• 한국수정란이식학회지
• /
• 제24권3호
• /
• pp.145-150
• /
• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

• 한국발생생물학회지:발생과생식
• /
• 제5권2호
• /
• pp.159-165
• /
• 2001

사람 정자에 대한 유인능과 운동성에 미치는 난포액의 영향을 밝히기 위하여 난관 폐색으로 내원한 환자에서 채취한 난포액 sample A, 남성 배우자의 불임으로 내원한 환자에서 채취한 난포액 sample B, Sample A를 가열처리한 난포액 그리고 modified human tubal fluid(m-HTF) 중 어느 하나를 함유한 각각의 모세관을 1, 2 및 4시간 동안 배양하여 유인된 정자의 수와 운동성을 가진 정자의 비율을 조사하였다. 유인된 정자 및 운동성을 가진 정자의 비율은 난포액 sample A에서 m-HTF, 난포액 sample B 및 가열 처리한 난포액보다 유의 적으로(P<0.05) 높게 나타났다. 불활성화 난포액보다 신선 난포액에서 유인능 및 운동성이 높게 나타나는 경향을 보였으나 유의적인 차이는 없었다.

• Clinical and Experimental Reproductive Medicine
• /
• 제27권4호
• /
• pp.359-366
• /
• 2000

The present studt was performed to investigate the effect of treatment and samples of human follicular fluid (hFF) on the development in vitro of mouse embryos. The two cell stage embryos collected at 40 h post-hCG injection were cultured in the modified human tubal fluid (m-RTF) containing 15% synthetic serum substitute (SSS) or human tubal fluid (hFF) for up to 3 days at $37^{\circ}C$ in 5% $CO_2$ incubator. Also the composition of hormone, total protein and protein pattern of hFF samples were analyzed. The developmental rate of mouse embryos developed to blastocyst were not significant difference in the m-RTF containing 15% hFF filtered with 0.22 or 0.8 ${\mu}m$ syringe filter, however, the embryos cultured in the m-RTF containing inactivated hFF were significantly (p<0.05) developed at the high rate to blastocyst than those containing fresh hFF and SSS. The in vitro developmental rate to blastocyst and hatched blastocyst in the m-RTF containing 15% hFF sample A (90.5 and 85.4%, respectively) and SSS (79.4 and 75.3, respectively) were significantly (p<0.05) increased, compared with hFF sample B (64.2 and 54.1 %, respectively). The hFF sample A tended to be higher concentration of LH, FSR, total protein and the ratio of progesterone/$E_2$ and lower concentration of $E_2$ and progesterone than the hFF sample B, but there were no differences in the protein pattern between the two hFF samples. The results of these study suggest that the addition of hFF to the culture medium enhances the development in vitro to blastocyst and hatched blastocyst, but the in vitro developmental rate of mouse embryos is different between hFF samples.

• Clinical and Experimental Reproductive Medicine
• /
• 제19권2호
• /
• pp.125-131
• /
• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• 한국가축번식학회지
• /
• 제18권1호
• /
• pp.71-81
• /
• 1994

These experiments were carried out to investigate the effects of human follicular fluid (hFF) as a protein supplement on development of mammalian embryo as well as to find out ways toward effective use of hFF. The developmental rates of mouse embryos to the blastocyst and implantation stages were significantly higher in T6 ＋hFF than T6＋hFCS. Classified hFF according to the maturity of contained oocytes (M-hFF and Im-hFF), and compared the rates of development of mouse embryo cultured in M-hFF or Im-hFF to culture medium T6. Total protein, albumin and estradiol concentrations were higher in M-hFF than Im-hFF (P<0.05). The developmental rates of mouse embryos to the blastocyst and hatching blastocyst stages cultured in Im-hFF were significantly lower than those in M-hFF and the basic medium. In accordance of the results of human IVF, hFF has been divided into 4 groups. The developmental rates of mouse embryos to the blastocyst stage in presense of hFF from pregnant patients, who have good grade embryos, were significantly higher than those in hFF from patients who have poor grade embryos or were not pregnant. In addition, the rates of development of human embryo were compared in presense of BSA, hFF or hFCS. The developmental rates of human embryos cultured in Ham's F10＋hFF were significantly higher than those in the Ham's F10＋BSA. These results suggests that the culture system using hFF could improve the development ability of mammalian embryos and the viability of blastocysts cultured in vitro.

