• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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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.

• 한국수정란이식학회지
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• 제15권1호
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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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• 제27권3호
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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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• 제44권6호
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• pp.701-710
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• 2002

본 연구는 소 난자의 초자화 동결 방법을 설정하기 위하여 체외성숙 소 난자에서 난구세포가 부착된 상태 또는 제거한 상태로 microdrop (MD) 방법과 straw (Straw) 방법을 이용하여 초자화 동결하여 생존율을 검사하였다. 동결 융해난자는 a) 단위발생을 유도하였고 b) 체외수정 후 전핵 형성을 관찰하였으며 c)체외수정 후 수정란 발달을 검사하였다. 초자화 동결 난자의 생존율은 MD 방법을 이용하였을 때가 Straw를 이용하였을 때 보다 높았다 (92.50 vs. 74.19%, p<0.05). MD 방법을 이용하였을 때 대부분의 난자가 생존을 하였다. 단위발생을 유도하였을 때 난할율과 배반포 발달율은 MD (45.05%, 10.81%, p<0.05)가 Straw 방법 보다 높았다 (27.17%, 6.52%, p<0.05). 난구세포의 부착 유무에 따라 동결 융해 후 체외수정 하여 자웅 전핵 형성을 각각 검사하였다. 난구세포 제거 난자에서는 MD와 Straw 방법으로 동결 융해하였을 때 차이가 없었다(80.36% vs. 67.31 %, p<0.05). 정상 수정율 (2PN)에서는 세 처리간에 차이가 없었다 (Fresh; 54.55% vs. MD; 42.22% vs. Straw; 37.14%, p>0.05). 그러나 미수정란 (<1PN)은 Fresh 난자가 동결융해 난자보다 유의적으로 낮았다 (Fresh; 32.47% vs. MD; 57.78% and Straw 62.86%, p<0.05). 다정자 침입은 (3PN) 신선란 (12,99%)에서 발생하였으나 동결 융해 난자에서는 발생하지 않았다. 난구세포가 제거된 난자에서, 정상 수정율 (2PN)은 Fresh와 동결 융해 난자간에 유의적인 차이를 보였다 (Fresh; 59.38% vs. MD; 17.31% and Straw; 30.43%, p<0.05). 또한 미수정율 (<1PN)에 있어서도 신선란과 동결 융해난은 유의적인 차이를 보였다 (Fresh; 23.44% vs. MD; 73.08% and Straw 58.70%, p<0.05). 다정자 침입 (3PN, >4PN)은 신선란과 동결 융해란 모두에서 나타났다. 체외수정 후, 난구세포가 부착된 난자의 2세포기 발달율은 신선란에 비하여 MD 또는 Straw 처리구에서 유의적으로 낮았다 (Fresh; 81.76% vs. MD; 22.22% and Straw; 11.36%, p<0.05). 동결융해난자는 배반포 발달율에 있어서도 신선란에 비하여 유의적으로 낮았다 (Fresh; 28.38 vs. MD; 1.71% and Straw 0%, p<0.05). 난구세포가 제거된 난자에서 2세포기 발달율은 신선란과 MD에서 차이가 없었다 (27.59% vs. 19.25%, p<0.05), 그러나 배반포 발달율에 있어서는 신선란이 MD 또는 Straw 처리구보다 유의적으로 높았다 (4.31% vs. 0.62% and 0%, respectively; p<0.05). 이상의 결과에서, 초자화 동결융해한 소 난자는 체외수정 후 배반포로 발달이 가능함을 나타내주고 있다.

• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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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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• 제50권8호
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• pp.740-745
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• 2007

목 적 : 인공수정으로 태어난 신생아는 쌍태아가 많고, 저체중아나 미숙아로 태어날 가능성이 많고, 사망률이 높다는 의견이 지배적이다. 한편 그동안의 인공수정의 기술적 발전과 신생아 집중치료실의 치료적 발전을 통해 신생아 생존율은 높아지고 있다. 따라서 본 연구는 인공수정으로 태어난 쌍생아와 자연 분만한 쌍생아의 임상 양상의 비교를 통해 이를 증명하고 인공수정아의 생존율을 높이기 위한 방법을 알아보고자 한다. 방 법 : 2000년 1월 1일부터 2006년 12월 31일까지 7년 동안 한림대학교 강남성심병원 신생아실 및 신생아 집중 치료실에 입원한 쌍생아를 대상으로 인공수정으로 태어난 쌍생아를 인공수정군(n=92)으로 자연수정을 통해 태어난 쌍생아를 자연임신군(n=265)으로 의무기록지를 후향적으로 검토, 비교하였다. 산모력으로는 산모의 나이, 산과적 질환유무, 산과적 합볍증 등에 대해 조사하였고, 신생아 분만력으로는 재태 주령, 출생체중, 분만방법, 1분 및 5분 아프가 점수, 사망 여부, 기타 합병증 발생 유무, 신체기형 유무 등에 대하여 비교, 분석하였다. 결 과 : 자연수정군에 비해 인공수정군에서 평균 재태 주령(자연임신군 $36.3{\pm}2.4$주 vs 인공수정군 $34.6{\pm}3.5$주)과 출생체중($2,367.0{\pm}517.9g$ vs $2,203.9{\pm}617.2g$)이 유의하게 낮았다. 또한 37주 미만 미숙아의 비율(51.3% vs 68.5%)과 극소 저출생 체중아의 비율(6.4% vs 15.2%)도 유의하게 높았다. 신생아기 예후에 있어서는 두 군 간에 유의한 차이를 보이지 않았다. 산과적 측면으로는 산모의 나이($32.6{\pm}3.3$세 vs $30.3{\pm}3.9$세)와 제왕절개 비율(79.9% vs 95.7%)이 인공수정군에서 유의하게 높았으며 자궁경부 무력증 빈도(3.6% vs 36.2%) 및 이로 인한 자궁경부 봉축술 빈도(4.3% vs 38.3%)가 실험군에서 유의하게 높았다. 다른 산과적 합병증 발생은 두 군 간에 차이가 없었다. 결 론 : 인공수정으로 태어난 쌍생아는 자연수정으로 태어난 쌍생아에 비해 출생 체중과 재태 주령이 낮았으나, 모체의 산과적 합병증 빈도와 신생아기 예후에 있어서는 큰 차이가 없었다. 따라서 인공수정 산모의 경우 철저한 산전 관리와 함께 태어난 신생아를 의료진이 보다 관심을 두고 치료한다면 자연 수정으로 태어난 신생아와 다름없이 건강한 아이로 키울 수 있을 것으로 사료된다.

• 한국가축번식학회지
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• 제17권4호
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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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• 제26권1호
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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.

• 한국수정란이식학회지
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• 제25권4호
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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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• 제24권1호
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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.