Collaborative study to evaluate a reporter gene assay for recombinant human follicle-stimulating hormone bioactivity / 药学学报

The goal of this work was to explore the prospect of standardized application of an in-vitro bioactivity assay for recombinant human follicle-stimulating hormone based on a reporter gene. The relative accuracy, intermediate precision, linearity and applicable range of the method were validated according to the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Three laboratories used this method to determine the in-vitro biological activities of six batches of drug product and three batches of drug substance manufactured by two different companies. The consistency of the potency determined by three laboratories, the intra-laboratory precision and inter-laboratory precision were analyzed. The method was optimized during the collaborative validation. The results of method validation meet the requirements of the General Rules of Chinese Pharmacopoeia 2020 edition Volume IV (9401). Aiming to resolve the problems found in the collaborative validation, the medium for cell seeding, the pre-diluted buffer solution of standard and sample, and the means of removing and discarding supernatant after stimulation were optimized. After optimization, there was no significant difference in the bioactivity among the different laboratories (P > 0.05), indicating statistical equivalency. Intra-laboratory and inter-laboratory precision were good and the geometric coefficient of variation (GCV%) were both less than 15%. In conclusion, the reporter gene assay has good intra-laboratory repeatability and inter-laboratory reproducibility and is suitable for analyzing recombinant human follicle-stimulating hormone drug product and drug substance by different manufacturers. It is expected to be used as a standardized method for the determination of the in-vitro bioactivity of such products.

Comparison of M-CSF and its receptor mRNA expression in luteinized granulosa cells after using recombinant human follicle stimulating hormone / 中国临床药理学与治疗学

AIM:To explore the effect of MCSF and its receptor on the response of ovarian stimulation by comparing the expression of macrophage colony stimulating factor (M-CSF) and its receptor mRNA in luteinized granulosa cells in patients after using combinant human follicle stimulating hormone.METHODS:Ninety-six patients with polycystic ovary syndrome (PCOS) and 157 patients with non-PCOS underwent in vitro fertilization were divided into four groups,i.e.the PCOS fast and slow reaction group,and the non PCOS fast and slow reaction group,according to their response to recombinant human follicle stimulating hormone (r-FSH).Luteinized granulosa cells were then collected after mature follicular puncture.SYBR Green quantitative RT-PCR method was used to detect the expression of M-CSF,M-CSFR and GAPDH in the mRNA gene of the granulosa cells samples.The relative quantity of these genes were determined by comparing the threshold value (CT value of the target gene subtract CT value of housekeeping gene).The difference of gene expression between two groups was compared by t test,and Spearman correlation analysis was used to describe the data relationships.RESULTS:No significant difference was observed in the use of r-FSH among the different groups (P ＞ 0.05).Neither was there any significant difference in mRNA quantity of M-CSF or M-CSFR between the entire PCOS and non PCOS patients (P ＞ 0.05).After grouping,no significant difference was observed between any two groups in the expression of M-CSF (P ＞ 0.05).The expression of M-CSFR in PCOS slow response group was significantly lower than that of PCOS fast response group (P =0.006).Meanwhile,the Spearman analysis showed that the correlation between the quantification of M-CSFR mRNA and the days of r-FSH in PCOS group was statistically significant (P =0.023);the correlation coefficient was 0.511.CONCLUSION:The slow response to ovarian stimulation in PCOS patients is possibly related to the reduction of granulocyte MCSFR expression.The M-CSF and its receptor may be involved in the ovarian stimulation response process.

The Efficacy of Recombinant Human Follicle Stimulating Hormone (rhFSH) in Human IVF-ET Program / 대한불임학회지

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+/-5491.1 vs 2527.8+/-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.

Recombinant Human Follicle Stimulating Hormone (rFSH) versus Highly Purified Urinary FSH (uFSH): Oocyte and Embryo Quality / 대한산부인과학회잡지

OBJECTIVE: To estimate the efficacy of recombinant human follicle stimulating hormone (rFSH) versus highly purified urinary human FSH (uFSH) in women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET). METHODS: From 1 January 2001 to 31 August 2001, A total of 254 cycles from 241 patients who attended infertility clinic at Samsung cheil hospital was enrolled in this study. With pituitary down regulation using GnRH agonist by short protocol, rFSH (Puregon(R), Organon, Netherlands) was administered in 131 cycles and uFSH (Metrodin-HP(R), Serono, Switzerland) was administered in 123 cycles. We analyzed ovarian response, pregnancy rate, live birth rate, oocyte quality and embryo quality. RESULTS: The clinical characteristics of two groups were not different. Total FSH dosages (1322.3+/-526.2 IU versus 2124.4+/-881.9 IU, p<0.001) and dosages per retrieved oocyte (90.6+/-36.0 IU versus 138.0+/-57.2 IU, p<0.001) were significantly lower in rFSH group than uFSH group. Clinical pregnancy rate and live birth rate of two groups were not significantly different. The rate of good quality oocyte (Grade I and II) from retrieved oocytes was higher in rFSH group (68.2% versus 64.8%, p=0.024), but after preincubating oocytes for 4 to 6 hours and removing cumulus cells in intracytoplasmic sperm injection (ICSI) cycles, nuclear maturity of oocytes were not significantly different. The quality of transferred embryos were not significantly different too. CONCLUSION: rFSH offered more effective ovarian response in COH and better quality of retrieved oocytes, compared with uFSH.

