• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.15-21
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• 2000

These studies were undertaken to evaluate morphological normality and development competence in vitro of hyman oocytes following vitrificatioin using ethylene glycol and electron microscopic grid. Human immature oocytes retrieved from natural and stimulated cycles was vitrified at 0 or 48 h and 0, 8 to 15 or 24 to 28 h after maturation culture, respectively. In oocytes retrieved from unstimulated cycle, no signifciant differences were found in morphological normality (56 to 63%) and fertilization (31 to 37%) rates between the times of vitrification. In stimulated patients, however, more oocytes were morphologically normal when vitrified at 24 to 28 h than when vitrified at 0 or 8 to 15 h after maturation culture. Regardlesss of the hormonal stimulation, high cleavage rates(83 to 100%) were obtained in all treatment groups but did not differ significantly. Twenty to 43% of cleaved oocytes developed to the blastocyst stage at 6 days after IVF. These results suggest that vitrified oocytes from unstimulated and stimulated cycles could develop to the blastocyst stage, regardless of the stages of vitrification.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.261-265
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• 2000

Objective: This study was performed to evaluate the influence of maternal age on embryo quality and the frequency of multiple pregnancy in IVF-ET program. Method: 86 conventional IVF-ET cycles were divided into three groups according to the age by 5 year (group A: 26-30, group B: 31-35, group C: 36-40 yrs). The in vitro fertilization and development outcome (fertilization, cleavage and high quality embryo rate) and the pregnancy outcome (pregnancy, implantation, G-sac/high quality embryo and multiple pregnancy rate) were examined. And then, these results were compared among the groups. Results: The rates of fertilization (62.7, 68.5 and 65.4%, respectively) and cleavage (95.6, 97.6 and 98.0%, respectively) were not different among the groups. And the high quality embryo (HQE) rate also was not different among the groups (61.8, 62.9 and 62.8%, respectively). The pregnancy rate of group C (23.3%) was significantly lower than that of group A (41.2%) and B (48.7%). And the implantation rate was significantly decreased with advance in maternal age (group A; 17.3%, B; 12.6% and C; 6.0%). The G-sac/high quality embryo rate was significantly higher in group A (70.8%) when compared to group B (32.2%) and C (40.0%). On the other hand, the multiple pregnancy rate was significantly lower in group C (14.3%) when compared to group A (71.4%) and B (36.8%). Conclusion: The pregnancy rate was significantly decreased over 35 years. The G-sac/HQE and multiple pregnancy rate were significantly high below 31 years. Thus, these results suggest that the number of high quality embryo transferred should be limited by the age and another criteria for embryo quality evaluation were required for single embryo transfer.

• Journal of Animal Science and Technology
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• v.44 no.6
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• pp.701-710
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• 2002

Studies on the Viability of In Vitro-Matured Bovine Oocytes Vitrified by Microdrop and Straw Method To establish vitrification method for bovine oocytes, mature bovine oocytes were vitrified by microdrop (MD) or straw (Straw) method and the viability of vitrified oocytes with or without cumulus cells (CC) were examined by several methods; a) parthenogenetic activation; b) pronuclear formation after in vitro fertilization (IVF); and c) embryonic development after IVF. The survival rate of vitrified oocytes by MD was significantly higher than by Straw (92.50 vs. 74.19%, p<0.05). Most of the oocytes survived from vitrification using the MD methods. Cleavage and blastocyst development of parthenogenetically activated oocytes were higher in MD (45.05% and 10.81%, respectively; p<0.05)) than those in Straw method (27.17% and 6.52%, respectively; p<0.05). Male and female pronuclear formation of vitrified-thawed oocytes with or without cumulus cells (CC) after IVF were examined, respectively. The survival rate of vitrified oocytes by MD without CC was no difference between MD and Straw (80.368.14% vs. 67.31%). Normal fertilization (2PN) rates were not different among groups (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). While no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). The polyspermy (3PN) was appeared in the fresh (12.99%), but no appeared in the vitrified-thawed groups. In the without CC, normal fertilization (2PN) rates were significantly different between fresh and vitrified-thawed oocytes (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). Moreover, no fertilization (<1PN) rates were significantly different between fresh and vitrified-thawed groups (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). The polyspermy (3PN, >4PN) was appeared not only fresh but vitrified-thawed groups. After IVF, two-cell developmental rates of vitrified oocytes with CC by MD and Straw were significantly low compared to fresh oocytes (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). Blastocyst developmental rates of vitrified oocytes also were significantly low compared to fresh oocytes (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). In the without CC, two-cell developmental rates were no difference between Fresh and MD (27.59% vs. 19.25%, p<0.05), while blastocyst rates were difference between Fresh and MD or Straw (4.31% vs. 0.62% and 0%, respectively; p<0.05). In conclusion, the results indicate that the vitrified bovine oocytes have the ability to develop to the blastocyst stage after IVF.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.287-294
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• 2002

