• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.281-292
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• 1997

The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.257-262
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• 2009

This study was to investigate pregnancy rate of IVM/IVF/IVC Korean cattle (registered in government) embryos according to transport time course. For the production of embryos, oocytes recovered from slaughtered excellent grade cow and highly motile frozen-thawed bull semen (purchased from LIMC, KPN#497) was used. In vitro produced embryos were cultured in CR1aa medium for 8 days and some of them were frozen. The rate of average cleavage (>2-cell) was 83.0% (308/371) and blastocyst rate at day 8 was 34.7% (107/308). Among in vitro produced blastocyst embryos at day 8, most healthy embryos were freshly transferred on production day and some frozen embryos were direct transferred on appropriate day. These embryos were produced in a laboratory, embryo transfer (ET) was planned in 10 areas of the remote island (Jeju) from the laboratory by airplane. Thus, we examined the pregnancy rate in recipient cow according to embryo of transport time course before ET. From embryo transferred 44 recipient cows, overall pregnancy was 40.9% (18/44), these 18 cows were all calved [single, 94% (17/18); twin, 6% (1/18)] and total embryo implantation rate was 26% (19/66). Comparing transport time in the base of 6 hr, pregnancy rate in ET group required less 4 hr (60%, 9/15) was significantly higher than that required more 6 hr (26.3%, 5/19). In direct ET of freezing embryos, the pregnancy rate was 40% (4/10). However, it was difficult to find the meaning of temperature, pH and corpus luteum quality of recipients on comparison of pregnancy rate. When the cell death level of embryos according to storage time in thermos (straw container) before ET was measured by TUNEL staining, apoptotic index was increased with storage time-dependent. These results demonstrated that long distance transfer of IVM/IVF/IVC embryos is possible and the time of embryo transport is very important for the pregnancy rate on field trial.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.9-16
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• 1990

This study was carried out in order to investigate effects of cryoprotectant concentration and equilibration time on survival of ultrarapidly frozen 2-cell mouse embryos. Mouse 2-cell embryos, following dehydration by exposure to DMSO and sucrose, were directly immersed into liquid nitrogen and thawed in 37$^{\circ}C$ water. Viability was defined by development rate to the blastocyst stage after in vitro culture for 72 hours. The results are summarized as follows ; 1. When 0.25M of sucrose was added into the freezing medium at various concentrations of DMSO and dilution medium, higher development rate of embryo was obtained in 3.0M DMSO concentrations (82.6%). However, when sucrose concentraitons of 0.25 and 0.5 M were added to the freezing medium with 3.0 M DMSO and dilution medium, development rate of embryos were 81.7% and 24.1%, respectively. 2. In the equilibration time at room temperature, higher development rate was attained after short period of time (2.5min) in 3.0 M DMSO＋0.25 M sucrose (85.9%). 3. The development rate of embryos at in vitro 2-cell, in vitro 2-cell, solution control and untreated control was 84.6%, 90.9%, 89.9%,, and 89.7%, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.273-277
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• 1997

This case report describes the pregnancy following the transfer of cryopreserved embryos generated from intracytoplasmic sperm injection (ICSI) using frozen-thawed sperm obtained by microepididymal sperm aspiration (MESA) in patient with congenital absence of the vas deferens (CAVD).

• 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.

• Korean Journal of Animal Reproduction
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• v.26 no.2
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• pp.105-110
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured oocytes after intracytoplasmic sperm injection(ICSI) with epididymal spermatozoa. The ovaries were obtained from slaughtered small species dogs. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with epididymal spermatozoa. After ICSI, one group of oocytes was activated with 2.0 mM dimethylaminopurine or 7% ethanol for 5 min. and second group was not activated. The follicular oocytes were cultured in synthetic oviductal fluid(SOF) and TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. 1. Results of IVM showed that the percentage of oocytes reaching MII after 24 h and 48 hrs of incubation were significantly higher(p<0.05) after culture with 48 hrs(9/30, 30.0%) than that after culture with 24hrs(a/30, 26.7%). 2. Results of IVM showed that the percentage of oocytes reaching MII after 48 hrs of incubation were significantly higher(p<0.05) after culture with SOF media(10/30, 30.3%) than TCM-199 media (7/30, 23.3%). 3. The rate of cleavaged embryos to blastocyst obtained by ICSI treated activation oocytes was significantly higher(p<0.05) than that of nonactivation oocytes(5/16, 25.0% vs 1/13, 5.0%). 4. The rates of development of cleavaged embryos to blastocyst obtained by ICSI treated sperm of fresh, epididymal and frozen-thawed epididymal were 8/18(44.43%), 5/16(31.3%), 2/14(14.3%), respectively. and these values of frozen-thawed epididymal sperm injection were lower than fresh sperm injection.

