• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.45-56
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• 2002

Objectives: Recently, recombinant FSH (rFSH) has been manufactured using a Chinese hamster ovary cell line transfected with the gene encoding human FSH. Both rFSH and urinary gonadotropin (uFSH) could be used for controlled ovarian hyperstimulation (COH). However, uFSH implies a number of disadvantages, such as batch-to-batch inconsistency, no absolute source control, dependence on large amounts of urine, low specific activity, and low purity. The purpose of this study was to evaluate the efficacy of rFSH in human IVF-ET program. Materials and Methods: A total of 508 infertile women was enrolled in this study. They are classified into rFSH group (n=177) or uFSH group (n=331), and all of them were matched by age and cause of infertility in same period. The $Puregon^{(R)}$ (Organon, Holland) was used as rFSH, and the Metrodin-$HP^{(R)}$ (Serono, Switzeland) and $Humegon^{(R)}$ (Organon, Holland) was used as uFSH. We subdivided the patients into three age groups. The outcomes of IVF-ET program were analyzed using the statistical package for social sciences (SPSS). Results: There was no significant differences in the level of estradiol on hCG injection day, the numbers of retrieved oocytes, matured oocytes, fertilized oocytes, transferred embryos, frozen embryos between the two groups. The total dose (IU) of gonadotropin for COH was significantly lower in the rFSH group compared to uFSH group ($1339{\pm}5491.1$ vs $2527.8{\pm}1075.2$ IU, p<0.001). Clinical pregnancy rate per embryo transfer in the rFSH group showed increasing tendency, compared to the uFSH group, but there was no statistical significance (35.2% vs 29.3%). Our results demonstrated that the relative efficiency of rFSH compared with uFSH is higher in older patients. Conclusions: The ovarian stimulatory effect and clinical outcome of recombinant FSH was similar to that of the urinary gonadotropin. The IVF-ET cycles with significantly lower dose of gonadotropin in rFSH group showed comparable results. Therefore, we suggest that recombinant FSH is more potent and effective than urinary gonadotropin.

• 한국발생생물학회지:발생과생식
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• 제5권2호
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• pp.123-129
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• 2001

포유동물의 난포내 난자의 성숙 시에는 난자를 둘러싸고 있는 난구세포의 확장 현상이 일어나는데 이 현상에는 hyaluronic acid 뿐만 아니라 다른 성분도 관여하는 것으로 알려져 있다. 본 연구는 조직 재구성 과정에서 중요한 역할을 하는 matrix metalloproteinase(MMP)가 사람의 성숙한 난자-난구 복합체의 extracellular matrix(ECM)에 존재하는지의 여부를 알아보고자 하였다. 체외수정 시술 시에 얻어지는 사람의 난자-난구 복합체를 재료로 zymography와 western blotting 방법으로 조사한 결과 난자-난구 복합체의 ECM에는 300kDa, 240kDa, 200kDa, 180kDa, 116kDa, 97kDa, 그리고 84kDa의 분자량을 갖는 적어도 7종류의 gelatinase들이 존재하는 것이 관찰되었다. 이들 gelatinase가 MMP인지를 확인하기 위해 zymography 동안에 ethylenediaminetetraacetic acid 혹은 phenanthroline 등의 MMP 억제제를 처리한 결과 7종류 모두의 gelatinase 효소활성이 사라졌다. 또한 MMP의 활성제인 aminophenylmercuric acetate를 zymography를 시행하기 전에 ECM에 처리한 결과 200kDa, 180kDa, 97kDa, 84kDa의 gelatinase활성이 사라지고, 대신에 80kDa, 65kDa, 60kDa의 분자량을 갖는 새로운 gelatinase 단백질의 효소활성이 나타났다. 이로 미루어 사람 난자-난구 복합체의 ECM에는 여러 종류의 gelatinase들이 있으며 이들 중 일부는 MMP-2와 MMP-9의 동위효소들인 것으로 여겨진다.