Objectives: Pelvic tuberculosis (TB) causes infertility despite of anti-TB chemotherapy and IVF-ET is effective treatment to achieve pregnancy. The aim of this study is to assess the outcomes of IVF-ET in pelvic TB, especially according to main Tb lesion, and to investigate the factors affecting the successful outcome. Methods: A total of 135 IVF-ET cycles were performed in 54 patients with pelvic TB and the outcome was compared with that of control group with tubal factor not associated with TB in 301 cycles, 227 patients. Anti-TB chemotherapy was performed in the patients with pelvic TB. Pregnancy rate was compared according to main TB lesion as salpingitis, peritonitis, and endometritis. In the patients with endometrial TB, when complicated with uterine synechia, hysteroscopic lysis was done before IVF-ET and pregnancy rate was compared according to the presence of uterine synechia. Results: There was no significant difference in peak E2 ($2,790{\pm}280.1$ vs $2,554{\pm}101.2$, p>0.05), the number of retrieved oocytes ($13.5{\pm}0.7$ vs $12.5{\pm}0.4$, p>0.05) and fertilized oocytes ($7.7{\pm}0.5$ vs $7.8{\pm}0.3$, p>0.05) between patient and control group. Clinical pregnancy rate per transfer in pelvic TB group was 22.9% and showed no difference from that of control group (24.3%, p>0.05). Although it was not statistically significant, pregnancy rate in the endometrial Tb (18%) was lower than that in the salpingitis (28.5%) or peritonitis (26.5%) (p>0.05). In the endometrial TB with uterine synechia, pregnancy rate was significantly lower than that of the patients without synechia even after hysteroscopic lysis (9.7% vs 31.6%, p<0.05). Conclusion: IVF-ET after anti-TB chemotherapy is the most effective treatment to achieve pregnancy in infertile patients with pelvic TB. Because the presence of endometrial TB and resulting uterine synechia affects the outcome of IVF-ET, thorough evaluation for endometrium with endometrial biopsy and hysteroscopy is important to predict the prognosis of IVF-ET treatment.

• Clinical and Experimental Pediatrics
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• v.50 no.8
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• pp.740-745
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• 2007

Purpose : There is a dominant opinion that in vitro fertilization (IVF) leads to an increased incidence of twins, low birth weight (LBW) infants, prematurity and mortality. On the other hand, technical development of IVF and improvement of neonatal intensive care have increased the survival rate of neonates. The purpose of this study was to verify the tendency by comparing the clinical aspects of IVF and spontaneously conceived twins, and to establish methods to increase the survival rate of neonates after IVF. Methods : Retrospective reviews were performed on all twin infants who were admitted to the nursery and NICU at Kangnam Sacred Heart Hospital, Hallym University from January 1, 2000 to December 31, 2006. Medical records of IVF twins (study group, n=92) and spontaneously conceived twins (control group, n=265) were analyzed and compared. Neonatal outcomes and complications, as well as obstetric outcomes, were analyzed and compared. Results : Mean gestational age and birth weight of the study group ($34.6{\pm}3.5$ weeks, $2,203.9{\pm}617.2g$) were considerably lower than those of the control group ($36.3{\pm}2.4$ weeks, $2,367.0{\pm}517.9g$). The frequency of prematurity less than 37 weeks (68.5% vs 51.3%) and extremely LBW (15.2% vs 6.4%) were also significantly higher in the study group. Other neonatal outcomes were all insignificant. The obstetric characteristics, maternal age ($32.6{\pm}3.3$ years vs $30.3{\pm}3.9$ years) and the frequency of cesarean delivery(95.7% vs 79.9%) were significantly higher in the study group. Other obstetric outcomes were insignificant except for the frequency of incompetent internal os of cervix (36.2% vs 3.6%) and cerclage operation (38.3% vs 4.3%). Conclusion : Based on the above results, clinical outcomes of twin infants will be further improved by careful attention and thorough antenatal care of the IVF twins.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.287-294
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• 1994

