마우스 배반포 배의 Differential staining에서 Propidium Iodide와 Bisbenzimide의 노출이 미치는 영향

박기상,박성백,이택후,전상식,송해범,Park, Kee-Sang,Park, Sung-Baek,Lee, Taek-Hoo,Chun, Sang-Sik,Song, Hai-Bum 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective: These experiments were conducted to investigate the optimal expose length of propidium iodide (PI) and bisbenzimide on differential staining of mouse blastocysts. Materials and methods: A total 964 blastocysts (early${\sim}$hatched) was exposed to PI (n=831) (group I: $\leq$ 10; II: $11{\sim}15$; III: $16{\sim}20$; IV: $\geq$21 sec) and bisbenzimide (n=133) (group A: $\leq$1; B: $1{\sim}$3; C: $\geq$ 4 hr) in several periods for differential staining. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: In case of PI exposure, differential staining rates were significantly higher (p<0.05) in group I (89.8%) than in any others (group II: 77.6%; III: 29.6%; IV: 22.2%) and higher (p<0.05) in group II than in group III and IV. In case of bisbenzimide exposure, differential staining rates were not statistically differences in three groups (group A: 97.4%; B: 87.8%; C: 93.3%). Conclusion: The differential staining rates of mouse blastocysts are not affected by the exposure length of bisbenzimide. However, blastocysts were exposed to PI with period of shorter than 15 sec show best outcomes of differential staining rates.

초임계 탄산가스/유기용매/이색성 염료의 혼합계를 이용한 폴리(비닐 알코올) 편광필름의 제조 및 편광특성

박기상(Ki Sang Park),최이준(E Joon Choi),장진호(Jin Ho Chang),박일현(Il Hyun Park) 한국고분자학회 2011 폴리머 Vol.35 No.5

초임계 탄산가스/유기용매 혼합계 상에서 이색성 염료인 C. I. direct black 22(DB22)로 고검화도의 폴리(비닐 알코올)(PVA)을 염색하여 내구성이 강한 편광필름을 제조하였다. 특히, 초임계 탄산가스 상으로 이색성 염료를 녹이기 위한 분산제로 에틸렌 글리콜과 디메틸 설폭사이드의 무게비=4:6의 혼합용매계를 사용하였을 때 염색 압력을 200 bar까지 낮출 수 있었다. 초임계 유체계의 사용으로, 최대 염색은 연신 전 투과도로 1% 미만까지, 폐수 발생량은 1/10 수준까지, 감소가 가능하였고, 이와 같이 염색된 PVA 필름을 500% 연신한 후 측정된 편광효율은 94%, 평균 투과도(single piece transmittance)는 30%로 각각 얻어졌으며, 또한 이색성 염료로서의 DB22의 한계 및 개선 방안 등도 검토하였다. In the mixed system of supercritical carbon dioxide/organic solvents, poly(vinyl alcohol)(PVA) film of high degree of hydrolysis could be dyed with a dichroic dye of C. I. direct black 22(DB22) and as a result, high durability was obtained. Especially, as a dye dispersant in a supercritical fluid phase, a mixed solvent system of ethylene glycol: dimethyl sulfoxide=4:6 weight ratio was investigated. Then the optimum pressure for dyeing could be reduced down to 200 bar. Using this supercritical fluid system, the maximum dyeing appeared as the transmittance of less than 1% and the waste amount was reduced to the level of 1/10. After 500% drawing of this PVA film, both the polarizing efficiency of 94% and the single piece transmittance of 30% were obtained. The limitation of DB22 and further improvements were also discussed.

Modified Bristow씨 술법을 이용한 재발성 견갑관절 탈구 14례

박기상(Ki Sang Park),박홍근(Hong Kun Park),이동화(Dong Hwa Lee),유총일(Chong Ill Yoo) 대한정형외과학회 1981 대한정형외과학회지 Vol.16 No.4

IVF, ICSI 또는 TESE-ICSI에서 수정을 유도한 난자의 배아 발생능력 및 임신율

박기상,박윤규,송해범,이택후,전상식,Park, Kee-Sang,Park, Yoon-Kyu,Song, Hai-Bum,Lee, Taek-Hoo,Chun, Sang-Sik 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.3

