Fertilization Promoting Peptide가 사람 정자의 운동양태, 수정능력획득 및 첨체반응에 미치는 영향

강희규,김묘경,김동훈,한성원,최도연,이호준,김문규,Kang, Hee-Gyoo,Kim, Myo-Kyung,Kim, Dong-Hoon,Han, Sung-Won,Choi, Do-Hyun,Lee, Ho-Joon,Kim, Moon-Kyoo 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.2

Objective: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. Methods: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. Results: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. FPP ($25{\sim}100$ nM) induced a significant increase in the proportion of B-pattem capacitated spermatozoa, and a significant decrease in the proportion of F-pattem uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maint3ined higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.

반응성 산소족이 사람 정자의 운동성에 미치는 영향

강희규,김동훈,한성원,김묘경,권혁찬,이호준,김문규,Kang, Hee-Gyoo,Kim, Dong-Hoon,Han, Sung-Won,Kim, Myo-Kyung,Kwon, Hyuck-Chan,Lee, Ho-Joon,Kim, Moon-Kyoo 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.4

Objective: To investigate the effects of ROS on kinematic parameters in human spermatozoa. To verify the changes in above parameters, human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spemlatozoa were incubated under low $O_2 (5%) condition. Methods: CASA was employed to analyze sperm motion parameters. Results: Under $H_2O_2 treatment, all kinematic parameters of spermatozoa were dramatically increased during 30 min, but gradually decreased thereafter. Under the low concentration of $H_2O_2 (125 ${\mu}M$ and 250 ${\mu}M$), the movement velocity (VAP, VCL, VSL) decreased, but forward movement increased. Under the 1mM $H_2O_2, sperm showed reduced kinematic parameters except during first 30 min of incubation. In the cases of X-XO and SNP treatment, the movement velocity increased but the forward movement reduced. After incubation for 3 hr treatment, the kinematic parameters gradually decreased in high concentration of X-XO. However these parameters maintained or increased in low concentration of X-XO. There was no obvious changes in the above parameters in the high concentration of SNP. In the presence of high concentration of lymphocytes, all parameters decreased. Under the 5% $O_2 condition, the parameters of the movement velocity and movement pattern increased, but forward movement decreased. Taken togethers, it suggested that ROS increased the movement velocity but decreased the forward movement and lateral head replacement. $H_2O_2, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within I h of incubation. There was no significant difference in capacitation between low- and high $O_2 group. Conclusion: The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation. These results demonstrate that low concentration ROS facilitates the movement velocity but high concentration ROS was inhibitory.

정자의 형태가 IVF와 ICSI의 결과에 미치는 영향

권윤정,강희규,김수경,양현원,최규완,차영범,이승재,박종민,Kwon, Yoon-Jung,Kang, Hee-Gyoo,Kim, Soo-Kyung,Yang, Hyun-Won,Choi, Kyoo-Wan,Cha, Young-Beom,Lee, Seung-Jae,Park, Jong-Min 대한생식의학회 1995 Clinical and Experimental Reproductive Medicine Vol.22 No.3

Objective: To investigate the effects of sperm morphology and their co-incubation with oocytes on the outcome of IVF and ICSI. Design: Strict morphology of washed sperm was assessed by Diff-Quick staining method before or after insemination. And the relationships between strict morphology and outcome (fertilization, embryo development and pregnancy) of IVF(with co-incubation) and ICSI (without co-incubation) were determined. Patients: Two-hundreds-and-sixty-three cycles of IVF and ninety-six cycles of ICSI were analyzed in order to clarify the influence of strict sperm morphology of spermatozoa on outcome of IVF and ICSI. These were divided into four groups. according to fertilization method and sperm morphology(Group 1: IVF, ${\geq}$12%, n:227; Group 2: IVF, <12%, n:36; Group 3: ICSI, ${\geq}$ 12%, n=48; Group 4: ICSI, <12%, n=48). Results: The fertilization rates of better morphology groups were higher than those of poor groups: Group 1(68.1%) > Group 2(62.1%), Group 3(78.1%) > Group 4(71.5%). There was no difference in embryo cleavage rates among four groups (>90%), Regarded with the good embryo rates, Group 1(56.8%) was significantly higher than Group 2(42.3%)(P<0.01), but there was no difference between Group 3(64.7%) and Group 4(61.2%). The pregnancy rates were also higher in better morphology groups as well as fertilization rates: Group 1(34.8%)> Group 2(16.7%)(p<0.05), Group 3(40.0%) > Group 4(23.0%)(p=0.08). Conclusion: Co-incubation with poor morphology sperm might adversely affect the quality of embryos. And strict sperm morphology may represent the ability to establish successful pregnancy. In short, the strict sperm morphology can be a good predictor of IVF and ICSI outcome.