This study was to investigate the effects of electrofusion, activation and developmental stage of donor embryos on in vitro development of nuclear transplant bovine embryos. A single blastomere nucleus from 8-cell to morula stage embryos produced by in vitro fertilization(IVF) was transferred into a recipient oocyte enucleated at 23∼25 h after in vitro maturation(IVM) or into a recipient oocyte enucleated and cultured for 14∼15 h. In one experiment the nuclear transplant embryos were subjected to additional activation treatments. Fusion rate of nuclear transplant eggs was high at direct current(D.C) voltages of 1.0 and 1.5 kV/cm 991.5 and 93.3%, respectively), but decreased at 2.0kV/cm (81.8%). Additional activation treatments by electric pulases or 7% ethanol did not affect the cleavage and development of nuclear transplant embryos. Development of nuclear transplant embryos slightly increased by delayed nuclear transfer and fusion (42∼43 h after IVM). With this system, blastocysts were obtained from transfer of 8-cell to morula stage donor nuclei (9.6%∼2.4%). The result of this study suggests that nucleo-cytoplasmic interactins, expecially activation of ooplast are very important for the development of nuclear transplant embryos, and donor cell stage does not affect the development of nuclear transplant embryos.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.1
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.267-272
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• 2010

The present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen in our laboratory for in vitro fertilization (IVF) experiments due to lack of fresh semen. Antibiotics were added in the frozen semen extender (kanamycin and gentamicin) and in vitro culture (IVC) medium (gentamicin) for further inhibiting growth of microorganisms. Nevertheless, proliferations of microorganisms were observed in IVC culture drop during culturing of IVF embryos using frozen semen. Randomly 3 samples were taken from the liquid semen, frozen semen and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen and egg yolk were cultured in de Man, Rogosa and Sharpe (MRS) agar medium. Whitish colonies were detected in contaminated IVC drop, frozen-thawed semen samples and egg yolk but no colonies were formed in liquid semen samples. Gram-negative and rod-shaped identical bacteria were found in both frozen-thawed semen sample and contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to manufacturer's instruction with identification value (% ID) 94.3% and T index 0.88. Antibiotic susceptibility tests were done according to Clinical and Laboratory Standards Institute (CLSI) by using ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin test. Among them Enterobacter cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, kanamycin but susceptible to tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin. From these findings it could be suggested that this contamination sources might be from egg yolk.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.111-118
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• 1997

Intracytoplasmic sperm injection (ICSI) recently has been utilized widely as the most successful technique to overcome the unfertilization problem in cases of severe male infertility in couples who could not be treated by conventional IVF. Recently, indications of ICSI have been extended further and more fertilized oocytes become available. Thus, it is necessary to examine the efficiency of freezing the surplus embryos obtained from ICSI. We compared the survival rate and the future outcome of cryopreserved embryos obtained either after conventional IVF or ICSI during the same period. After ICSI or IVF, five best-quality embryos from each patient were transferred in the stimulation cycle and the surplus pronuclear (PN) stage oocytes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. A total of 792 embryos from ICSI trial were thawed and 65.2% (516/792) survived. The survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 63.5%, 68.2%, 64.0%, respectively. After 111 transfers, 34 pregnancies were achieved, corresponding to a clinical pregnancy rate of 30.6% per transfers. We thawed 1033 embryos from IVF trials and 57.5% (594/1033) survived. In IVF cycle, the survival rates of PN stage oocyte, multicellular embryo and PN + multicellular embryo were 58.2%, 65.2%, 40.2%, respectively. Thirty eight clinical pregnancies were established after 134 transfers, corresponding to a pregnancy rate of 28.4% per transfer. The cleavage rate of thawed PN stage oocytes from ICSI trial (61.3%) was significantly higher than those from conventional IVF (53.4%). The developmental rates of good embryo (${\geqq}$ grade II) in thawed PN stage oocytes obtained from conventional IVF and ICSI were 63% and 65%, respectively. We concluded that PN stage oocytes, multicellular embryos resulting from ICSI procedure can be successfully frozen/thawed with reasonable clinical pregnancy rates comparable to those of IVF.