ}C$ and 3$0^{\circ}C$ resulted in a significantly higher embryos survival rate after 72 hrs in culture than did at 35$^{\circ}C$. 4. The equilibration time on the survival rate of porcine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time(10~20 min.).

• Journal of Embryo Transfer
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• v.5 no.2
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• pp.38-44
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• 1990

This study was carried out to produce superior dairy cattle by embryo transfer. Seven dairy cows were superovulated with divided injection of FSH 4Omg for 5 days started on day 9 to 14 of the estrus cycle and injection of PGF$_2$$\alpha$ 45mg on day 4 of FSH injection. Donor cows were flushed to collect embryos on day 7 or 8 of the estrus cycle. Fresh embryos collected were transferred to synchronized dairy recipients or frozen using glycerol 3 step method to he equilibrated. And 35 embryos which were frozen using glycerol 6 step method were imported from U.S.A. After glycerol dilution of frozen embryos was done by reverse density during freezing. frozen-thawed embryos were transferred to synchronized dairy or beef recipients. The results obtained were as follows; 1. Total of 24 embryos were collected from 7 donor cows flushed and transferable embryos were 18 (75.0%). 2. Among 24 embryos. morula, early blastocyst, blastocyst, expanded blastocyst and unfertilized ova were 3 (12.5%), 1 (4.2%), 10 (41.6%), 4 (16.7%) and 6 (25.0%), respectively. 3. Heat inducing rate after 1st and 2nd injections of PGF$_2$$\alpha$ in Holstein and beef cattle was 83.3% and 71.4% and 62.5% and 69.2%, respectively. 4. Among 56 recipients, 23 head were pregnant (41.1%). The pregnancy rate of fresh embryos was 50.0% (1/2 heads) and the pregnancy rate of frozen embryos which were frozen using glycerol 3 step and using glycerol 6 step imported from U.S.A. was 52.6%(l0/19 heads) and 34.3%(12/35 heads), respectively. 5. The pregnancy rate of blastocyst (60.0%) was higher than that of morula (39.0%), early blastocyst (25.0%) and expanded blastocyst (0%). 6. The pregnancy rate of grade I embryos (52.2%) was higher than that of grade 2 (34.6%) and grade 3 (28.6%). 7. The pregnancy rate according to synchrony of recipient with donor was higher in simultaneous recipient (55.0%) and +l2hrs' (53.8%) than -24hrs' (23.5%), -l2hrs' (20.0%) and +24hrs' (0%).

• Clinical and Experimental Reproductive Medicine
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• v.29 no.3
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• pp.201-207
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• 2002

Objective: To observe the capability of fertilization and embryo development including blastocyst formation of the oocytes in simple media after thawing of the cryopreserved cumulus-free mouse oocytes by vitrification method. Methods: Oocytes were collected from 5 to 6 weeks old ICR female mice, and were denuded from the cumulus cells by 0.1% hyaluronidase. Recovered mature oocytes in study group were cryopreserved by vitrification method using EM grid for $5{\sim}7$ days. In brief, oocytes were exposed in dPBS containing 1.5 M EG and 5.5 M EG+1 M sucrose for 2.5 minutes and 20 seconds each, and then executed vitrification by plunging in LN2 after loading on EM grid. Thawing treated by exposure of 1, 0.5, 0.25 and 0.125 M sucrose solution for 2.5 minutes each in order and used for experiments. Spermatozoa aspirated form the epididymis of 12 weeks old ICR male mice were used for insemination after capacitation. T6 media containing 0.4% BSA were used for fertilization and development. Results: Survival and fertilization rates after thawing were 76.9% and 79.6% respectively. Fertilization rate was lower (p<0.005) than that of control group (92.9%). There was no difference in embryo developmental rates from 2-cell to morula, however, the blastocyst formation rate and mean cell numbers of blastocysts in study group (63.3%, $58.9{\pm}9.2$) were lower compared with those of control group (76.1%, $63.5{\pm}8.9$). Conclusion: Vitrification is an effective method for mouse mature oocyte cryopreservation with high survival and fertilization rate after thawing. And in simple media, fertilization rates and embryo development of frozen-thawed mouse oocytes are satisfactory.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.3
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• pp.193-202
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• 2003

