난자내 정자 직접주입술에서 난자의 처리방법이 난자의 발생능력에 미치는 영향

박기상,이택후,송해범,전상식,Park, Kee-Sang,Lee, Taek-Hoo,Song, Hai-Bum,Chun, Sang-Sik 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.3

Objective: In the preparation of ICSI, cumulus and corona cells should be removed from the oocytes by using a combination of enzymatic (hyaluronidase) and mechanical (pipetting) methods. But little is known about the effects of different degrees of oocyte denudation and incubation time between denudation and sperm injection on the outcomes of ICSI. The aim of this study was to evaluate the effects of varying the degrees of oocyte denudation and the lengths of incubation time from denudation to sperm injection on the outcomes of ICSI. Methods: In experiment 1, patients (oocytes) were grouped into group A and B according to the degree of denudation, complete and partial, respectively. In experiment 2, patients (oocytes) were grouped into group I, II and III according to the length of incubation time of denuded oocytes until sperm injection as < 1, $1{\sim}2$ and >2 hours, respectively. Results: There was no significant difference between the degree of oocyte denudation on the survival, fertilization and development rates after ICSI procedure. In case of the incubation time of denuded oocytes until ICSI, survival rates was higher in group III (83.1 %) than in group I (61.5%, p<0.05) or group II (64.3%). However no statistically significant differences were found between incubation time and fertilization or development rates. Conclusions: This study reveals that the outcomes of ICSI are not affected by the degree (complete or partial) of oocyte denudation. However, the denuded oocytes with incubation period of more than 2 hours show better outcomes of ICSI than those with the incubation period of less than 2 hours.

난자채취 2일과 5일에 연속으로 실시한 배아이식의 안전성과 효과

박기상,송해범,이택후,전상식,Park, Kee-Sang,Song, Hai-Bum,Lee, Taek-Hoo,Jeon, Sang-Sik 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.2

Objective: In vitro fertilization (IVF) and a prolonging the time of culture may be helpful in establishing a viable pregnancy through a selection effect. Some embryos do not develop beyond the 4-cell stage and some may not develop to the blastocyst stage. We have evaluated the safety of SET and the outcomes of pregnancy. Methods: Sperms were treated with Ham's F-10 supplemented with 10% human follicular fluid (hFF). oocytes or fertilized oocytes were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% or 20% hFF respectively. Up to five oocytes were inseminated with approximately 200,000 sperm cells/2 ml in each well. Fertilization was examined in the following morning and fertilized oocytes were co-cultured until embryo transfer. Vero cells for co-culture were prepared in Tissue Culture Medium - 199 (TCM-199) with 10% fetal bovine serum. At the two to four cell and blastocyst on day 2 and day 5, embryo and blstocyst grading were evaluated. Pregnancy rate was determined after transfer of human embryos at the two to four cell stage on day 2 (Group I) or subsequent transfer of embryos on day 2 and at the blastocyst stage on day 5 (Group II). For statistical analysis, Student's t-test and Chi-square (${\chi}^2$_test) were used. Results were considered statistically significant when p value was less than 0.05. Results: No differences was found in the fertilization between Group I (81.0%, 98/121) and Group II (81.8%, 180/220). In case of cleavage rate, no difference was found in Group I (95.9%, 94/98) and Group II (97.8%, 174/178). However, the rate of-clinical pregnancy was significantly higher (p=0.014) in Group II (66.7%, 12/18) than in Group I (26.3%, 5/19). Conclusion: The results of this study showed that SET is safe and effective, and significantly increases the pregnancy rate.

인간양수에 의한 생쥐 난자 투명대의 정자수용능력 억제의 관찰

박기상,이택후,송해범,전상식,Park, Kee-Sang,Lee, Taek-Hoo,Song, Hai-Bum,Chun, Sang-Sik 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.1

