• 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.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.1-5
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• 2001

A total of 92 unfertilized human oocytes were treated with ethanol (EtOH), calcium ionophore A23187 (CI) or electric pulse (EP) for activating pronuclear formation and subsequent development. In Experiment 1, there was a significant (P=0.0001) treatment effect on the activation of unfertilized oocytes. No spontaneous activation was occurred in the control, but activation treatments induced PN formation with various efficacy. More unfertilized oocytes (UFOs) were activated after EtOH or EP treatment than after CI treatment. EP was as effective (63.6 %) as EtOH, but fragmentation was observed in 43% of UFOs activated by EP. Proportion of UFOs that formed presumptive haploid PN (2 PNs+1 PB or 1 PN +2 PBs) was 33.3, 0 and 28.6% after EtOH, CI and EP treatments, respectively. In Experiment 2, a significant (P=0.0362) effect of immature oocytes (IOs) status on activation was fecund. IOs at the GVBD-MI oocytes had higher potential to form PN than those at the GV stage or with abnormal morphology (25 vs. 77.8%). The results of this study clearly demonstrated that the treatment of 10% ethanol for 5 min effectively induced the activation of UFOs. IOs could form pronucleus with high efficacy by ethanol treatment, as long as they grew beyond the GVBD stage.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.135-142
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• 2003

본 연구는 사람의 시험관아기 프로그램에서 수정 후 1일째의 전핵 등급이 체외 수정란의 발달에 미치는 영향을 조사하였다. 정상적인 시험관 아기 시술을 시행한 실례 환자를 대상으로 과배란을 유기하여 배란 직전의 난자를 채취하여 정자를 주입한 다음 체외 수정을 유도하였다. 수정 유도 18시간 후 전핵과 핵인의 형태에 따라 전핵의 등급을 1 및 2등급으로 나누어 각각 3일 동안 체외 배양을 실시하여, 체외 수정란의 형태에 따라 1, 2 및 3등급으로 분류하였든 바, 전핵의 등급에 따라 각각 1등급 체외수정란의 발달율은 1등급 전핵란에서는 83.5%로서 2등급 전핵란의 5.5%보다 유의적으로 (P<0.05) 높았다. 1등급 및 2등급 전핵란에서 수정 후 3일째에 5-세포기 이상의 단계로 발달하는 경우는 각각 85.1% 및 24.5%를 나타내었고, 평균 할구수는 7.4$\pm$2.1 및 4.1$\pm$3.5개를 나타내어 발달 능력에서 1등급 전핵란이 2등급 전핵란보다 유의적으로 (P<0.05) 높게 나타났다.

• 대한생식의학회:학술대회논문집
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• 2003.12a
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• pp.123-124
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• 2003

• 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.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.125-134
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• 2003

본 연구는 사람의 시험관아기 프로그램에서 체외 수정란의 질을 향상시키는 한 방법으로 난포액으로 처리된 정자를 체외 수정에 사용하여 생산된 체외 수정란의 전핵 등급과 발달 능력을 조사하였다. 정상적인 시험관 아기 시술을 시행한 실례 환자를 대상으로 과배란을 유기하여 배란 직전의 난자를 채취하여, 난포액으로 처리된 정자와 체외 수정시킨 후 사람 체외 수정란의 전핵 등급과 체외발달율을 조사하였다. 체외수정을 위한 정자의 처리 방법으로 synthetic serum substitute (SSS)를 15% 첨가한 modified human total fluid (m-hTF) 혹은 난포액에서 정자를 2시간 동안 swimming-up 처리 후 각각 체외수정에 사용한 결과, 수정율은 75.3 및 82.1%를 나타내어 유의적인 차이는 없었으나, 1등급 전핵란은 각각 48.0 및 65.5%를 나타내어 난포액에서 유의적으로 (P<0.05) 높았고, 배양 후 3일째에 수정란의 등급을 조사한 결과, 1등급 체외수정란은 각각 44.9 및 60.5%를 나타내어 난포액에서 유의적으로 (P<0.05) 높았다. 또한 체외수정란을 배양 후 3일째에 수정란의 발달 단계를 조사 한 결과, 5-세포기 단계 이상을 발달하는 비율은 각각 51.0 및 70.5%를 나타내어 난포액에서 유의적으로 (P<0.05) 높았다.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.4
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• pp.244-252
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• 2023

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.4
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• pp.329-338
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• 2010