생쥐모델을 이용한 동결보존제의 독성조사

양관철,강희규,이회창,이향흔,고덕성,양현원,박원일,박은주,김세웅,Yang, Kwan-Cheal,Kang, Hee-Gyoo,Lee, Hoi-Chang,Lee, Hyang-Heun,Ko, Duck-Sung,Yang, Hyun-Won,Park, Won-Il,Park, Eun-Joo,Kim, S. Samuel 대한생식의학회 2004 Clinical and Experimental Reproductive Medicine Vol.31 No.1

Objectives: The aim of this study was to assess toxicities of cryoprotectants. Methods: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. Results: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH ($75.9{\pm}27.0$) or the control ($99.0{\pm}18.3$) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO ($14.2{\pm}1.5$) and PROH ($11.2{\pm}1.4$) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control ($6.2{\pm}0.9$, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). Conclusions: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

착상기간의 자궁내 환경이 생쥐 난자 및 배아의 투명대 미세구조에 미치는 영향

한성원,정호삼,강희규,이호준,계명찬,김성례,김문규,Han, Sung-Won,Chung, Ho-Sam,Kang, Hee-Gyoo,Lee, Ho-Joon,Gye, Myung-Chan,Kim, Sung-Rye,Kim, Moon-Kyoo 대한생식의학회 1999 Clinical and Experimental Reproductive Medicine Vol.26 No.3

In the studies on the hatching mechanisms in mammals, many investigators focused on the embryonic intrinsic factor(s) in in vitro culture, but the uterine environment as the extrinsic factor(s) is thought to play an important role in hatching mechanism. Therefore, to evaluate the effect of uterine environment on the hatching event in vivo, the immature(GV) and ovulated(MII) oocytes, and the late 2-cell embryos of mouse were transferred to pseudopregnant foster mother's uterus during peri-implantation period. So it was verified whether there would happen hatching by only uterine environment independently on embryonic stage. The ultrastructural changes of the zona surface of transferred group were compared with those 01 in vivo and vitro group by SEM. 36 hrs after transfer, the immature and ovulated oocytes almost degenerated, and the late 2-cell embryos developed to various embryonic stages. However, the embryos which didn't develop to blastula stage did not hatch. The ultrastructural network of ZP in transferred group seemed to be smoothed uniformly, which was different from in vitro group. In conclusion, it is suggested that the uterine environment during peri-implantation period enhances the embryo hatching by provoking the structural change of ZP.

시험관 아기 시술에서 여성의 연령이 수정란의 질과 다태 임신 발생에 미치는 영향

이명섭,박장옥,정지학,박준숙,강희규,김동훈,이호준,Lee, Myeong-Seop,Park, Jang-Ok,Jung, Ji-Hak,Park, Jun-Suk,Kang, Hee-Gyoo,Kim, Dong-Hoon,Lee, Ho-Joon 대한생식의학회 2000 Clinical and Experimental Reproductive Medicine Vol.27 No.3

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.

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF)의 첨가가 생쥐 수정란의 발생과 착상관련 유전자 발현에 미치는 영향

김동훈,고덕성,이회창,이호준,강희규,김태전,박원일,김세웅,Kim, Dong-Hoon,Ko, Duck-Sung,Lee, Hoi-Chang,Lee, Ho-Joon,Kang, Hee-Gyoo,Kim, Tai-Jeon,Park, Won-Il,Kim, Seung-Samuel 대한생식의학회 2002 Clinical and Experimental Reproductive Medicine Vol.29 No.2

Objective : The purpose of the current series of experiments were to assess the effect of GM-CSF, as a medium supplement, on the development of mouse embryos and the expression of LIF and IL-1? mRNA. Materials and Methods: Mouse 2-cell embryos were collected from the oviducts of 6 weeks old ICR mice at 48 hours after hCG injection. Embryos were cultured in P-1 medium supplemented with mouse GM-CSF (0, 1, 5, 10 ng/ml). The embryo development to blastocysts and hatching blastocysts was assessed and the cell number in blastocyst was also examined. Using RT-PCR, the expressions of LIF and IL-1? mRNA in blastocyst were evaluated in the GM-CSF supplemented group and control group. Results: In mouse, the addition of GM-CSF increased the percentage of blastocysts (65.5%, 68.6%, 73.0% and 76.1% for control and 1, 5 and 10 ng/ml, respectively), and increased the proportion of hatching blastocysts (35.2%, 36.4%, 43.2% and 53.0% for control and 1, 5 and 10 ng/ml, respectively). The mean cell numbers in blastocyst were significantly increased in GM-CSF supplemented groups compared to control group. LIF and IL-1? expression in blastocyst were significantly higher in GM-CSF supplemented group than in control group. Conclusion: The results of experiment by mouse embryos showed beneficial effects of GM-CSF as a medium supplement. Furthermore, the addition of GM-CSF significantly increased the expression of LIF and IL-1? in mouse embryos. These results suggest that GM-CSF might be a important molecule in embryo implantation.