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

배양액에 첨가하는 에너지원이 생쥐 배 발생 능력에 미치는 영향

박기상,이현정,박성백,김지철,이택후,전상식 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.1

본 연구는 인간 난관액 또는 자궁액 내에 존재하는 에너지원이 생쥐 2-세포기 배의 체외 발달에 미치는 영향을 조사하기 위하여 실시하였다. ICR 암 생쥐에 5 IU hCG 주사 후 46~50시간에 2-세포기 배를 회수하였다. 회수된 배는 3가지 배양 조건 [대조군: 0 mM, Group A: glucose(G) 0.5 mM + pyruvate(P) 0.32 mM + lactate(L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM]에서 72시간 배양하였다. 배양 24 시간에 상실배 출현율은 group A (72.3%)와 group B (56.6%)가 대조군(34.9%)보다 유의하게 높았다(p<0.05). 그러나 48시간에 배반포기 배 출현율은 대조군(51.8%)이 group A (39.8%)와 group B (28.9%)보다 유의하게 (p<0.05) 높았다. 72시간에 투명대 부착 (ZiB, 41.0~51.8%), 투명대 탈출 (ZeB, 18.1~32.5%) 및 총 배반포기 배 출현율 (68.7~73.5%)은 실험군 간에 통계적인 차이가 없었다. 배반포기 배의 평균 세포수와 ICM 세포수는 group A (70.8, 13.4)와 group B (64.4, 11.8)가 대조군 (53.1, 5.7)보다 유의하게(p<0.05) 많았고, 통계적인 유의차는 없었으나 group A가 group B보다 많은 경향이었다. 총 세포수에 대한 ICM 비율은 group A(22.9%)와 group B(23.7%)가 대조군(14.2%)보다 유의하게(p<0.05) 높았다. 영양배엽(TE) 세포수(34.1~45.1)는 실험군 간에 통계적인 차이가 없었다. ICM에 대한 TE 비율(ICM:TE ratio)은 대조군(1:6.0)이 group A(1:3.4)나 group B(1:3.4)보다 유의하게(p<0.05) 높았다. 생쥐 2-세포기 배를 배양하여 72시간까지의 배 발달율을 살펴보면 배양액에 에너지원을 첨가하는 것이 효과적이었으며, 자궁액 농도보다는 난관액 농도로 에너지원을 조절했을 때 배 발생 능력이 높은 경향을 보였다. This study was conducted to examine the effects of energy substrates in different concentration of carbohydrates in the human oviduct and uterus on the in vitro development of mouse 2-cell embryos. Two-cell embryos were collected from ICR female mice at 46~50 hr after 5 IU hCG injection, and cultured in three different media [Control: 0 mM, Group A: glucose (G) 0.5 mM + pyruvate (P) 0.32 mM + lactate (L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM] for 72 hr. Rates of morula formation of group A (72.3%) and B (56.6%) were significantly higher (p<0.05) than that of control (34.9%) at 24 hr. However, blastocyst rate was significantly higher (p<0.05) in control (51.8%) than group A (39.8%) and B (28.9%) at 48 hr. At 72 hr, no differences were found in the number of zona-intact, zona-escape and total blastocysts among groups. Mean and ICM cell numbers were significantly higher (p<0.05) in group A (78.0, 13.4) and B (64.4, 11.8) than control (53.1, 5.7), respectively. The percent of ICM were significantly higher (p<0.05) in group A (22.9%) and B (23.7%) than control (14.2%). No differences were found in the TE cell numbers (34.1~45.1). The ICM:TE ratio was significantly higher (p<0.05) in control (1:6.0) than group A (1:3.4) and B (1:3.4). This study shows that energy substrates added to culture media especially, the oviductal level of carbohydrates increase the developmental capacity of 2-cell mouse embryos.

Culture conditions of gametes and their evaluating systems in mouse /

박기상,송해범 대구대학교 생명과학연구소, 2003 생명과학연구 Vol.2 No.2

성숙배양액에 첨가하는 인간체액 (Human Body Fluids) 및 성선자극호르몬이 생쥐 미성숙난자의 핵성숙과 수정능력에 미치는 영향

박기상,손원영,김진희,이경아,한세열,고정재,차광열,Park, K.S.,Son, W.Y.,Kim, J.H.,Lee, K.A.,Han, S.Y.,Ko, J.J.,Cha, K.Y. 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.2

Purpose of the present study was to find the optimal culture conditions for the maturation and fertilization of immature oocytes by the use human body fluids and gonadotropins (Gn) in the mouse model. Cumulus-enclosed mouse immature oocytes were incubated in the medium containing various human body fluids with or without Gn in vitro, and examined to confirm nuclear maturation (NM) and fertilization. Female ICR mice were stimulated with 7.5 IU pregnant mares' serum gonadotropin (PMSG). Cumulus-enclosed immature oocytes were isolated at 48-52 hr post PMSG injection and cultured in TCM 199 supplemented with various concentrations (20, 50, and 70%) of human body fluids such as fetal cord serum (hCS), follicular fluid (hFF), peritoneal fluid (hPF) and amniotic fluid (hAF) in the presence or absence of 10 IU/ml PMSG and 10 IU/ml human chorionic gonadotropin (hCG) for 18 hr. Fetal calf serum (FCS) was used as a control for the supplements. Matured oocytes were fertilized with sperm collected from the epididymis of male mice. Fertilization was conducted in T6 medium containing 15 mgl ml bovine serum albumin, and confirmed at 6 hr post-insemination. Evaluation of nucler maturation and fertilization was carried out by rapid staining using fuchin. There was no significant difference between the effects of human body fluids and FCS supplements on nuclear maturation of cumulus enclosed mouse immature oocytes. When maturation medium was supplemented with 20% hPF or 20% hAF, fertilization rates were significantly (P<0.01) lower than that of 20% FCS, hCS and hFF groups. However, higher concentrations of body fluids during IVM were not more beneficial on fertilizability of oocytes. The addition of Gn significantly increased the fertilization rates in hPF and hAF groups (hPF without Gn; 51.5%, compared with 85.1% for addition of Gn, and hAF without Gn; 30.1% compared with 85.8% for addition of Gn) at 20% concentration. These results suggest that human body fluids at 20% concentration and gonadotropins can be used as supplements for the maturation of mouse immature oocytes in vitro. When gonadotropins supplemented with the human body fluids in the maturation medium, fertilizability of mouse immature oocytes was increased in hPF and hAF groups. These results can be applied to maturation of human immature oocytes in vitro.