• 한국수정란이식학회지
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• 제28권3호
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• pp.193-198
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• 2013

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide($H_2O_2$) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

• 한국가축번식학회지
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• 제14권3호
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• pp.183-190
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• 1990

These studies were carried out ot investigate the effects of estrous cow serum(ECS), fetal calf serum(FCS), bovine follicular fluid(BFF) and matured cumulus cell(MCC) on in vitro maturation and fertilization of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluid from the visible follicles of diameter 3-5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones, FCS, ECS, BFF and MCC for 24~48 hrs. in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 18$^{\circ}C$20 hrs. with motile capacitated sperm in the TCF(Tyrode calcium-free) solution containing 200$\mu\textrm{g}$/ml of heparin. The results obtained in these experiments were summarized as follows : 1. The oocytes classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 61.4%, 12.1%, 19.2%, 4.2% and 3.0% of the total oocytes harvested, respectively. The maturation and fertilization rate of the A, B, C class follicular oocytes, cultured in the TCM-199 medium supplemented with 10% FCS were 89.1%, 78.0%, 52.6% and 78.1%, 33.3%, respectively. 2. The maturation and fertilization rate of the follicular oocytes cultured in TCM-199 medium supplemented with 5%~20% ECS and FCS were 74.0%~80.6, 26.2%~30.0% and 71.7%~76.9%, 51.9%~58.0%, and those values were higher the supplement of ECS than FCS. 3. The maturation rate(68.0%~64.6%) and fertilization rate(59.6%~60.4%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 20~30% BFF were higher than those of follicular oocytes cultured TCM-199 medium supplemented with 10% FCS and 10% and 50% BFF. 4. The maturation rate(76.5%) and fertilization rate(61.7%) of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$106/ml cumulus cells were higher than those of follicular oocytes cultured in TCM-199 medium supplemented with 10% FCS and 1$\times$104~5/ml and 1$\times$108/ml cumulus cells.lus cells.

• Reproductive and Developmental Biology
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• 제30권1호
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• pp.59-64
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• 2006

본 연구는 돼지정액의 보다 간편하고 손쉽게 동결시킬 수 있은 방법을 확립하기 위하여 수행하였다. 돼지정액은 3시간에 걸쳐 $5^{\circ}C$까지 냉각 후 Straw에 봉입하고 다양한 방법 및 step에 의해 스티로폼 용기 내에 들어있는 $LN_2$ 중에서 동결하였다. 정자의 생존성은 $LN_2$ 표면으로부터 10 cm 위에서 10분간 정치 후 침적할 경우 가장 높게 나타났다(54.0%). Straw를 $-102^{\circ}C$에서 10분간 정치시킨 처리구가 여타구보다 높은 생존성이 얻어졌다(74.0%, P<0.05). 응해 방법에 따른 동결정액의 생존성 실험에서는 $37^{\circ}C$의 융해구가 $52^{\circ}C$ 융해구보다 유의적으로 높은 결과를 나타냈다(P<0.05). 1단계 동결 방법과 3단계 동결방법으로 돼지 정액을 동결시킨 결과 정액의 일반적 특성 및 첨체의 이상 유무를 평가하는 CTC 검사에서는 커다란 차이가 인정되지 않았다. 동결정액을 이용한 체외수정 결과에서는 상실배기 이상 발육율에서 1-step이 3-step보다 높은 발육율을 나타내었다(27.5 vs 14.7%, P<0.05). 본 실험의 결과 돼지정액 동결 시 $-102^{\circ}C$에서 10분간 정치시키는 1단계 동결방법이 간편하면서 유리한 동결 방법으로 활용될 수 있음을 보여준다.

• 한국수정란이식학회지
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• 제27권1호
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• pp.29-35
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• 2012

The purpose of this study was to examine the effect of growing stages of the Korean Native Striped Bull (KNSB) on the freezability and fertility of frozen-thawed semen. First, we investigated the total motility (TM) and progressive motility (PM) according to the diluent used for semen freezing. Second, we examined the effect of the age of KNSB on semen volume, TM and PM of fresh and frozen-thawed semen. Third, we examined the effect of frozen semen from the different age of KNSB on the $in-vitro$ fertilization rate, and the artificial insemination pregnancy rate. The diluents used in this experiment were Triladyl$^{(R)}$ and Tris-egg yolk extender (EYE). Semen was collected from 5 KNSB in the growing stage (15 months) and 5 adult KNSB (36 months). When Triladyl or Tris-EYE extender was used for semen freezing, there was no difference of the mean TM and the mean PM. However, the mean TM was significantly higher in Bull No. 1885 than Bull No. 4283 ($p$ <0.05). The mean volume of semen collected from the 15-month-old bulls (2.3 ml) was significantly lower ($p$ <0.05) than that from the 36-month-old bulls (5.0 ml). The mean semen concentration was similar for the 15-month-old ($2.1{\times}10^9$ spermatozoa/ml) and 36-month-old ($1.8{\times}10^9$ spermatozoa/ml) bulls. For the 15-month-old and 36-month-old bulls, the mean TM of fresh semen were 93.7% and 88.3%, respectively, and the mean PM were 97.0% and 88.3%, respectively; the 15-month-old bulls showed a particularly high PM ($p$ <0.05). For the 15-month-old and 36-month-old bulls, the mean TM (56.0% and 58.0%, respectively) and the mean PM (64.0% and 70.7%, respectively) of frozen-thawed semen did not differ. The development rates of embryos after $in-vitro$ fertilization and the pregnancy rate after artificial insemination using frozen-thawed semen did not differ according to the bull's age. In summary, semen volume differed according to the bull's age, but semen concentration and survival rate, the $in-vitro$ fertilization rate, and the pregnancy rate did not differ according to the stripe bull's age. Accordingly, semen from bulls in the growing stage can be collected and frozen for the preservation and multiplication of rare livestock.