• 한국수정란이식학회지
• /
• 제19권1호
• /
• pp.11-18
• /
• 2004

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37$^{\circ}C$/with hFF, Group II: 32$^{\circ}C$/withGroup III: 37$^{\circ}C$/without, Group IV:32$^{\circ}C$ /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.9$\pm$1.9%. After 24 hours culture, sperm motility was 23.5$\pm$2.1% (Group I), 8.1$\pm$1.1% (Group II), 10.4$\pm$ 1.4% (Group III) and 4.0$\pm$0.8% (Group IV), respectively. After 48 hours, the motility had been changed as 32$\pm$2.3% (Group I), 14.3$\pm$1.7% (Group II), 5.3 $\pm$1.4% (Group III) and 4.3$\pm$0.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37$^{\circ}C$ was higher than those of group II at 32$^{\circ}C$. But, sperm viability of group I cultured at 37$^{\circ}C$ was lower than those of group II at 32$^{\circ}C$ (54.4$\pm$4.1% vs. 59.4$\pm$3.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37$^{\circ}C$. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.

• Clinical and Experimental Reproductive Medicine
• /
• 제44권2호
• /
• pp.63-72
• /
• 2017

Objective: Hyperstimulation methods are broadly used for in vitro fertilization (IVF) in patients with infertility; however, the side effects associated with these therapies, such as ovarian hyperstimulation syndrome (OHSS), have not been well studied. N-glycoproteomes are subproteomes used for the remote sensing of ovarian stimulation in follicular growth. Glycoproteomic variation in human follicular fluid (hFF) has not been evaluated. In this study, we aimed to identify and quantify the glycoproteomes and N-glycoproteins (N-GPs) in natural and stimulated hFF using label-free nano-liquid chromatography/electrospray ionization-quad time-of-flight mass spectrometry. Methods: For profiling of the total proteome and glycoproteome, pooled protein samples from natural and stimulated hFF samples were selectively isolated using hydrazide chemistry to obtain the total proteomes and glycoproteomes. N-GPs were validated by the consensus sequence N-X-S/T (92.2% specificity for the N-glycomotif at p<0.05). All data were compared between natural versus hyperstimulated hFF samples. Results: We detected 41 and 44 N-GPs in the natural and stimulated hFF samples, respectively. Importantly, we identified 11 N-GPs with greater than two-fold upregulation in stimulated hFF samples compared to natural hFF samples. We also validated the novel N-GPs thyroxine-binding globulin, vitamin D-binding protein, and complement proteins C3 and C9. Conclusion: We identified and classified N-GPs in hFF to improve our understanding of follicular physiology in patients requiring assisted reproduction. Our results provided important insights into the prevention of hyperstimulation side effects, such as OHSS.

• 한국발생생물학회지:발생과생식
• /
• 제5권1호
• /
• pp.23-33
• /
• 2001

포유류의 난자가 수란관내로 배란될 때는 난포액 성분도 같이 수란관내로 들어간다. 본 연구에서는 처음으로 난포액의 일부 성분이 수란관액에 의해서 변화하는 것을 관찰하였다. 사람의 난포액을 gelatin zymogram으로 분석한 결과 621kDa gelatinase 이외에 110kDa gelatinase (GA110) 등의 여러 gelatinase 활성이 나타났다. 이 활성들은 EDTA나 phenanthroline에 의해 억제된 반면 PMSF 처리에 의해서는 아무런 변화가 없었다. 소의 수란관액에서는 62kDa gelatinase의 활성만이 주로 관찰되었다. 소의 수란관 액과 사람의 난포액을 1:1로 섞고 이를 37$^{\circ}$C에서 3시간 동안 둔 결과 사람의 난포액의 GA110 활성은 사라졌다. 사람의 난포액에 APMA를 첨가한 결과 GA110의 활성은 대부분 감소하고 대신 62kDa gelatinase의 활성은 오히려 증가하였다. 반면에 사람의 난포액에 EDTA를 3시간 동안 처리한 결과 GA110의 활성은 오히려 현저히 증가하였고 이 때 다른 gelatinases의 활성은 영향을 받지 않았다. PMSF나SBTI는 난포액내의 gelatinases활성에 아무런 변화를 일으키지 않았다. EDTA, PMTA 혹은 SBTI 등의 proteinase inhibitor를 미리 처리한 사람의 난포액에 소의 수란관 내액을 섞은 경우에도GA110의 활성은 여전히 감소하였다. 사람의 혈청에서도 EDTA에 의해 활성이 현저히 증가하는 GA110이 발견되었다. 사람의 난포액과 유사하게 혈청내의 GA110도 소의 수란관액에 의하여 활성이 사라졌다. 그러나 사람의 난포과립세포의 추출물에서는 단지 92kDa gelatinase만 관찰이 되었다. 마지막으로 anti-human gelatinase A 항체를 사용하여 사람의 난포액과 혈청 그리고 난포과립세포의 추출액을 western blotting한 결과 621kDa과 GA110 만이 항원-항체 반응을 나타내었다. 이 같은 결과로 미루어 사람의 난포액과 혈청에는 gelatinase A의 독특한 isoform인 GA110이 있으며 특히 난포액내의 GA110은 수란관액성분에 의해 선택적으로 분해되는 것으로 여겨진다.