• Clinical and Experimental Reproductive Medicine
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• v.45 no.3
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• pp.129-134
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• 2018

Objective: In frozen and thawed embryos, the zona pellucida (ZP) can be damaged due to hardening. Laser-assisted hatching (LAH) of embryos can increase the pregnancy rate. This study compared thinning and drilling of the ZP before frozen embryo transfer (FET). Methods: Patients were randomly allocated into two groups for LAH using thinning or drilling on day 2 after thawing. Twenty-five percent of the ZP circumference and 50% of the ZP thickness was removed in the thinning group, and a hole $40{\mu}m$ in diameter was made in the drilling group. Results: A total of 171 in vitro fertilization/intracytoplasmic sperm injection FET cycles, including 85 cycles with drilling LAH and 86 cycles with thinning LAH, were carried out. The thinning group had a similar ${\beta}$-human chorionic gonadotropin-positive rate (38.4% vs. 29.4%), implantation rate (16.5% vs. 14.4%), clinical pregnancy rate (36.0% vs. 25.9%), miscarriage rate (5.8% vs. 2.4%), ongoing pregnancy rate (30.2% vs. 23.5%), and multiple pregnancy rate (7.0% vs. 10.6%) to the drilling LAH group. There were no significant differences in pregnancy outcomes between subgroups defined based on age (older or younger than 35 years) or ZP thickness (greater or less than $17{\mu}m$) according to the LAH method. Conclusion: The present study demonstrated that partial ZP thinning or drilling resulted in similar outcomes in implantation and pregnancy rates using thawed embryos, irrespective of women's age or ZP thickness.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.5-5
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• 2001

This lecture will begin by tracing some of the history behind techniques that we nowadays take for granted in the practice of embryo transfer, and in the application of the technique to various animal biotechnologies. It will be argued that an appreciation of such history can teach us a great deal about how we need to study and teach the subject, and about the best ways to conduct and finance the research that is essential to further progress. Examples in support of this argument will be taken from the changes that have occurred in the way embryos, particularly bovine embryos, have been collected, maintained in vitro, subjected to a variety of manipulations (sexing, division to produce identical animals, combination into chimeras, transfection with foreign genes), frozen and thawed, and transferred over the past 50 years. (omitted)

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.53-63
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• 1995

The effects of different protein sources (serum vs bovine serum albumin), growth factors (EGF and PDGF) and co-culture with various type of somatic cel1s (BOEC, MEF and BRL) on the in vitro development of in vitro matured / in vitro fertilized bovine oocytes were examined, and the viability of frozen/thawed embryos derived from IVM /IVF was examined. Cell numbers of blastocysts were also counted. In Experiment 1, CR$_1$aa with serum was superior to CR$_1$aa with BSA in producing morulae plus blastocysts from IVM /IVF oocytes(24.4% vs 30.4%, p>0.05). In Experiment 2, more morulae plus blastocysts(42.3%) were produced in CR$_1$aa containing long /ml EGF than in the control CR$_1$aa(33.3%). In Experiment 3, 2- to 8-cell embryos derived from IVM /IVF oocytes were randomly allotted to one of 4 culture groups : a) CR$_1$aa ; b) CR$_1$aa + ing /ml PDGF ; CR$_1$aa + Sng /ml PDGF ; CR$_1$aa + lOng /ml PDGF ; culture resulted in 21.3, 51.2, 41.4 and 45.9%(p<0.05), respectively, developing into morulae and blastocysts. In Experiment 4, 0 and Sng /ml PDGF added to CR$_1$aa coculture with BRL or BOEC yielded 47.5, 42.5, 33.8 and 41.6% morulae and blastocysts, respectively. In Experiment 5, the proportion of embryos into morulae and blastocysts was highest in CR$_1$aa with MEF coculture group(50.9%) compared to any other group(CR$_1$aa, 22.3%; CR$_1$aa+BRL, 32.9%; CR$_1$aa+BOEC, 33.8%, p>0.05). In Experiment 6, survival rate of blastocysts produced by in vitro fertilization when cryoprotectant was removed in 0.7M glycerol+0.7M sucrose and 0.7M sucrose solution for 10 min. after thawing at 2$0^{\circ}C$ (Exp. H, 58.8%) was slightly higher than when cryoprotectant was removed 10%, 6.7% and 3.3% glycerol for 10 min. after thawing at 37$^{\circ}C$ (Exp. I, 54.3%). These study indicate that growth factors and somatic cell co-culture can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number and frozen /thawed method employed this experiment was not different.

• Journal of Veterinary Clinics
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• v.4 no.2
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• pp.449-455
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• 1987

Quick freezing of bovine embryos was attempted after they were predehydrated at room temperature. Combined solutions of 2M glycerol or 2M ethylene glycol in the presence of either 0.5 or 1.0M sucrose in phosphated buffered saline+20% calf serum were compared. The quick freezing method in which embryos were directly transferred in liquid nitrogen vapor for 2 minutes at - l70$^{\circ}C$ before being plunged into liquid nitrogen was used. Post-thaw survival rates in 2M glycerol and 2M ethylene glycol were high with 0.5 M (55.6% and 53.3%) versus 1.0M(38.1% and 31.6%) sucrose(P < 0.05). But survival rates with 2M glycerol and 2M ethylene glycol were not significantly different. Transfer thawed embryos frozen with 2M glycerol and 2M ethylene glycol by 0.5M sucrose resulted in birthrates of 40.9% and 40.0%, respectively compared to 26.3% and 27.2%, respectively, for 1.0 M sucrose(p<0.05). This was 56.0% for fresh control.