면봉시료에서 세균의 보존을 위한 최적 보관 온도와 채취 시약의 비교

이영주 ( Yeong Ju Lee ),유희상 ( Hee Sang You ),이송희 ( Song Hee Lee ),이소립 ( So Lip Lee ),이한 ( Han Lee ),성호중 ( Ho Joong Sung ),강희규 ( Hee Gyoo Kang ),현성희 ( Sung Hee Hyun ) 대한임상검사과학회 2021 대한임상검사과학회지(KJCLS) Vol.53 No.4

면봉을 사용한 샘플링 방법은 의학, 생태학, 생명공학, 법의 학 및 오염 정도 모니터링 시스템과 같은 다양한 연구 분야에서 유용하다. 샘플링에서 채취 시약은 중요한 요소 중 하나이다. 시료를 장기간 보관해야 하는 경우에는 시료 채취 키트와 시료 보관 온도가 매우 중요하다. 이 연구의 목적은 별도의 배지 없이, 세 가지 채취 시약과 세 가지 보관 온도가 살아있는 세균의 생균수에 미치는 영향을 확인하는 것이다. 대표적인 환경 세균으로 E. coli 와 S. aureus를 선정하였다. 증류수(DW), 멸균된 인산염 완충액(PBS), Tris-EDTA (TE) 버퍼를 채취 시약으로 사용하여 샘플링한 후, 22, 4, -70°C에서 보관을 진행했다. 각 채취 시약 및 보관 온도가 시료에 미치는 영향은 RLU와 CFU로 결과로 비교하였다. -70°C 보관 온도와 TE 버퍼를 사용할 때 CFU와 RLU 값은 다른 조건에서보다 일정하게 유지되는 경향이 보였다. 따라서 이 연구는 시료가 채취 직후 -70°C에서 보관되어야 하며, 채취 시약으로 TE 버퍼와 함께 사용하는 것이 좋다는 것을 시사한다. Swabs are useful and common sampling tools in various research fields, such as medicine, ecology, biotechnology, forensic medicine, and pollutant monitoring systems. Collection reagents are one of the essential components in sampling. It is important to develop a sample collection kit and designate an appropriate storage temperature because samples need to be stored for a long time. The purpose of this study was to identify the effects of three collection reagents and three storage temperatures on the recovery of living bacteria without media. We selected Escherichia coli and Staphylococcus aureus as representative environmental bacteria. Distilled water (DW), phosphate buffered saline (PBS), and Tris-EDTA (TE) buffer were used as collection reagents and stored at 22℃, 4℃, and -70℃ after sampling. The results of using each collection reagent and storage temperature on the bacteria were compared using relative light units (RLU) and the number of colony forming units (CFU). When using -70℃ storage temperature and the TE buffer, the number of living bacteria and the RLU values remained constant. It is therefore recommended that the sample be stored at -70℃ immediately after collection and a TE buffer solution be used as the collection reagent.

산소분압과 발생부위에 따른 켈로이드 배양섬유모세포의 혈관내피 성장인자(VEGF) 발현

문형식 ( Hyung Sik Moon ),손숙자 ( Sook Ja Son ),박건 ( Kun Park ),강희규 ( Hee Gyoo Kang ),임희정 ( Hee Joung Lim ),박향준 ( Hyang Jun Park ) 대한피부과학회 2009 대한피부과학회지 Vol.47 No.5

