Recombinant Human FSH Research Articles

A randomized controlled trial investigating the use of a predictive nomogram for the selection of the FSH starting dose in IVF/ICSI cycles

The number of oocytes retrieved is a relevant intermediate outcome in women undergoing IVF/intracytoplasmic sperm injection (ICSI). This trial compared the efficiency of the selection of the FSH starting dose according to a nomogram based on multiple biomarkers (age, day 3 FSH, anti-Müllerian hormone) versus an age-based strategy. The primary outcome measure was the proportion of women with an optimal number of retrieved oocytes defined as 8–14. At their first IVF/ICSI cycle, 191 patients underwent a long gonadotrophin-releasing hormone agonist protocol and were randomized to receive a starting dose of recombinant (human) FSH, based on their age (150 IU if ≤35 years, 225 IU if >35 years) or based on the nomogram. Optimal response was observed in 58/92 patients (63%) in the nomogram group and in 42/99 (42%) in the control group (+21%, 95% CI = 0.07 to 0.35, P = 0.0037). No significant differences were found in the clinical pregnancy rate or the number of embryos cryopreserved per patient. The study showed that the FSH starting dose selected according to ovarian reserve is associated with an increase in the proportion of patients with an optimal response: large trials are recommended to investigate any possible effect on the live-birth rate.

143 EFFECT OF FSH OR EPIDERMAL-GROWTH-FACTOR-LIKE PEPTIDE SUPPLEMENTATION TO MATURATION MEDIUM ON DEVELOPMENTAL COMPETENCE OF BOVINE OOCYTES DERIVED FROM FULLY DEVELOPED FOLLICLES INDUCED BY SUPER-STIMULATION

Bovine oocytes obtained by ovum-pick-up (OPU) following follicle growth treatment (FGT) have improved quality and competence (Imai et al. 2008 Reprod. Fertil. Dev. 20, 182). However, the effect of the presence of FSH or epidermal growth factor (EGF) like peptide during in vitro maturation (IVM) on the developmental competence of FGT oocytes has not been well known. This study was undertaken to examine the developmental competence of FGT oocytes following IVM in the presence of FSH (recombinant human FSH) or EGF-like peptide (amphiregulin; Areg) and IVF. Japanese Black cows (n = 17) were used as donors. Five days after arbitrary OPU (opu group), follicles ≥8 mm in diameter were aspirated again, a controlled internal drug release (CIDR) was inserted into the vagina, and then pFSH was injected twice a day from the evening of Day 6 to the morning of Day 10 with decreasing doses (total of 20 AU; 4, 4, 3, 3, 2, 2, 1, 1 AU/day). On the evening of Day 8, PGF2α (0.5 mg of cloprostenol) was administered. On Day 11, oocytes were aspirated from follicles with ≥5 mm in diameter of the treated donors by OPU (fgt group). The cumulus-oocyte complexes (COC) were cultured in the absence (opu-cont and fgt-cont groups) or presence of 0.1 IU mL−1 FSH (opu-fsh and fgt-fsh groups) or 100 ng mL−1 Areg (opu-areg and fgt-areg groups) in IVM medium (mTCM199 containing 5 mg mL−1 BSA) for 20 to 22 h (1 COC/5 µL, total of 162–171 COC per group), and then co-cultured with 3 × 106 sperm/mL for 6 h. The presumptive zygotes were continued to culture in mCR1aa supplemented with 5% newborn calf serum for 216 h (1 zygote/5 µL) using micro-well culture dishes (Dai-Nippon-Print). When repeating this opu-fgt session in the same cow, an interval at least for 50 days was kept, and the session was performed 28 times. Statistical analysis was carried out by Mann-Whitney’s U-test (between opu and fgt groups) or Steel-Dwass test after Kruskal-Wallis test (among all groups). The number of follicles ≥5 mm increased in the fgt than opu group (17.8 v. 2.9; P < 0.01). The number of COC collected was not different between the opu and fgt groups (23.1 v. 19.6; P > 0.05). The blastocyst formation rate was higher in the fgt than opu group (36.9 v. 23.1%; P < 0.01). Within 6 groups, the blastocyst formation rate was higher in the fgt-fsh (43.3%; P < 0.01) and fgt-areg (39.5%; P < 0.05) groups than the opu-cont (16.3%) group. The rate in the fgt-fsh group was also higher than that in the opu-fsh group (43.3 v. 18.7%; P < 0.01). These results suggested that FGT improved the developmental competence of bovine oocytes, probably through improving the ability of the COC to react against FSH/Areg.