Objective: This study was performed to evaluate and compare the embryonic developmental capacity and pregnancy rates in conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with ejaculated sperm or testicular sperm cycles. Materials and Methods: Fertilization was examined in the following morning after IVF (group I), ICSI (group II) or TESE-ICSI cycles (group III). Fertilized oocytes were co-cultured with Vero cells until embryo transfer (ET). On day 2 and $5{\sim}7$, grades of embryos (<4- or $\geq$4-cell) and blastocysts (BG1, 2, 3 or early) were evaluated. Clinical pregnancy rate was determined by detecting G-sac with transvaginal ultrasonogram. We analyzed the results by $X^2$ and Student's t-test and considered statistically significant when P value was less than 0.05. Results: Fertilization rate was significantly higher (p<0.05) in group I ($79.0{\pm}21.2%$) than in group II and III ($56.8{\pm}21.6%$ and $36.7{\pm}25.3%$). Cleavage and blastulation rate of group I ($95.8{\pm}13.8%$ and $59.5{\pm}25.3%$) were significantly higher (p<0.05) than those of group III ($83.4{\pm}18.6%$ and $40.4{\pm}36.5%$). Clinical pregnancy rate was significantly higher (p<0.05) in group I and II (40.7% and 41.7%) than that in group III (12.5%). No differences were found in the rates of multiple pregnancy and abortion among three groups. Embryonic implantation rate was higher in group I ($15.1{\pm}20.2%$, p<0.05) and II ($14.7{\pm}20.6%$, NS) than that in group III ($5.1{\pm}15.6%$). However, embryonic implantation rate was increased in ET with blastocyst(s) among three groups. Conclusions: Fertilized oocytes obtained from TESE-ICSI were harder to be successfully cultured to blastocyst stage for 5$\sim$7 days than that from IVF cycles. However, all blastocyst(s) ET increased the embryonic implantation rate equally in IVF, ICSI and TESE-ICSI cycles.

마우스에서 배반포 형성, 세포 수 및 ICM의 비율에 미치는 배양액의 효과

박기상,이택후,전상식,송해범,Park, Kee-Sang,Lee, Taek-Hoo,Chun, Sang-Sik,Song, Hai-Bum 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.4

Objective : The aim of this study was to evaluate the influence of different media on blastulation, mean cell number, percentage of inner cell mass (ICM) of total cells and ICM : trophectoderm (TE) ratio in mice. Materials and methods: A total 552 two cell embryos were retrieved from ICR female mice (4 weeks old) at 48 hr after hCG injection (mated just after hCG injection) and cultured in MEM (n=276) or TCM (n=276) supplemented with 20% hFF. The grading of blastocysts from zona-intact (ZiB) to -escape (hatching and hatched, ZeB) was performed at 72 hours after culture. Total, TE and ICM cell numbers were analyzed by differential staining of blastocyst. Statistical analysis was performed using t-test with SigmaPlot-2001. P-values < 0.05 were accepted as statistically significant. Results: The blastulation rate in MEM ($64.9{\pm}4.95%$) was significantly higher (p=0.0031) than that in TCM ($57.2{\pm}5.22%$). No differences were found in the number of ZiB and ZeB between MEM ($31.9{\pm}2.62$, $33.0{\pm}4.58%$), and TCM ($27.2{\pm}4.28$, $30.1{\pm}4.58%$). A total 314 blastocysts (MEM=166; TCM=148) were stained differentially. Mean cell number of blastocysts was significantly higher (p=0.0002) in TCM ($73.1{\pm}3.3$) than in MEM ($61.7{\pm}2.5$). Differential staining was successfully performed in 155 blastocysts (MEM=77; TCM=78). The percentage of ICM was significantly higher in MEM than in TCM ($20.9{\pm}1.3$ vs. $17.1{\pm}1.2%$, p=0.0281). The ICM : TE ratio was higher in TCM than in MEM (1 : $4.85{\pm}0.68$ vs. 1 : $3.78{\pm}0.78$, NS). Conclusion: These results show that MEM increase the blastocyst formation and percentage of ICM of total cells comparing with TCM in mice.

체외성숙배양 조건이 마우스 난자의 체외수정 및 다정자침입에 미치는 영향

박기상,이상호,송해범,Park, Kee-Sang,Lee, Sang-Ho,Song, Hai-Bum 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.2

ICR female mice aged 3 to 4 weeks, were stimulated with 7.5 IU PMS injection. At 48-52h post-PMS injection, ovaries were dissected out and oocytes-cumulus complexes(OCCs) were divided into three groups, cumulus-free oocytes(O), cumulus-free oocyte cocultured with cumulus cells(O+C) and OCC. The oocyte were cultured in TCM199 containing various protein sources, FCS, BSA or PVP with gonadotropins(Gns) for 24h. Spermatozoa were collected from cauda epididymis and capacitated in T6 + BSA for 2h. After oocyte maturation in vitro(IVM) in different experimental groups, matured oocytes were inseminated with the capacitated spermatozoa in T6 + BSA for 6h. In the groups of IVM in TCM + BSA or PVP, fertilization(IVF) did not occur efficiently. However, increased fertilization was found in TCM+ FCS group. The oocytes groups, with cumulus cells showed decreased polyspermy in FCS group (O; 31.8 %, O + C; 12.2 %, OCC; 16%), the addition of Gns did not prevent polyspermy in all three groups. The rates of fertilization increased in zona-free oocytes in PVP group. This results showed that culture system for IVM and IVF could be improved. Furthermore, PVP can be used for the substitution of protein source during maturation, and its low rate of fertilization has been found due to zona hardening which occurred in FCS-free medium.

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.

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.

호르몬 무 첨가 배양액에서 생쥐 Pre-antral Follicles의 체외성장과 난포강 형성

박기상,김주환,이택후,송해범,전상식,Park, Kee-Sang,Kim, Ju-Hwan,Lee, Taek-Hoo,Song, Hai-Bum,Chun, Sang-Sik 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.2

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

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.