The Efficacy of Recombinant Human Follicle Stimulating Hormone (rhFSH) in Controlled Ovarian Hyperstimulation for In Vitro Fertilization and Embryo Transfer / 대한산부인과학회잡지

OBJECTIVE: To compare the efficacy and safety of recombinant human follicle stimulating hormone (rhFSH) versus highly purified urinary human FSH (uhFSH-HP) in women undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET). METHODS: Ninety-three women with tubal infertility, stage I/II endometriosis or unexplained infertility were admitted to this study. After pituitary desensitization using GnRH agonist, rhFSH (n=45) or uhFSH-HP (n=48) was administered with a step-down regimen in all patients. RESULTS: Patient's characteristics were comparable in both groups. Low responders were 20 in rhFSH group and 22 in uhFSH-HP group. The total dose of administered FSH was significantly lower in rhFSH group than that in uhFSH-HP grup (p<0.001). The days of stimulation were also significantly shorter in rhFSH group than those in uhFSH-HP group (p<0.05). However, there were no differences in IVF results such as the numbers of oocytes retrieved, oocytes fertilized, grade I/II embryos, embryos transferred between the two groups. There were also no differences in clinical pregnancy rate, miscarriage rate, and multiple pregnancy rate. Even in the low responder subgroup, COH using rhFSH was also associated with significant decreases in the total dose of FSH and the duration of stimulation required. IVF results and pregnancy outcomes were comparable in rhFSH and uhFSH-HP groups. CONCLUSION: These data suggest that the total dose of FSH and the duration of stimulation can be reduced by using rhFSH.

Randomized Controlled Trial of Follicle-Stimulating Hormone Versus Human Menopausal Gonadotropin in Controlled Ovarian Hyperstimulation for in Vitro Fertilization and Embryo Transfer / 대한산부인과학회잡지

Recent evidence suggests that a high level of serum LH during follicular recruitment and development is associated with poor reproductive outcome. Consequently, exogenous LH administration for controlled ovarian hyperstimulation(COH) in in vitro fertilization and embryo transfer(IVF-ET) may be harmful to folliculogenesis. The purpose of this clinical study was to evaluate and compare the efficacy of human menopausal gonadotropin(hMG) and human follicle-stimulating hormone(hFSH) for COH with long protocol of gonadotropin-releasing hormone(GnRH) agonist in IVF-ET program. Randomized clinical trial was performed in 125 patiens undergoing IVF-ET at Seoul National University Hospital from May to Septebmer, 1995. The inclusion criteria of patients included age < 40 years and normal semen analysis, and the study population was also classified into two groups by the etiology of infertility : Group T - 95 patients with only tubal factor and Group O - 30 patients with endometriosis or anovulatory factor. There were no statistically significant differences in dosage(29.0+/-7.9 vs 26.0+/-6.8 ampoules) and duration(12.3+/-1.3 vs 12.2+/-1.5 days) of gonadotropin administration, serum E2 level on hCG day(1,943+/-1,255 vs 1,580+/-1,067 pg/mL), cancellation rate(7.5% vs 6.7%), number of oocytes retrieved(9.9+/-6.0 vs 11.3+/-6.0), fertilization rate(68.4% vs 64.5%), number of embryos transferred(4.7+/-2.0 vs 4.7+/-2.0), and preganancy rate per cycle(26.3% vs 24.4%) and per ET(28.4% vs 26.2%) between hMG(N=80) and hFSH(N=45) Groups. In Group T, no significant differnces in results of IVF-ET were also detected between hMG (N=61) and hFSH(N=34) Groups. In Group O, serum E2 level on hCG day was significantly higher in hMG Group (N=19) compared with hFSH Group(N=11), but other results of IVF-ET were similar in both Groups. As this study could not demonstrate any significant differences in results of IVF-ET between hMG and hFSH when used for COH in IVF-ET program, it could be concluded that hFSH is at least as efficacious as hMG for COH.