82 HOW LOW CAN YOU GO? DEFINING THE MINIMAL NUTRIENT REQUIREMENTS FOR BOVINE EMBRYOS IN CULTURE

Recent metabolomic studies in our laboratory have indicated that bovine embryos only use a small amount of the nutrients provided to them in culture media. Our objective was to determine the effects of reducing nutrient concentrations in our media to 75, 50, and 25% (experiment 1) or 25, 12.5, and 6.25% (experiment 2) of those in our control medium (100%) on the development of in vitro-matured/IVF bovine embryos in a serum-free medium. Cumulus–oocyte complexes were matured in a defined maturation medium (0.1 IU mL−1 recombinant human FSH, 50 ng mL−1 recombinant mouse epidermal growth factor, and 2.5 mg mL−1 recombinant human serum albumin) and co-incubated with frozen–thawed spermatozoa (2 × 106 mL−1, 20 h). Embryos were cultured (7.5% CO2, 6.5% O2, 38.7°C) in a sequential media system (0–72 h and 72–168 h). Concentrations of salts, bicarbonate, and protein (2.5 mg mL−1 fatty acid–free BSA) were the same in all treatments. All nutrients (glucose, lactate, pyruvate, amino acids, and vitamins) were diluted to the same extent (e.g. 25%) relative to the control medium for each culture period. Blastocyst formation and hatching (per cleaved embryo) were evaluated on Day 7 of culture. Hatching blastocysts were stained to determine the number of inner cell mass (ICM; SOX2+), trophectoderm (TE; CDX2+), and total cells (ICM+TE) in the embryo. All data were analysed by ANOVA. The proportion of zygotes that cleaved on Day 3 was not affected (P > 0.05) by the concentration of nutrients present. In experiment 1, dilution of nutrients to 25% did not affect (P > 0.05) blastocyst development (40.1 ± 3.7%) or hatching (16.3 ± 2.8%) compared with 100% (45.2 ± 3.8% blastocyst and 24.9 ± 3.3% hatching). In experiment 2, dilution of nutrients to 12.5% tended (P = 0.08) to reduce hatching (12.9 ± 2.6%) compared with 100% (20.0 ± 3.1%) but did not affect (P > 0.05) blastocysts formation (12.5%, 41.7 ± 3.9% v. 100%, 40.0 ± 3.8%). It was not until nutrient concentrations were reduced to 6.25% that blastocyst formation (18.3 ± 3.0%) and hatching (3.0 ± 1.3%) were inhibited (P < 0.05). Hatching blastocysts cultured with 25 or 12.5% nutrients had fewer total (P < 0.05; 150.7 ± 9.7 and 121.6 ± 7.6, respectively), TE (P < 0.05; 124.1 ± 8.5 and 90.5 ± 7.1), and ICM (P = 0.06; 26.6 ± 3.4 and 30.7 ± 4.0) cells compared with control embryos (195.2 ± 15.9 total, 156.1 ± 14.1 TE, and 39.1 ± 4.0 ICM). To determine whether the embryo’s ability to develop with reduced concentrations of nutrients was dependent on lipid metabolism, embryos were cultured with 50, 25, 12.5, and 6.25% nutrients in the presence or absence of an inhibitor of fatty acid oxidation (50 μM etomoxir). The presence of etomoxir reduced (P < 0.05) blastocyst development at all nutrient concentrations, but this effect was more pronounced when nutrients were limited (≤25% nutrients, 28.7 to 40.9% reduction) compared with 50% (12.5% reduction). Although blastocyst cell numbers decrease when nutrient concentrations are reduced to 25% of those in control media, the proportion of embryos reaching the blastocyst stage is not affected until nutrients are reduced to 6.25%. The ability to develop under nutrient-restricted conditions appears to be related to fatty acid metabolism.

Individualized versus conventional ovarian stimulation for in vitro fertilization: a multicenter, randomized, controlled, assessor-blinded, phase 3 noninferiority trial

To compare the efficacy and safety of follitropin delta, a new human recombinant FSH with individualized dosing based on serum antimüllerian hormone (AMH) and body weight, with conventional follitropin alfa dosing for ovarian stimulation in women undergoing IVF. Randomized, multicenter, assessor-blinded, noninferiority trial (ESTHER-1). Reproductive medicine clinics. A total of 1,329 women (aged 18-40years). Follitropin delta (AMH <15 pmol/L: 12μg/d; AMH ≥15 pmol/L: 0.10-0.19μg/kg/d; maximum 12μg/d), or follitropin alfa (150 IU/d for 5days, potential subsequent dose adjustments; maximum 450 IU/d). Ongoing pregnancy and ongoing implantation rates; noninferiority margins -8.0%. Ongoing pregnancy (30.7% vs. 31.6%; difference -0.9% [95% confidence interval (CI) -5.9% to 4.1%]), ongoing implantation (35.2% vs. 35.8%; -0.6% [95% CI -6.1% to 4.8%]), and live birth (29.8% vs. 30.7%; -0.9% [95% CI -5.8% to 4.0%]) rates were similar for individualized follitropin delta and conventional follitropin alfa. Individualized follitropin delta resulted in more women with target response (8-14 oocytes) (43.3% vs. 38.4%), fewer poor responses (fewer than four oocytes in patients with AMH <15 pmol/L) (11.8% vs. 17.9%), fewer excessive responses (≥15 or ≥20 oocytes in patients with AMH ≥15 pmol/L) (27.9% vs. 35.1% and 10.1% vs. 15.6%, respectively), and fewer measures taken to prevent ovarian hyperstimulation syndrome (2.3% vs. 4.5%), despite similar oocyte yield (10.0 ± 5.6 vs. 10.4 ± 6.5) and similar blastocyst numbers (3.3 ± 2.8 vs. 3.5 ± 3.2), and less gonadotropin use (90.0 ± 25.3 vs. 103.7 ± 33.6μg). Optimizing ovarian response in IVF by individualized dosing according to pretreatment patient characteristics results in similar efficacy and improved safety compared with conventional ovarian stimulation. NCT01956110.