Objective: To evaluate the difference of implantation rate (IR) and clinical pregnancy rate (CPR) between two protocols of endometrial preperation in women undergoing frozen-thawed embryo transfer (FET) cycles. Methods: This study was performed during the different time periods: A retrospective study from January 2000 to June 2001 (phase I) and a prospective study from July 2001 to March 2002 (phase II). All the patients received estradiol valerate (6 mg p.o. daily) starting from day 1 or 2 of the menstrual cycle without pituitary down regulation. Progesterone was administered around day 14 after sonographic confirmation of endometrial thickness $\geq$7 mm and no growing follicle. In Group A (n=88, 99 cycles) of phase I, progesterone was administered i.m. at a dose of 50 mg daily from one day prior to thawing of pronuclear (PN) stage frozen embryo or three days prior to thawing of 6-8 cell stage frozen embryo and then each stage embryos were trasnsferred 2 days or 1 day later after thawing. In Group B (n=246, 299 cycles) of phase I, patients recieved progesterone 100 mg i.m. from one day earlier than group A; two days prior to PN embryo thawing, four days prior to of 6-8 cell embryo thawing. During the phase II, to exclude any differences in embryo transfer procedures, in Group 1 (n=23, 28 cycles) of phase II embryo was transfered by one who have used the progesterone protocol since the phase I. In Group 2 (n=122, 139 cycles) of phase II embryo was transfered by one who use the progesterone protocol from the phase II. Results: When compared across the phase and group, there were no significant differences in the characteristics. During the phase I, there were significant increase in IR (14.4% vs 5.9%, p=0.001) and CPR (28.3% vs 14.5%, p=0.000) in group A. During the phases II, IR (11.8% vs 10.6%) and CPR (27.6% vs 27.3%) show no differences between two groups. Conclusions: In FET cycles, IR and CPR are increased significantly by the change of dosage and timing of progesterone administraton. And the timing is considered to be more important factor because the dosage of progesterone did not affect implantation window in previous studies. Therefore, we suggest that progesterone administration in FET cycle should begin from one day prior to PN stage embryo thawing and three days prior to 6-8 cell stage embryo thawing.

• 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.34 no.4
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• pp.253-273
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• 2007

Objective: This study was to find ways to let a manager or superintendent rationally and consistently inspect as well as let a embryologist precisely record [The Book of Supernumerary Embryo Preservation] and [The Book of Supernumerary Embryo Donation]. Methods: Based on the data collected between 1994 and 2004 in Clinic 44 (Maria Fertility Hospital), [The Present State about Production and Use of Embryos], [The Preservation of Supernumerary Embryos and Their Thaw State], [The Present State about Thaw and Use of Frozen Embryos], [The Present State about Donation and Charge of Frozen Embryos], [The Book about Frozen Embryo Discard], and [The Summarization Book about Management and Use of Frozen Embryos] were designed and recorded. Results: The production, use, preservation, discard and donation quantity of human embryos, the use and discard quantity of thawed embryos, and the cumulative embryo preservation quantity could be totalized in [The Present State about Production and Use of Embryos in Clinic 44]. Also, [The Preservation of Supernumerary Embryos and Their Thaw State in Clinic 44] supported "the supernumerary embryo preservation quantity" etc. In addition, [The Present State about Thaw and Use of Frozen Embryos in Clinic 44] or [The Book about Frozen Embryo Discard in Clinic 44] supported "the use and discard quantity of thawed embryos" etc. Moreover, "The embryo donation quantity" could be totalized in [The Present State about Donation and Charge of Frozen Embryos in Clinic 44]. Finally, [The Summarization Book about Management and Use of Frozen Embryos in Clinic 44] could be used for rational and consistent management or inspection. Conclusion: The present results suggest that the documents not only be standard data to record [The Book about Supernumerary Embryo Preservation in Clinic] and [The Book about Supernumerary Embryo Donation in Clinic] but can also be preserved as treatment references.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.1
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• pp.7-12
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• 2001