Background: The pathophysiological events resulting in keloid formation remain unclear. Overabundant levels of VEGF have been reported to contribute to excessive wound healing. There have been many studies describing the relationship between keloids and VEGF expression. However, there have been no reports about VEGF expression related to donor sites. Objective: We investigated VEGF expression of cultured normal and keloid fibroblasts obtained from different body areas under normoxic and hypoxic culture conditions. Methods: Normal fibroblasts from the earlobe (n=2), shoulder (n=2) and chest (n=2) as well as keloid fibroblasts from the earlobe (n=3), shoulder (n=3) and chest (n=3) were collected and cultured. VEGF expression of fibroblasts at 6 hours, 12 hours, 24 hours and 48 hours for cells maintained under normoxic and hypoxic conditions was measured by the use of RT-PCR. Paraffin-embedded tissues (normal and keloid tissue) were assayed by immunohistochemical staining. Results: For the cultured normal fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition, irrespective of the donor site and time. However, for the cultured keloid fibroblasts, VEGF expression for cells in the hypoxic condition was higher as compared to VEGF expression in cells in the normoxic condition for cultured shoulder fibroblasts. For each donor site, VEGF expression was highest in the shoulder, followed by the chest and earlobe for cultured normal fibroblasts, irrespective of time. For the cultured keloid fibroblasts, the highest VEGF expression occurred at 6 hours for cells in the normoxic condition and the highest VEGF expression occurred at 6 hours and 12 hours for cells in the hypoxic condition. Based on immunohistochemical staining, VEGF expression of paraffin-embedded normal tissue was lower as compared to paraffin-embedded keloid tissue. For each donor site in paraffin-embedded keloid tissue, VEGF expression was highest in the shoulder, followed by the chest and earlobe. Conclusion: Oxygen tension and the nature of fibroblasts from different donor sites are involved in keloid pathogenesis. (Korean J Dermatol 2009;47(5):539~546)

골조직의 신속한 진단을 위한 탈회방법의 비교 연구

김성철 ( Sung Chul Kim ),백운철 ( Oun Chul Back ),김태전 ( Tai Jeon Kim ),배형준 ( Hyung Joon Bae ),강희규 ( Hee Gyoo Kang ) 대한임상검사과학회 2005 대한임상검사과학회지(KJCLS) Vol.37 No.1

These studies were done to know decalcification methods to reduce the time of decalcification for quick bone tissue diagnosis. When bone tissue was decalcified with 10 % formic acid at room temperature, decalcification and hematoxylin & eosin (H&E) stains were complete and satisfactory after 12 hours, but some of the tissue sections fell off during staining. In this way, decalcification, H&E stains were complete and satisfactory after 24 hours, 36 hours and 48 hours, tissue sections didn``t fall off during staining. When bone tissue was decalcified with 10 % formic acid in a 60 ℃ paraffin oven, decalcification and H&E stains were complete and satisfactory after 6 hours, but some tissue sections fell off during staining. In this way, decalcification and tissue sections were complete, with no falling off during staining after 8 hours, 10 hours, 12 hours, 14 hours, 24 hours, or H&E stains were satisfactory from 8 hours to 12 hours, but H&E stains appeared to reddish nucleus after 14 hours and 24 hours. Bone tissue was decalcified with 10 % formic acid for 6 hours, 12 hours and 24 hours at DECAL machine frequencies of 15 Hz and 45 Hz, and for 6 hours, 12 hours and 24 hours at a DECAL machine frequency of 90 Hz. Decalcification and H&E stains were complete and satisfactory after 6 hours at the 15 Hz and 45 Hz DECAL settings. Some of the tissue sections fell off during staining at the 15 Hz DECAL machine setting. At the 90 Hz setting, decalcification, H&E stains, and tissue sections were complete and satisfactory with no falling off during staining after 4 hours. In this way, decalcification, H&E stains, and tissue section were complete and satisfactory with no falling off during staining after 12 hours, 24 hours at all machine settings. Bone tissue was decalcified with 10 % formic acid for 6 hours, 12 hours and 24 hours at 37 ℃ 3 hours, 6 hours and 12 hours at 45 ℃ and 1 hours, 5 hours and 10 hours at 60 ℃ with the RHS-1 machine setting at 60Hz. At the temperatures of 37 ℃, 45 ℃, and 60 ℃ decalcification, H&E stains, and tissue sections were complete and satisfactory, with no falling off during staining except for after 6 hours at 37 ℃. 3 hours, 1 hours, or decalcification, H&E stains, and tissue sections were complete and satisfactory with no falling off during staining after 12 hours and 24 hours at 37 ℃, 6 hours and 12 hours at 45 ℃, and 5 hours at 60 ℃. But H&E stains appeared to reddish nucleus after 10 hours at 60 ℃. From the above reults, the authors were able to deduce that decalcification is accelerated by heat and frequency. We therefore think that it is necessary for machines which are similar to the RHS-1 machine to be maintained at the temperature evenly with agitation effect for quick decalcification.