Anti-Müllerian hormone for prediction of ovarian response in Chinese infertile women undergoing IVF/ICSI cycles: a prospective, multi-centre, observational study

This study explored the correlation between serum anti-Müllerian hormone (AMH) concentration and the number of retrieved oocytes after ovarian stimulation in Chinese infertile women undergoing assisted reproductive technology treatment and AMH cut-off values predicting low and high ovarian response. This was a prospective, multi-centre, observational study. A total of 615 subjects were included in nine assisted reproductive centres in China for outcome analysis. Subjects received assisted reproductive technology treatment and used recombinant human FSH (r-HFSH) or r-HFSH plus recombinant LH (rLH) for ovarian stimulation according to conventional treatment regimens. The main outcome variables were correlations between AMH and the number of retrieved oocytes and the cut-off values of AMH predicting low and high ovarian response. Serum AMH concentration was positively correlated with the number of oocytes retrieved in Chinese infertile women treated with IVF/intracytoplasmic sperm injection (ICSI) (Pearson correlation coefficient = 0.4754, P < 0.0001). The optimal AMH cut-off value was 2.6 ng/ml (sensitivity: 81.28%, specificity: 59.51%) in predicting high and normal response, and 1.1 ng/ml (sensitivity: 52.27%, specificity: 87.23%) in predicting low and normal response. In conclusion, serum AMH concentration can be used as a biomarker to predict ovarian response in Chinese infertile women treated with assisted reproductive technology.

Comparison of ultrasound-guided endometrial polypectomy carried out on the oocyte retrieval day and the first day of ovarian stimulation in IVF–ICSI cycles

In this retrospective cohort study, the effect of endometrial polypectomy carried out on the day of oocyte retrieval and on the first day of ovarian stimulation in patients with a large (≥10 mm) endometrial polyp undergoing IVF and intractyoplasmic sperm injection (ICSI) was investigated and compared. A total of 74 eligible IVF–ICSI cycles in 74 women who underwent endometrial polypectomy on either the day of oocyte pick-up (late polypectomy group, 39 cycles) or the first day of ovarian stimulation (early polypectomy group, 35 cycles) between January 2007 and July 2012 were included in this study. Patient characteristics between early and late polypectomy groups were similar. Total dose and days of recombinant human FSH administered, numbers of retrieved oocytes, mature oocytes, fertilized oocytes, grade 1 or 2 embryos and embryos transferred between the two groups were also similar, as was clinical pregnancy rate per cycle, embryo implantation rate and spontaneous abortion rate between the two groups. Therefore, endometrial polypectomy on the day of oocyte retrieval could be a more patient-friendly option for patients with a large endometrial polyp undergoing IVF–ICSI.

Treatment with human, recombinant FSH improves sperm DNA fragmentation in idiopathic infertile men depending on the FSH receptor polymorphism p.N680S: a pharmacogenetic study

Does the sperm DNA fragmentation index (DFI) improve depending on the FSH receptor (FSHR) genotype as assessed by the nonsynonymous polymorphisms rs6166 (p.N680S) after 3 months of recombinant FSH treatment in men with idiopathic infertility? FSH treatment significantly improves sperm DFI only in idiopathic infertile men with the p.N680S homozygous N FSHR. FSH, fundamental for spermatogenesis, is empirically used to treat male idiopathic infertility and several studies suggest that DFI could be a candidate predictor of response to FSH treatment, in terms of probability to conceive. Furthermore, it is known that the FSHR single nucleotide polymorphism (SNP) rs6166 (p.N680S) influences ovarian response in women and testicular volume in men. A multicenter, longitudinal, prospective, open-label, two-arm clinical trial was performed. Subjects enrolled were idiopathic infertile men who received 150 IU recombinant human FSH s.c. every other day for 12 weeks and were followed-up for a further 12 weeks after FSH withdrawal. Patients were evaluated at baseline, at the end of treatment and at the end of follow-up. Eighty-nine men with idiopathic infertility carrier of the FSHR p.N680S homozygous N or S genotype, FSH ≤ 8 IU/l and DFI >15%, were enrolled. A total of 66 patients had DFI analysis completed on at least two visits. DFI was evaluated in one laboratory by TUNEL/PI (propidium iodide) assay coupled to flow cytometry, resolving two different fractions of sperm, namely the 'brighter' and 'dimmer' sperm DFI fractions. Thirty-eight men (57.6%) were carriers of the p.N680S homozygous N and 28 (42.4%) of the homozygous S FSHR. Sperm concentration/number was highly heterogeneous and both groups included men ranging from severe oligozoospermia to normozoospermia. Total DFI was significantly lower at the end of the study in homozygous carriers of the p.N680S N versus p.N680S S allele (P = 0.008). Total DFI decreased significantly from baseline to the end of the study (P = 0.021) only in carriers of the p.N680S homozygous N polymorphism, and this decrease involved the sperm population containing vital sperm (i.e. brighter sperm) (P = 0.008). The dimmer sperm DFI fraction, including only nonvital sperm, was significantly larger in p.N680S S homozygous patients than in homozygous N men (P = 0.018). Total DFI was inversely related to total sperm number (P = 0.020) and progressive sperm motility (P = 0.014). When patients were further stratified according to sperm concentration (normoozospermic versus oligozoospermic) or -211G>T polymorphism in the FSHB gene (rs10835638) (homozygous G versus others), the significant improvement of sperm DFI in FSHR p.N680S homozygous N men was independent of sperm concentration and associated with the homozygous FSHB -211G>T homozygous G genotype. The statistical power of the study is 86.9% with alpha error 0.05. This is the first pharmacogenetic study suggesting that FSH treatment induces a significant improvement of total DFI in men carriers of the p.N680S homozygous N FSHR; however, the results need to be confirmed in larger studies using a personalized FSH dosage and treatment duration. The evaluation of sperm DFI as a surrogate marker of sperm quality, and of the FSHR SNP rs6166 (p.N680S), might be useful to predict the response to FSH treatment in men with idiopathic infertility. The study was supported by an unrestricted grant to M.S. and H.M.B. from Merck Serono that provided the drug used in the study. MS received additional grants from Merck Serono and IBSA as well as honoraria from Merck Serono. The remaining authors declare that no conflicts of interest are present. EudraCT number 2010-020240-35.