• 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.

• 한국패류학회지
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• 제23권1호
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• pp.39-49
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• 2007

우리나라 고성군 송지호의 기수역에서 채집된 2배체 일본재첩 (Corbicula japonica) 과 춘천 의암호 담수에서 채집된 3 배체 재첩 (C. fluminea) 을 대상으로 핵형 및 번식특성의 차이점을 확인하기 위해 조사하였다. 2배체인 일본재첩은 25 개 세포들에서 세포당 38 개의 염색체가 관찰되었다. 이들의 핵형은 상동염색체 2 개가 19 쌍을 이루고 있다. 염색체는 11 쌍의 telocentric과 3 set의 subtelocentric 그리고 3 쌍의 submetacentric 그리고 1 쌍의 metacentric 염색체로 구성되어 있었다. 일본재첩은 난생이며 자웅이체로 내반새와 외반새를 갖는 두 쌍의 아가미를 가지나, 이들은 보육낭으로 기능을 하지는 않는다. 난들과 정자들은 기수역에서 체외수정을 한다. 3배체인 재첩은 세포당 54 개의 염색체가 관찰되었다. 이들의 핵형은 상동염색체 3 개가 18 set를 이루고 있었다. 이들 염색체 18 set는 1 개의 metacentric group과 5 개의 submetacentric group, 그리고 12 개의 subtelo 또는 telocentric group으로 이루어져 있었다. 재첩은 난태생이며 기능적 자웅동체이다. 3배체 조건은 자웅동체성과 밀접한 관련을 가진다. 재첩의 경우는 주로 보육낭으로 기능을 하는 내반새 (inner-demibranch) 를 갖는 2 쌍의 아가미를 가진다. 아가미의 내반새 구조에서는 임란기 중에는 완숙란들이 소모되고, 비임란기 중에는 완숙난들이 생식소의 자웅동체 소낭 내에서 생산되어 주기적 변화가 나타났다. 일본재첩과 재첩의 생식소발달 단계들은 조직학적 관찰에 의해 초기활성기, 후기활성기, 완숙기, 부분산란기, 퇴화 및 비활성기의 5 단계로 나눌 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.129-138
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• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.239-249
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• 1999

The role of amino acids in culture media for IVF-ET was examined in a total of 76 cycles. Patients received clomiphene citrate (CC) followed by hMG or GnRH-a combined with gonadotropins (FSH/hMG) for controlled ovarian hyperstimulation. Severe male (<$4{\times}10^6$ motile sperm) or age factor (>39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. No significant differences were found in age, duration of infertility, follicle size, the level of $E_2$ on the day of hCG injection, the mean number of oocytes retrieved, total motile sperm count, fertilization rate and the mean number of embryos transferred between bHTF (without amino acids) and mHTF (with amino acids) groups. However, total ampules of gonadotropins were higher (p<0.01) in mHTF group than bHTF group. Significantly (p<0.05) more clinical pregnancies were recorded in mHTF group (13/30) compared with bHTF group (9/46). The multiple pregnancy rates were 11.1% in bHTF group and 7.7% in mHTF group. There were one ectopic pregnancy in mHTF group and one heterotopic pregnancy in bHTF group. Abortion rates were 22.2% in bHTF group and 7.7% in mHTF, respectively. The ongoing pregnancy or livebirth rate was significantly (p<0.05) higher in mHTF group (12/30) than bHTF group (7/46). These results suggest that the addition of amino acids in culture media is essential for culture of zygotes in vitro and adjustment of energy substrates in phosphate-free culture media appears to be beneficial for human IVF-ET procedure.