• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.395-402
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• 2005

Biologically active recombinant human follicle stimulating hormone (rhFSH) was produced in Chinese hamster ovary cells and purified by a series of chromatographic steps. The chromatographic steps included anion-exchange chromatography (DEAE Sepharose F/F, Q Sepharose F/F), hydrophobic interaction chromatography (Source 15 PHE), and hydroxyapatite chromatography (Macro-Prep ceramic hydroxyapatite type I). A distinctive step of the purification process developed was the use of ZnCl$_2$ for the removal of non-glycosylated or lowly-glycosylated FSH and impurities through co-precipitation with Zn$^{2+}$. Purified rhFSH was identified and characterized by several physicochemical and biological methods such as gel electrophoresis, high-performance liquid chromatography, amino acid analysis, carbohydrate analysis, and biological activity. The overall yield of the purification was ~$30\%$. The rhFSH preparation obtained showed high purity (>$99\%$) and high in vivo potency (>16,000 IU/mg). Carbohydrate analysis suggested that the purified rhFSH contained approximately $40\%$ (w/w) carbohydrate with di­or tri-antennary structure on average, which is somewhat more heavily sialylated than commercially available rhFSH. In conclusion, the results of these analyses established an identity of the purified rhFSH with natural FSH from human pituitary glands, and furthermore, the purified rhFSH preparation showed higher in vivo potency and was slightly more heavily sialylated than commercially available rhFSH.

• Archives of Pharmacal Research
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• 제25권3호
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• pp.357-363
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• 2002

The effect of a new rhFSH, PG-0801, on oocyte quality, ovulation and in vitro fertilization (IVF) was examined in androgen-sterilized mice. Experimental sterility was induced by a single subcutaneous injection of testosterone propionate (TP, 1 mg/head) into 5 day old female mice. Ovulation was generated in the 10 to 13-week old TP-injected mice by a subcutaneous rhFSH injection (1, 5 or 10 IU/head) followed 48 hours later by a second rhFSH injection (1, 5 or 10 IU/head). For comparison, a subcutaneous PMSG (5 IU/head) injection was used for folliculogenesis and a hCG (5 IU/head) injection was used for ovulation. These were administered using the same protocol. The eggs were harvested from the oviducts and counted 17 to 20 hours after the second injection. IVF was performed by adding sperms ($2{\times}10^{5}/ml{\;}to{\;}2{\times}10^{6}/ml$) to determine the functional activity of the eggs, and the fertilization rate was measured. In addition, the pregnancy rate and fetal development were examined after 15-17 days of gestation. The number of oocytes recovered from the rhFSH/rhFSH group increased dose-dependently and was slightly higher than that of the PMSG/hCG group. The pregnancy rates of the group receiving 1, 5, and 10 IU of rhFSH/rhFSH were 50%, 66.7%, and 75%, respectively, which were significantly higher than that of the control (untreated) group (0%). The numbers of viable fetuses in the 1, 5, and 10 IU/head of the rhFSH/rhFSH group ($8.0{\pm}1.50$, $8.9{\pm}1.02$, and $8.9{\pm}1.12$ fetuses/dam, respectively) were comparable to that of the 5 IU/head PMSG/hCG group ($9.4{\pm}0.94$). The mice receiving rhFSH/rhFSH and PMSG/hCG showed similar fertilization rates (around 65%) via the IVF procedure. These results demonstrate that a new rhFSH, PG-0801, may be useful for inducing ovulation in functionally infertile patients and for superovulation in ovulatory patients participating in assisted reproductive technology (ART) programs.

• 한국발생생물학회지:발생과생식
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• 제1권1호
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• pp.25-28
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• 1997

The present study was conducted to determine the effect of recombinant human follicle stimulating hormone (rhFSH) on the estrogen synthesis by human fetal ovarian tissues. Fetal ovaries were 18-19 weeks old (18 wks:n=1, 19 wks:n=2). One case of 19-week-old fetal ovaries were obtained from anencephalic fetus. Obtained ovarieswere cleaned and diced around $600\mu\textrm{m}$ pieces, and cultured in the sandwich agar bed system for 21-23 days. Estrone ($E_{1}$) and estradiol-17 $\beta$($E_{2}$) in the medium was measured by radioimmunoassay. Amount of $E_{2}$ synthesis was greater than $E_{1}$ in both normal cases. In contrast, fetal ovaries from anencephalic fetus did not produce neither $E_{1}$ nor $E_{2}$ in the presence or absence of rhFSH. Results suggest that the fetal ovaries have a capacity of estrogen production at 18-19 weeks of gestation Existence of FSH receptor is also supposed. Different results by anecephalic fetal ovaries suggest the difference in the development between normal and anencephalic fetal ovaries.

• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.387-392
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• 2000

Objectives: 1) To compare the efficacy of urofollitropin (Follimon) to that of recombinant human FSH (rhFSH) on the growth and maturation of mouse early preantral follicles in vitro, and 2) effect of vitrification on the growth and maturation of preantral follicles and oocytes . Methods: Isolated early preantra1 follicles (100-130 ${\mu}m$ diameter) were cultured for 12 days in 20 ${\mu}l$ ${\alpha}$-MEM media drop under the mineral oil. Follimon or rhFSH was added to the culture medium at various concentrations (0, 10, 100, and 1000 mIU/ml). Results: With Follimon, the dose of 10 mIU/ml showed the best follicle survival, growth, and MIl rate of oocyte than the other concentrations. Whereas the optimal dose of rhFSH was 100 mIU/ml. Despite the different optimal doses, the efficacy of two different FSHs on the follicle growth and maturation was similar. Isolated mouse preantral follicles were cryopreserved by vitrification and cultured in vitro for 12 days with 100 mIU/ml rhFSH. Despite the decreased follicular survival rate after thawing, the follicular growth and maturation rate of its oocyte were comparable to those of the fresh follicle. Conclusion: Results from the present study revealed that 1) the optimal doses of Follimon and rhFSH for in-vitro culture of mouse follicles are different, and 2) the frozen-thawed follicles develop normally after vitrification.

• 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.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 1996년도 추계 학술대회 및 정기총회
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• pp.67.1-67.1
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• 1996

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.228-233
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• 2011

Objective: To investigate the effectiveness of GnRH antagonist multiple-dose protocol (MDP) with oral contraceptive pill (OCP) pretreatment in poor responders undergoing IVF/ICSI, compared with GnRH antagonist MDP without OCP pretreatment and GnRH agonist low-dose long protocol (LP). Methods: A total of 120 poor responders were randomized into three groups according to controlled ovarian stimulation (COS) options; GnRH antagonist MDP after OCP pretreatment (group 1), GnRH antagonist MDP without OCP pretreatment (group 2) or GnRH agonist luteal low-dose LP without OCP pretreatment (group 3). Patients allocated in group 1 were pretreated with OCP for 21days in the cycle preceding COS, and ovarian stimulation using recombinant human FSH (rhFSH) was started 5 days after discontinuation of OCP. Results: There were no differences in patients' characteristics among three groups. Total dose and days of rhFSH used for COS were significantly higher in group 3 than in group 1 or 2. The numbers of mature oocytes, fertilized oocytes and grade I, II embryos were significantly lower in group 2 than in group 1 or 3. There were no significant differences in the clinical pregnancy rate and implantation rate among three groups. Conclusion: GnRH antagonist MDP with OCP pretreatment is at least as effective as GnRH agonist low-dose LP in poor responders and can benefit the poor responders by reducing the amount and duration of FSH required for follicular maturation.

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

• Clinical and Experimental Reproductive Medicine
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• 제36권4호
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• pp.249-254
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• 2009

목 적: 고식적 체외수정시술과 세포질내정자주입법을 사용하여 체외성숙 난자를 수정시키고 수정률과 배발달율을 비교하고자 하였다. 연구방법: 2007년 1월부터 2008년 8월까지 난소과자극과 체외수정시술을 받은 59명의 여성에서 135개의 배아소포 단계의 미성숙난자만을 얻어 75 mIU/mL rFSH, 0.5 IU/mL rhCG, 10 ng/mL rEGF가 포함된 체외성숙용 배양액에서 최대 48시간까지 배양하였다. 체외성숙된 난자는 고식적 체외수정방법 (n=41) 또는 세포질내정자주입법 (n=94)을 이용하여 수정시켰으며 이후 배발달율을 관찰하였다. 결 과: 고식적 체외수정군과 세포질내정자주입군에서 체외성숙률은 각각 51.2%와 59.6%였고, 수정률은 각각 71.4%와 80.4%로 두 군간에 통계적으로 유의한 차이는 없었다. 배발달율도 두 군에서 비슷하게 관찰되었다. 결 론: 난소과자극을 통해 얻은 미성숙난자는 고식적 체외수정법을 사용하더라도 세포질내정자주입법과 동등한 수정률과 배발달율을 보임을 확인하였다.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2001년도 제41차 추계학술대회
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• pp.80.2-80.2
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• 2001