Functional Expression of FSH Receptor in Endometriotic Lesions.

FSH receptor (FSHR), besides being expressed in gonads, is also expressed in some extragonadal tissues at low levels. We examined the functional expression of FSHR in different types of endometriotic lesions. Extensive studies were carried out to detect functional FSHR expression and FSH-stimulated estrogen production in ovarian endometriomas and recto-vaginal endometriotic nodules (RVEN). Normal endometrium, ovary, and myometrium tissues from nonpregnant cycling women served as controls. This laboratory-based study was carried out on tissue specimens from patients with endometriosis and healthy donors. Endometriotic lesions and normal secretory-phase endometrium showed FSHR expression at both mRNA and protein level. RVEN and ovarian endometrioma demonstrated up-regulated CYP19A1, dependent on the activation of CYP19A1 proximal promoter II. Estrogen receptor-β (ESR2) expression was significantly increased in RVEN vs normal endometrium. Recombinant human FSH stimulation of RVEN explants significantly increased estradiol production and CYP19A1 and ESR2 expression. FSHR was up-regulated in recombinant human FSH-stimulated endometrial and decidualized stromal cells with increased CYP19A1 expression. We described a novel functional FSHR expression, where FSH-stimulated CYP19A1 expression and estrogen production in RVEN are demonstrated. This locally FSH-induced estrogen production may contribute to the pathology, development, progression, and severity of RVEN.

Anti-Müllerian hormone promotes pre-antral follicle growth, but inhibits antral follicle maturation and dominant follicle selection in primates.

What are the direct effects and physiological role of anti-Müllerian hormone (AMH) during primate follicular development and function at specific stages of folliculogenesis? AMH actions in the primate ovary may be stage-dependent, directly promoting pre-antral follicle growth while inhibiting antral follicle maturation and dominant follicle selection. AMH is expressed in the adult ovary, particularly in developing follicles. Studies in mice suggest that AMH suppresses pre-antral follicle growth in vitro, and inhibits primordial follicle recruitment and FSH-stimulated antral follicle steroidogenesis. For in vitro study, secondary follicles were isolated from ovaries of 12 rhesus macaques and cultured for 5 weeks. For in vivo study, intraovarian infusion was conducted on five monkeys for the entire follicular phase during two spontaneous menstrual cycles. For in vitro study, individual follicles were cultured in a 5% O2 environment, in alpha minimum essential medium supplemented with recombinant human FSH. Follicles were randomly assigned to treatments of recombinant human AMH protein or neutralizing anti-human AMH antibody (AMH-Ab). Follicle survival, growth, steroid production, steroidogenic enzyme expression, and oocyte maturation were assessed. For in vivo study, ovaries were infused with control vehicle or AMH-Ab during the follicular phase of the menstrual cycle. Cycle length, serum steroid levels, and antral follicle growth were evaluated. AMH exposure during culture weeks 0-3 (pre-antral stage) promoted, while AMH-Ab delayed, antrum formation of growing follicles compared with controls. AMH treatment during culture weeks 3-5 (antral stage) decreased (P < 0.05) estradiol (E2) production, as well as the mRNA expression of cytochrome P450 family 19 subfamily A polypeptide 1, by antral follicles relative to controls, whereas AMH-Ab increased (P < 0.05) follicular mRNA levels of the enzyme. Intraovarian infusion of AMH-Ab during the follicular phase of the menstrual cycle increased (P < 0.05) the average levels of serum E2 compared with those of the control cycles. Three of the five AMH-Ab-treated ovaries displayed multiple (n = 2-9) medium-to-large (2-8 mm) antral follicles at the mid-cycle E2 peak, whereas only one large (4-7 mm) antral follicle was observed in all monkeys during their control cycles. The average levels of serum progesterone were higher (P < 0.05) during the luteal phase of cycles following the AMH-Ab infusion relative to the vehicle infusion. The in vitro study of AMH actions on cultured individual macaque follicles was limited to the interval from the secondary to small antral stage. A sequential study design was used for in vivo experiments, which may limit the power of the study. The current study provides novel information on direct actions and role of AMH during primate follicular development, and selection of a dominant follicle by the late follicular phase of the menstrual cycle. We hypothesize that AMH acts positively on follicular growth during the pre-antral stage in primates, but negatively impacts antral follicle maturation, which is different from what is reported in the mouse model. NIH NICHD R01HD082208, NIH ORWH/NICHD K12HD043488 (BIRCWH), NIH OD P51OD011092 (ONPRC), Collins Medical Trust. There are no conflicts of interest. Not applicable.