• 한국발생생물학회지:발생과생식
• /
• 제7권2호
• /
• pp.113-118
• /
• 2003

포유동물의 성숙한 난포의 난포액 속에는 여러 종류의 단백질 분해효소가 있으며 이들은 난포의 형성과 퇴화 및 난자의 성숙과 배란 등의 다양한 변화에 중요한 역할을 하는 것으로 여겨진다. 난포액 속의 단백질 가수분해효소 중에는 serine proteinase가 비교적 잘 알려져 있으나 다른 효소 특히, caseinolytic enzyme에 대해서는 거의 알려져 있지 않다. 본 연구에서는 사람의 난포액을 재료로 하여 caseinolytic enzyme의 존재 여부 및 동 효소의 특성을 알아보고자 하였다. 사람의 난포액과 혈청 그리고 사람의 제대혈을 $\alpha$-casein을 기질로 하는 zymography 방법으로 분석 한 결과 분자량 80 kDa의 매우 강한 caseinolytic activity를 지니는 효소 단백질과 분자량 78 kDa의 비교적 약한 caseinolytic activity를 갖는 단백질 등 두 개의 caseinolytic enzyme이 관찰되었다. 이 caseinase들의 특성을 알아보기 위해 phenylmethylsulfonyl fluoride(PMSF), soybean trypsin inhibitor(SBf), 1,10-phenanthroline, E-64 그리고 ethylenediamine tetraacetic acid(EDTA)를 zymouaphy의 substrate buffer에 처리한 결과 EDTA와 SBTI에 의해서 80 kDa와 78 kDa caseinase의 활성이 억제되었다 이로 미루어 80 kDa와 78 kDa caseinase는 trypsin-like enzyme인 것으로 추측된다. 한편 사람 난포액의 zymouaphy 수행시에 5 mM의 EDTA가 첨가된 substrate buffer에 CaC $l_2$, MgC $l_2$, MnC $l_2$ ZnC $l_2$를 각각 0에서 10 mM의 농도별로 처리한 결과 모두 5 mM 농도에서 가장 높은 caseinase 활성을 보였다. 금속 이온의 첨가없이 EDTA만 처리한 대조군의 경우 caseinase의 활성은 나타나지 않았다. 이로 미루어 80 kDa 및 78 kDa caseinase는 효소 활성을 위해 이가 금속 양이온을 필요로 하는 것으로 여겨진다.

• Clinical and Experimental Reproductive Medicine
• /
• 제33권1호
• /
• pp.53-60
• /
• 2006

목 적: 보조생식술에서의 정자처리법으로서 인간 난포액을 이용한 swim-down 방법의 유용성을 확인해 보고자 하였다. 연구방법: 분당서울대학교병원 산부인과에서 불임평가 목적으로 정액검사를 시행할 때 정자무력증 (asthenozoospermia, sperm motility < 50%)을 보이는 12명의 남성을 대상으로 하였다. 이들에서 검사 후 남은 정액을 100% 인간 난포액을 이용한 swim-down법과 SpermGrad를 이용한 밀도차 분리법을 적용하여 각각 처리하고 컴퓨터 정액분석기를 이용하여 정액검사를 시행하였다. 결 과: 두 군 모두에서 운동성과 빠른 운동성 정자의 비율, VCL (curvilinear velocity), ALH (amplitude of lateral head displacement)치 및 과활동성 정자의 비율이 통계적으로 유의하게 상승하였고 LIN (mean linearity)치는 유의하게 감소하였다. 100% 인간 난포액을 이용한 swim-down법에서 정자의 운동성이 SpermGrad를 이용한 밀도차분리법에 비하여 유의하게 높게 나타났으며 ($81.2{\pm}4.7$ vs. $67.6{\pm}2.3$, p=0.02), 다른 변수들은 두 군에서 차이를 보이지 않았다. 결 론: 100% 인간 난포액을 이용한 swim-down법은 정자무력증을 보이는 경우 유용한 정자 처리법으로 사료된다.