Factors Affecting the Survival of Frozen Thawed Bovine In Vitro Produced Blastocysts. The effect of some factors on the post-thaw survival of a total of 240 in vitro produced bovine blastocysts was investigated using logistic regression analysis. The explanatory variables tested were: type of culture medium before freezing (TCM 199 supplemented with BSA, BSAITS (BSA+insulin+transferrin+selenium), ECS (estrous cow serum) with or without BOEC (bovine oviductal epithelial cells), age of the blastocyst (Day 7, Day 8+9), morphological appearance before freezing (distinct=Q1 or indistinct=Q2 inner cell mass) and type of cryoprotectant (glycerol, 1.0 M or ethylene glycol, 1.6 M). The survival after thawing based on the post-thaw quality and the development after co-culture with BOEC for 24 and 48 hours. Day 7 blastocysts had an almost three times better chance of survival than Day 8+9 blastocysts. Q1, Day 8+9 blastocysts had higher odds to survive after 48 hours in culture than Q2 blastocysts (p<0.05). Blastocysts produced in BSAITS medium had the best chances of survival; however, the odds were not always significant. Blastocysts frozen in glycerol had a better post-thaw quality rating than those frozen in ethylene glycol; however, the difference in post-thaw development at culture was not significant. The relationship between post-thaw quality and post-thaw development at culture was significant (p<0.05). The developmental stage and/or age of the embryo and culture medium where development up to blastocyst takes place affect the post-thaw survival of the bovine embryos.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.301-310
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• 2009

Objective: To compare the embryonic development and pregnancy results using sperms retrieved from fresh and frozen-thawed testicular tissue in patients with obstructive (OA) and non-obstructive azoospermia (NOA). Methods: A total two hundred twenty-two cycles of TESE-ICSI were performed in OA and NOA. Sperms were retrieved from fresh and frozen-thawed testicular tissue. ICSI was performed patient's own sperm. Fertilization was assessed 16~18 hrs after ICSI. Embryo development and pregnancy rates were analysed. Results: The fertilization rates were significantly different between OA and NOA patients (75.2% vs. 56.7%, p<0.05), however, embryo development did not differ between the groups (96.9% vs. 98.0%). Likewise, OA and NOA groups had no differences in their clinical pregnancy and delivery rates, 33.9% vs. 36.0% and 28.1% vs. 28.0%, respectively. With regard to sperm retrieved from fresh testicular tissue, fertilization rates were significantly different between the OA and NOA groups (76.4% vs. 52.9%, p<0.05); however, embryo development, clinical pregnancy and delivery rates were not different. For sperm retrieved from thawed testicular tissue, the fertilization rates were significantly different between the two groups (74.7% OA group vs. 65.6% NOA group, p<0.05); however, embryo development, clinical pregnancy and delivery rates were not different. Conclusions: Embryo development and clinical pregnancy did not differ in patients with obstructive and non-obstructive azoospermia, whether sperm retrieved from fresh and thawed testicular tissue were used, although the fertilization rates were different. Therefore, ICSI with sperm retrieved from fresh and thawed testicular tissue could achieve relevant clinical pregnancy results in patients with azoospermia.

• Journal of Veterinary Clinics
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• v.17 no.1
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• pp.234-237
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• 2000

The dornor, 2 years old, 20kg and mixed breed, was bred naturally on day 1 and day 3 of estrus and eight gastrulae were collected by flushing the uterus of the donor after laparatomy on day 13 after the second mating. The embryos were frozen by programmable freezer and preserved for about 3 months in liquid nitrogen. Another bitch in natural estrus, 2 years old, 30kg, mixed breed, was selected as the recipient and the frozen embryos(8 gastrulae) were thawed and each 4 embryos were transferred into upper partr of left and right uterine horn, respectively, on day 13 after the proper mating day determined by vaginal smear. The ecipient delivered 6 offspring 48 days after embryo transfer. Of 6 puppies, one was still birth and two puppies died one month after birth. Parentage test was performed by DNA analysis using microsatellite sequences for 3 puppiers, the recipient, the donor, the male dog bred with the donor, and the male dog raised near to the recipient. The markers selected for the test were CXX 873(133-157 base pair) and CXX 894(141-165 base pair). Using primers manufactured according to the markers, the blood samples were processed for polymerase chain reaction and the PCR products were treated for electrophoresis. The three puppies showed identical band to that of recipient, consequently, it was concluded that the puppies were offspring of the recipient mated naturally by the male dog, not the offspring by embryo transfer.