A randomized, single-blind, parallel-controlled and multicentre study: compare the efficacy and safety of domestic and imported human recombinant FSH in WHO group Ⅱ anovulatory infertility

To evaluate the efficacy and safety of domestic human recombinant FSH(rhFSH)in women with anovulation of WHO group Ⅱ. A randomized, blind, parallel-controlled, non-inferiority and multicenter study was performed. A total of 534 admitted to 13 hospitals from May 2008 to August 2009. There were 531 women with ovulatory disorder was included in the statistical analysis, were randomly divided into test group(domestic rhFSH, n=352)and control group(imported rhFSH, n=179). Percentage of cycle with mature follicle, ovulation rate, clinical pregnancy rate, multiple pregnancy rate, ovarian hyperstimulation syndrome(OHSS)and adverse events were observed. No statistical significant differences(P>0.05)were observed between the two groups in terms of the efficiency on mature follicle[91.8%(323/352)versus 88.8%(159/179)], ovulation rate[91.3%(295/323)verus 90.6%(144/159)], clinical pregnancy rate[19.2%(62/323)verus 18.2%(29/159)], the number of the follicles<14 mm, the level of serum LH and progesterone, the thickness of endometrium on the day of hCG administration. The number of follicle≥18 mm and 14 mm≤follicle<18 mm and the level of serum estradiol on the day of hCG in the test group were significantly higher than those in the control group(P<0.05). The number of days of rhFSH administration in the test group was significantly less than that in the control group[(9.8±2.2)versus(11.4± 0.6)days, P<0.05], the dosage of rhFSH was significantly lower than that in the control group[(879 ± 419)versus(1 043±663)U, P<0.05]. The multiple pregnancy rate in the test group was significantly higher than that in the control group[21%(13/62)versu 10%(3/29), P<0.05]. The incidence of OHSS and adverse events were similar between the two groups(P>0.05), and no other adverse events were observed in test group during treatment. Ovarian stimulation with domestic rhFSH is effective, safe and economical in women with anovulation of WHO group Ⅱ.

Comparison between stimulation with highly purified hMG or recombinant FSH in patients undergoing IVF with GnRH antagonist protocol

Purpose: Highly purified Human Menopausal Gonadotropins (hp-hMG) and recombinant FSH (rFSH) are widely used in assisted reproductive technology (ART). The aim of this study was to compare ART results of the two preparations in GnRH antagonist cycles.Methods: In this retrospective cohort study, IVF antagonist cycles performed from 2011 through 2013 were reviewed. There were 508 antagonist cycles: 320 stimulated with rFSH and 188 with hp-hMG. For every hp-hMG, two rFSH were matched for patient's age and infertility diagnosis. Subgroup analysis of patients younger and older than 35 was done as well.Results: Both treatments were resulted in comparable pregnancy and live birth rates. However, cumulative pregnancy rates were higher for the rFSH group. In the matching analysis, the rFSH group had more mature oocytes and more embryos while using lower doses of gonadotropins. Pregnancy, cumulative pregnancy rates, and live birth rates were comparable. In the subgroup analysis, young patients in the rFSH group had better cycle outcomes compared with those in the hp-hMG group.Conclusion: In antagonist protocol, different gonadotropin products are equally effective. The choice of one or the other should depend on the availability, convenience of use, and cost.

Comparative Assessment of Glycosylation of a Recombinant Human FSH and a Highly Purified FSH Extracted from Human Urine

Glycosylation is an important PTM and is critical for the manufacture and efficacy of therapeutic glycoproteins. Glycan significantly influences the biological properties of human follicle-stimulating hormone (hFSH). Using a glycoproteomic strategy, this study compared the glycosylation of a putative highly purified FSH (uhFSH) obtained from human urine with that of a recombinant human FSH (rhFSH) obtained from Chinese hamster ovary (CHO) cells. Intact and subunit masses, N-glycans, N-glycosylation sites, and intact N- and O-glycopeptides were analyzed and compared by mass spectrometry. Classic and complementary analytical methods, including SDS-PAGE, isoelectric focusing, and the Steelman-Pohley bioassay were also employed to compare their intact molecular weights, charge variants, and specific activities. Results showed that highly sialylated, branched, and macro-heterogeneity glycans are predominant in the uhFSH compared with those in rhFSH. The O-glycopeptides of both hFSHs, which have not been described previously, were characterized herein. A high degree of heterogeneity was observed in the N-glycopeptides of both hFSHs. The differences in glycosylation provide useful information in elucidating and in further investigation the critical glycan structures of hFSH.