Objective: This study was carried out to know whether cryopreservation of sibling 2PN zygotes could increase the cumulative delivery rates in the patients who had less than 10 fertilized zygotes. Methods: A retrospective analysis was performed in 138 in vitro fertilization-embryo transfer (IVF-ET) cycles with less than 10 fertilized zygotes during January 2003 to December 2007 in Cheil General Hospital. These cycles were divided into two groups. In Group I (n=86), all fertilized embryos were cultured to transfer on day 3 without cryopreserved embryos at the 2PN stage. In Group II (n=52), among fertilized zygotes, some sibling zygotes were frozen at the 2PN stage, the remainder were cultured to transfer. Clinical outcomes in fresh ET cycles and cumulative ongoing pregnancy rates after subsequent frozen-thawed (FT)-ET cycles were compared. Results: There were no significant differences in female mean age, number of retrieved oocytes and total fertilized embryos between two groups, Number of cultured embryos was significantly lower in Group II ($5.2{\pm}0.5$) than in Group I ($8.4{\pm}0.7$) (p<0.01). Also, number of transferred embryos was significantly lower in Group II ($3.3{\pm}0.6$) compared with Group I ($3.6{\pm}0.6$) (p<0.01). ${\beta}$-hCG positive rates and delivery rates (51.2 vs. 46.2 % and 41.9 vs. 34.6 %, respectively) after fresh ET were slightly higher in Group I than in Group II. However, the differences were not statistically significant. Also, the cumulative delivery rates after subsequent FT-ET cycles were not significantly different between Group I (48.8%) and Group II (50.0%). Conclusion: This study showed that cryopreservation of sibling 2PN zygotes from patients who had less than 10 zygotes in the fresh ET cycles did not increase cumulative delivery outcomes. But, it could provide an alternative choice for patients due to offering more chance for embryo transfers if pregnancy was failed in fresh IVF-ET cycles.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.105-105
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• 2002

염색체의 구조적 이상으로 인한 습관성 유산과 기형아의 출산을 예방하기 위해 착상전 배아에서 할구를 분석하여 정상적인 핵형을 가진 배아만을 이식하는 착상전 유전자 진단 (preimplantation genetic diagnosis, PGD)의 성공적인 임상 적용이 보고되고 있으며, 그 적용 범위가 확대되고 있다. 그러나 일반적인 간기의 핵상을 이용한 PGD에서는 형광직접보합법 probe의 제약으로 보인자와 정상적인 핵형을 구분할 수 없는 단점이 있다. 따라서 본 연구에서는 보다 정확한 PGD를 위해 생쥐 배아를 이용하여 분리한 할구에서 중기 염색체상을 획득하기 위해 미세소관 (microtubule) 형성 저해제를 처리하였으며, 이를 통해 확립된 방법을 인간의 PGD에 적용하고자 하였다. 과배란이 유도된 ICR 생쥐에서 4- 또는 8-세포기 배아를 수획하여 colcemid, nocodazole, vinblastine을 각각 0.1, 0.5, 1.0, 5.0$\mu$M을 처리하고, hoechst 33342로 염색하여 핵상을 관찰하여 최적의 농도를 결정하였다. 또한 각 미세소관 형성 저해제를 혼합 처리하여 가장 높은 중기 염색체상을 획득할 수 있는 혼합 처리를 결정하였다. 이렇게 결정된 혼합 처리 방법을 인간의 체외 수정 및 배아 이식술에서 획득된 3PN 배아에 처리하여 중기 염색체를 획득하였다. Colcemid, nocodazole, vinblastine 모두 1 $\mu$M이 최적 농도임을 확인할 수 있었다 (각각 96.3%, 92.0%, 98,4%). 미세소관 형성저해제를 혼합 처리하였을 경우 nocodazole과 vinblastine (각각 1$\mu$M)을 혼합 처리했을 때 중기 염색체 획득률(97.3%)이 가장 높았다. 인간의 3PN 배아에 1$\mu$M의 nocodazole과 vinblastine을 혼합 처리한 후, 113개의 할구를 분석하여 44개(38.9%)의 할구에서 중기 염색체를 확인할 수 있었다. 본 실험 결과를 통해 중기 염색체를 획득하기 위하여 미세소관 형성 저해제를 처리하는 방법은 생쥐의 배아에서는 효과적이지만, 인간의 배아에서는 그 효율이 다소 낮음을 알 수 있었다. 그러나 이 방법을 개선하여 인간의 할구에서 중기 염색체의 획득률을 높이고, 이를 염색체의 구조적 이상에 대한 착상전 유전자 진단에 적용한다면, 보인자와 정상의 핵상을 구분하여 정상의 핵상만을 갖는 배아의 이식을 통하여 더욱 정확한 착상전 유전자 진단을 시행할 수 있으리라 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.2
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.