• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.118-118
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• 2003

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.65-70
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• 1998

This study was conducted to investigate the effects of Tissue Culture Medium 199 (TCM) and Dulecco's Modified Eagle Medium (DMEM) on the blastulation and grade of human oocytes on Vero cells in vitro. A cohort of 79 and 93 oocytes in metaphase II stage were used in TCM 199 and DMEM respectively. No differences were found in the numer of oocytes showing two-pronuclei between TCM (82.3%) and DMEM (86.0%). The number of fertilized oocytes reaching the blastocyst was not significant in TCM (60.0%) and DMEM (63.1%). A total of 89 blastocysts were categorized into the four grades (BG1, BG2, BG3 and early) depending on their morphology. The number of embryos achieving the blastocyst grade 1 (BG1) was significantly higher (p<0.05) in DMEM (50.8%) than TCM (15.0%). It is concluded that cultured oocytes in DMEM with glutamine on Vero cells should be significantly increased BG1.

• 한국가축번식학회지
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• 제21권1호
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• pp.61-69
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• 1997

본 연구는 EGF와 anti-EGF가 초기 생쥐배아의 발생 및 부화에 미치는 영향을 알아보고자 실행되었다. 초기 2세포기부터 상실배까지의 배아를 EGF와 anti-EGF를 각각 처리한 Ham's F10 배양액에서 배양하여 그 발생률과 부화율을 대조군과 비교하였다. EGF 처리시 배양시간에 따른 발생률은 증진되었으나 통계학적 유의성은 없었다. EGF 처리군에서의 부화율(57.5, 62.5, 65.0, 62.5%)은 대조군(35%)에 비하여 유의하게 (p<0.01) 높았다. Anti-EGF 처리시 각 발생시기별 1:1000 실험군의 발생률은 대조군과 차이가 없었다. 그러나, 1:100 실험군의 경우, 2∼4세포기의 배아는 모두 4∼8세포기에서 정지되었고, 8세포기와 상실배의 포배형성은 48시간 이상 지연되었으며 부화 역시 대조군에 비해 억제되었다(8세포기; 2%, 44%, 상실배; 6.2%, 58.3%). 이 실험에서, EGF는 생쥐 배아의 포배형성과 부화를 증진시키는 반면, anti-EGF는 이를 억제하였다. Anti-EGF 처리시 나타나는 발생정지 현상은 anti-EGF가 배아에서 만들어지는 EGF와 반응하여 EGF의 작용을 억제시키기 때문으로 사료된다. 그러므로 EGF는 paracrine mode로서 뿐만 아니라 antocrine mode로서 생쥐 초기배아의 발생에 중요한 인자로 작용함을 알 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.21-28
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• 2002

Objective: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). Methods: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and $x^2$. Results: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). Conclusions: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.

• Clinical and Experimental Reproductive Medicine
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• 제29권4호
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• pp.237-243
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• 2002

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.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2003년도 제45차 추계학술대회
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• pp.146.2-147
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• 2003

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.118-118
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• 2003

The aim of this study was to investigate the effect of glucose on embryonic development of mouse embryos. Two cell embryos were recovered from ICR female mice(3-4weeks) at 46~50 hrs after hCG 5 IU injection (mated just after hCG injection) and cultured in 50 $\mu m$ DMEM droplets supplemented with nothing (control: n=46), glucose 0.5mM (Group A; n=46) or glucose 3.15 mM(Group B; n=46) under mineral oil. All experimental media were supplemented with 20% human follicular fluid. Total blastocyst formation rates was lower (NS) in glucose groups (group A: 52.2% : B. 47.8%) than control group (60.9%). ZiB rates was the highest (P<0.05) in control (47.8%) than those in group A (21.7%) and B (28.3%). ZeB rates were the highest (NS) in group A (30.4%) than those in control (13.0%) and group B (19.6%). Blastocysts, cultured in group B (50.5), had the highest (NS) mean cell number compared with the others (control: 39.2 ; group A: (45.6). The ICM proportion (% ICM of total cells) in blastocysts cultured in group A (20.6%) was the highest (NS) than those of other tested groups (control: 15.2 ; group B: 13.9%). This study shows that a low dose of glucose added to culture medium increases the ICM proportion of blastocysts in mice.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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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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• 제37권2호
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• pp.125-134
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• 2010

목 적: 난관수종액내의 사이토카인 농도를 측정하고, 사이토카인 농도가 다른 난관수종액을 이용하여 생쥐 배아 발생에 미치는 영향을 비교하고자 하였다. 연구방법: 난관수종액은 자궁난관 조영술에서 난관수종이 진단되어 복강경을 통한 난관 절제술을 시행하는 경우 난관 절제술 전에 난관으로부터 채취한 다음 3,000 rpm에서 10분간 원심분리시킨 후 상층액만을 $-20^{\circ}C$에서 보관하였다. 난관수종액의 사이토카인의 조성 및 농도를 확인하기 위하여 interleukin (IL)-$1{\alpha}$, IL-$1{\beta}$, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-$\alpha$, interferon (IFN)-$\gamma$, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), monocyte chemotactic protein (MCP)-1 등을 ELISA 방법으로 측정하였다. 기본 배양액에 난관수종액을 5%, 10%, 30%의 비율로 첨가하여 각 군별로 배반포로의 발달을 관찰하였다. 결 과: 난관수종액내에서 IL-$1{\alpha}$, IL-$1{\beta}$, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-$\alpha$, IFN-$\gamma$, VEGF, EGF, MCP-1가 검출되었으며, 그 농도에 있어서는 큰 차이를 보였다. 정상 혈청 농도에 비하여 난관수종액-1은 IL-6, IL-10이 증가되어 있었고, 난관 수종액-2는 IFN-$\gamma$, MCP-1 및 VEGF가 증가되어 있었다. 각 난관수종액의 Th1/Th2 비는 HSF-1의 경우 IFN-$\gamma$:IL-10이 3.69로 정상인 데 비하여 HSF-2의 경우 IFN-$\gamma$:IL-10이 61.14로 크게 증가되어 있었다. 난관수종액을 포함하지 않는 배양액에서는 배반포기 발달률은 76.7%이었고, 난관수종액-1군은 74%로 대조군과 차이를 보이지 않았지만, 난관수종액-2군의 경우 27.7%로서 대조군 및 난관수종액-1군과도 유의한 차이를 보였다. 난관수종액-1의 경우 난관수종액 농도에 따른 차이는 없었으며, 난관수종액-2군의 경우 농도에 증가함에 따라 배반포로의 발달이 감소하기는 하였지만 통계적으로 유의하지는 않았다. 결 론: 난관수종액마다 사이토카인의 조성이 다르며 이에 따라 생쥐 배아발달에 미치는 영향이 다를 수 있다. 염증성 사이토카인이 증가된 난관수종액이 배아발달에 악영향을 미칠 것으로 추정된다. 특정 사이토카인에 의한 작용을 규명하기는 위해서는 향후 지속적인 연구가 필요할 것으로 생각된다.

• Clinical and Experimental Reproductive Medicine
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• 제31권3호
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• pp.169-176
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• 2004

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.