Effect of FSH supplementation on the GnRH antagonist suppressed growth of PC-3 cell xenografts in nude mice.

e630 Background: One of the fundamental differences between GnRH agonist and antagonist treatment is the permanent suppression of FSH by antagonist, while a rebound in FSH secretion has been documented upon agonist treatments. To assess the potential beneficial effects of permanent FSH suppression in the hormone ablation treatment of prostate cancer we studied the effect of the GnRH antagonist degarelix in the absence and presence of FSH supplementation on the growth of PC-3 human prostate cancer cell xenografts in nude mice. Methods: Intact (n = 20) and gonadectomized (n = 20) nude male mice were inoculated with 2 x 106PC-3 cells. Half of each group received degarelix treatment at 10 mg/kg body weight s.c. In a second experiment (n = 10/group), degarelix treatment was supplemented with recombinant human FSH at10 IU/kg/day using i.p. ALZET osmotic minipumps. Tumor growth in both experiments was monitored for 4 weeks by external inspection and caliper measurement, after which the mice were sacrificed. Results: The first experiment demonstrated that degarelix treatment significantly reduced tumor growth by 33% and 35%, respectively, in intact and gonadectomized mice as compared to non-treated controls (p&lt;0.0001 for each). In the second experiment, FSH reversed the inhibitory effect of degarelix in intact mice. Conclusions: These results demonstrated that the suppression of PC-3 xenograft growth by GnRH antagonist treatment was completely inhibited by concomitant FSH treatment. The findings prove experimentally the principle that combined FSH and LH suppression by GnRH antagonist could offer an advantage over the isolated LH suppression by GnRH agonist upon prostate cancer treatment. [Table: see text]

Successful fertility treatment with gonadotrophin therapy for male hypogonadotrophic hypogonadism

SummaryGonadotrophin therapy with human chorionic gonadotrophin and recombinant FSH is indicated for use in men with reduced spermatogenesis due to hypogonadotrophic hypogonadism (HH). Patients require regular monitoring for side effects and desired response to treatment. We present a man with HH, azoospermia and a history of previous anabolic steroid usage who had undergone gonadotrophin therapy, had subsequently achieved conception and has now fathered a child.Learning points In total, 15% of couples do not achieve pregnancy within 1 year and seek medical treatment for infertility: male factors contribute to 50% of these.The evaluation of male infertility should include a full history and examination, an endocrine profile and a quality-controlled semen analysis.HH with defective spermatogenesis is an important cause of male infertility in a small percentage of cases.Gonadotrophin therapy requires regular monitoring for side effects and desired response to treatment.Any sustained rise in prostate specific antigen levels should prompt urological assessment for possible prostate biopsy.A multidisciplinary approach is required for gonadotrophin therapy, especially if assisted fertilisation techniques are required once, spermatogenesis is achieved.

A Preliminary Report of A Low-Dose Step-Up Regimen of Recombinant Human FSH for Young Women Undergoing Ovulation Induction with IUI.

The aim of this study was to evaluate the efficacy and safety of a recombinant human follicle stimulating hormone (r-FSH) low-dose step-up regimen for controlled ovarian hyperstimulation in patients undergoing ovulation induction (OI) with intrauterine insemination (IUI). The study was conducted in the Department of Obstetrics and Gynecology, Far Eastern Memorial Hospital, New Taipei, Taiwan. In this prospective, observational study, consecutive infertile women (20-35 years) with regular menstrual cycles and a normal baseline FSH level were prospectively enrolled between January 2010 and September 2010. A starting dose of 112.5 IU/day r-FSH was administered on day 3 and increased by 37.5 IU/day every 2 days until a follicle ≥11 mm in diameter was present. Recombinant human chorionic gonadotropin (r-hCG) was administered when a follicle ≥18 mm was noted. Monifollicular development was defined as only one follicle with a diameter ≥16 mm. Clinical pregnancy was defined as a pregnancy diagnosed by ultrasonographic visualization of one or more gestational sacs. A total of 29 women and 30 cycles were included. The mean daily dose of r-FSH to achieve a follicle of ≥11 mm in diameter was 131.3 ± 23.6 IU and the mean total dose was 1030.0 ± 383.2 IU. Approximately 41% of the cycles were monofollicular. Clinical pregnancy was observed in 9 (30.0%) cycles, and a fetal heart beat was observed in 7 (23.3%). There were no multiple pregnancies. Mild ovarian hyperstimulation syndrome, which was resolved with conservative management, was observed in 3 (10.0%) cycles. This r-FSH low-dose step-up regimen seems to be a feasible and practical method for OI in younger infertile women undergoing IUI.