고관절 표면대치 인공관절술

박기상,변재윤,이근환,이종우,양재우 釜山醫師會 1980 釜山醫師會誌 Vol.16 No.10

Feasibility of Rapid Classification of Edible Salts by a Compact Low-Cost Laser-induced Breakdown Spectroscopy Device

박기상,유혜림,공용득,Sheng Cui,남상호,함경식,유종현,한송희,이용훈 대한화학회 2015 Bulletin of the Korean Chemical Society Vol.36 No.1

Weinvestigated feasibility of a compact, low-cost, laser-induced breakdown spectroscopy (LIBS) device made up of a Q-switched, diode-pumped, solid-state laser and a nongateable miniature spectrometer for the classification of edible salts. LIBS spectra of edible salts from 12 different geographic origins were obtained by this compact LIBS device. The detection limits of the compact LIBS device for potassium, magnesium, and calcium with effective discrimination power were sufficient to classify the edible salts. The classification model was developed by the multivariate analysis of the LIBS spectra. The comparison of the LIBS results with inductively coupled plasma-atomic emission spectroscopy analysis indicates that the clustering of principal component scores was well dominated by chemical compositions of the salts. The cross- and external validations of the classification model showed reasonable performance (98.3 and 87.5% correctness, respectively). Our results indicate that rapid classification of edible salts can be realized by a compact, low-cost LIBS device.

Mouse의 체외수정에 있어서 수정능획득시간 및 수정시각의 비교

박기상,김광식,송해범 大邱大學校出版部 (대구대학, 한사대학 1992 農業科學硏究論文集 Vol.6 No.-

This research was conducted to comparison of capacitation time of spermatozoa and insemination time on in vitro fertilization using out-bred ICR mice. The results obtained are as folows, 1) Optimum capacitation time of spermatozoa was 2 hours, when in vitro fertilization rate was 81%. 2) Optimum insemination time of sperm and oocyte was 6 hours, when in vitro fertilization rates were 81% and 76% in T6 and TYH media.

난자내 정자 직접주입술에서 난자의 처리방법이 난자의 발생능력에 미치는 영향

박기상,이택후,송해범,전상식 경북대학교 병원 2002 경북대학교병원의학연구소논문집 Vol.6 No.1

Objective: In the preparation of ICSI,cumulus and corona-cells should be removed from the oocytes by using a combination of enzymatic(hyaluronidase) and mechanical(pipetting) methods.But little is known about the effects of different degrees of oocyte denudation and incubation time between denudation and sperm injection on the outcomes of ICSI.The aim of this study was to evaluate the effects of varying the degrees of oocyte denudation and the lengths of incubation time from denudation to sperm injection on the outcomes of ICSI. Methods: In experiment 1, patients(oocytes) were group A and B according to the degree of denudation, complete and partial, respectively.In experiment 2, paients(oocytes) were grouped into groupⅠ,Ⅱ and Ⅲ according to the length of incudation time of denuded oocytes until sperm injection as<1,1∼2 and>2hours, respectively. Results: There was no significant difference between the degree of oocyte denudation on the survival, fertilization and development rates after ICSI procedure.In case of the incubation time of denuded oocytes until ICSI,survival rates was higher in groupⅢ(83.1%) than in groupⅠ(61.5%,p<0.05)or groupⅡ(64.3%). However no statistically significant differences were found between incubation time and fertilization or development rates. Conclusion: This study reveals that the outcomes of ICSI are not affected by the degree(complete or partial)of oocyte denudation. However, the denuded oocytes with incubation period of more than 2 hours show better outcomes of ICSI than those with the incubation period of less than 2 hours.