Development and characterization of a novel long-acting recombinant follicle stimulating hormone agonist by fusing Fc to an FSH-β subunit

Does a novel long-acting recombinant human FSH, KN015, a heterodimer composed of FSHα and FSHβ-Fc/Fc, offer a potential FSH alternative? KN015 had in vitro activity and superior in vivo bioactivity than recombinant human FSH (rhFSH), suggesting KN015 could serve as a potential FSH agonist for clinical therapy. rhFSH has very short half-life so that repeat injections are needed, resulting in discomfort and inconvenience for patients. The longest-acting rhFSH available in clinics is corifollitropin alpha (FSH-CTP), but its half-life is not long enough to sustain the whole therapy period, and additional injections of rhFSH are needed. Plasmids containing FSHα, FSHβ-Fc and Fc cDNA were transfected into Chinese hamster ovary (CHO) cells for KN015 production. The pharmacokinetics of KN015 was investigated in 6-week-old SD rats (n = 6/group) and healthy Cynomolgus monkeys in two different dose groups (n = 2/group). A series of experiments were designed for in vitro and in vivo characterization of the bioactivity of KN015 relative to rhFSH. The purity and molecular weight of KN015 were determined by reducing and non-reducing SDS-PAGE. To measure KN015 half-life, sera were collected at increasing time points and the remaining FSH concentration was measured by enzyme-linked immunosorbent assay. To assess the bioactivity of KN015 versus rhFSH in vitro, firstly cAMP production was assessed in CHO cells expressing FSH receptor (FSHR) with the treatment of Fc/Fc, rhFSH or KN015 at eight different doses (0.03, 0.09, 0.28, 0.83, 2.5, 7.5, 22.5, 67.5 nM), and secondly cumulus oocyte complexes (COCs; n = 20/group) of ICR mice (primed-PMSG 44 h before sacrificed) were collected and cultured in medium containing 1.25 pM Fc/Fc, rhFSH or KN015 at 37°C and then germinal vesicle breakdown (GVBD) and COC expansion were observed at 4 and 16 h, respectively. The in vivo activity of KN015 was compared with rhFSH by ovary weight gain and ovulation assays. In the former, ovary weight gains in 21-day-old female SD rats, after a single subcutaneous injection of KN015, were compared with those after several injections of rhFSH over a range of doses (n = 8/group). Sera were harvested for estradiol (E2) analysis, and the ovaries were processed for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL), RT-PCR and western blot. In the latter, 26-day-old female SD rats (n = 8/group) were injected with different doses of KN015 or rhFSH, and were sacrificed at 24 h after an injection of hCG (20 IU/rat). Moreover, the molecular responses stimulated by KN015 or rhFSH in the ovary were also analyzed through detecting expression of the FSH target genes (Cyp19a1, Fshr and Lhcgr) and phosphatidylinositide 3-kinase (PI3K) pathway activation. KN015 has a molecular weight of 82 kD and its half-life is 84 h in SD rats (10-fold longer than that of rhFSH) and 215 h in Cynomolgus monkeys. The EC50 value of the cAMP induction in CHO cells (KN015 versus rhFSH, 1.84 versus 0.87 nM), COC expansion and oocyte maturation assays showed KN015 had approximately half of rhFSH's activity in vitro. A single dose of KN015 (1.5 pmol/rat, 166.1 ± 19.7 mg, P < 0.01) stimulated significantly larger ovary weight gain than several injections of rhFSH (1.5 pmol/rat, 59.3 ± 28.1 mg, P < 0.01). The serum E2 level in the KN015 group was significantly higher than that in rhFSH group. The number of oocytes obtained by ovulation induction was comparable with or higher in the KN015 group than in the rhFSH group. KN015 was more effective than rhFSH in inducing FSH target genes (Cyp19a1, Fshr, Lhcgr) or activating the PI3K pathway in vivo. Moreover, a single injection of KN015 promoted granulosa cell proliferation and prevented follicle atresia to the same extent as several injections of rhFSH. All assays in this study were operated only in animals and clinical trials are needed to confirm they can be extrapolated to humans. KN015 is a valuable alternative to FSH and may have great potential for therapeutic applications. This study was supported by National Basic Research Program of China (2011|CB944504, 2012CB944403) and National Natural Science Foundation of China (81172473, 31371449). The authors have no conflicts of interest to declare.

Follicular and endocrine dose responses according to anti-Müllerian hormone levels in IVF patients treated with a novel human recombinant FSH (FE 999049).

To study the association between serum anti-Müllerian hormone (AMH) levels and follicular development and endocrine responses induced by increasing doses (5·2-12·1 μg/day) of a novel recombinant human FSH (rhFSH, FE 999049) in patients undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) in a GnRH antagonist protocol. Secondary analysis of a randomized controlled trial with stratified randomization according to AMH (lower stratum: 5·0-14·9 pmol/l; higher stratum: 15·0-44·9 pmol/l). Infertile women of good prognosis (n = 265). Follicular development and endocrine parameters during controlled ovarian stimulation (COS) with rhFSH. Serum FSH levels increased with increasing rhFSH doses and steady-state levels for each dose were similar in both AMH strata. In the whole study population, significant (P < 0·001) positive dose responses were observed for the number of follicles ≥ 12 mm, and serum levels of oestradiol, inhibin B, inhibin A and progesterone at end of stimulation. In comparison with the higher AMH stratum, patients in the lower AMH stratum had significantly different slopes of the dose-response curves for these hormones, and no clear dose-related increase was observed for the number of follicles in these patients. Dose-response relationships between rhFSH and follicular development and endocrine parameters are significantly different for IVF/ICSI patients with lower and higher serum AMH levels at start of COS.

Uso de la hormona folículo estimulante humana recombinante en la infertilidad anovulatoria

OBJETIVO: Estudiar los efectos usando dosis múltiples de la FSH humana recombinante en nueve mujeres con hipogonadismo hipogonadotrófico, analizando su capacidad de inducir el crecimiento folicular y síntesis de esteroides ováricos. DISEÑO: Trabajo clínico abierto. MATERIAL Y MÉTODOS: De nueve casos de hipogonadismo gonadotrófico, una tenía diagnóstico de síndrome de Kallman, una deficiencia aislada de gonadotropinas y siete panhipopituitarismo secundario a hipofisectomía. Se administró inyecciones 1M de hFSHr (Folitropina alfa Instituto Ares-Serono, Ginebra) en dosis crecientes (semana 1: 75 UI/dia, semana 2: 150 UI/dia, semana 3: 225 UI/día) durante un promedio de 17 dias (rango: 15-19). Se efectuó toma de muestras de sangre regularmente y estudios ultra sonográficos frecuentes para monitorizar el desarrollo folicular. Para las mediciones hormonales se recurrió al radioinmunoanálisis. RESULTADOS: Las concentraciones séricas iniciales de FSH y de hormona luteinizante (LH) fueron 0.48 (0.9-1.22) UI/L y 0.26(˂0.15, 0.41) UI/L, respectivamente. Los valores máximos de FSH durante la prueba llegaron a 10.3 (7.9-13.6) UI/L, en tanto que las concentraciones séricas de LH significativamente permanecieron estables 0.33 (0.19-0,52) UI/L. Los niveles séricos de androstenediona y testosterona no mostraron cambios significativos durante la administración de hFSHr y el estradiol sérico reveló sólo un aumento leve: 131.7 (51-239) pmol/L. Se desarrollaron folículos múltiples que llegaron a dimensiones preovulatorias (˃ 14 mm), con un índice de desarrollo folicular de 1.92 ± 0.7 mm/día. CONCLUSIONES: Del presente trabajo se deduce que la hFSHr resultó efectiva en mujeres con deficiencia de gonadotropinas para estimular el crecimiento folicular normal hasta la etapa preovulatoria e inducción de ovulación, sin impedirlo el aumento leve observado del estradiol. Asimismo, los niveles séricos de LH endógeno extremadamente bajos cursaron con secreción androgénica deficitaria, indicando la necesidad de contar con dicha gonadotropina para inducir una apropiada síntesis de esteroides.

Ongoing pregnancy rates in intrauterine insemination are affected by late follicular-phase progesterone levels

To determine the relationship between serum P levels on the day of hCG administration and ongoing pregnancy rates. Retrospective study. University-affiliated private IVF. A total of 2,458 couples undergoing IUI. Ovarian stimulation with human recombinant FSH. Ongoing pregnancy and miscarriage rates. Progesterone concentrations were significantly higher given that the E2 concentration increased. Ongoing pregnancy rates were significantly decreased in women with P levels higher than 1.1 ng/mL; similar results were obtained in relation to miscarriage rates. Significant differences in ongoing pregnancy rates when P levels were elevated on the day of hCG administration may help clinicians to counsel patients about the reduced success rates with IUI and manage the timing of insemination to optimize implantation.

Randomized trial comparing luteinizing hormone supplementation timing strategies in older women undergoing ovarian stimulation

In this open-label study, women aged 36–40 years undergoing ovarian stimulation were randomized to recombinant human FSH (rhFSH) plus recombinant human luteinizing hormone (rhLH) from stimulation day 1 (group A; n = 103), or rhFSH alone (days 1–5) followed by rhFSH plus rhLH from day 6 (group B; n = 99). The primary objective was equivalence in number of oocytes retrieved per patient. The mean (±SD) number of oocytes retrieved was 9.7 (±6.9) in group A and 10.9 (±6.5) in group B; the estimated difference between groups (−1.28 oocytes [95% confidence interval: −3.15 to 0.59]) did not reach the predefined limit of equivalence (±3 oocytes). The study's primary objective was therefore not met. In both groups, a mean (±SD) of 1.9 (±0.6) embryos were transferred per patient. Implantation rates were 24.7% in group A and 13.3% in group B. Clinical pregnancy rates per started cycle and per embryo transfer were 31.6% and 34.4% in Group A, 17.2% and 18.9% in Group B. Ovarian hyperstimulation syndrome was reported in four (group A) and five (group B) patients. The potential benefit of initiating LH supplementation earlier during ovarian stimulation in older women is of interest, warranting further exploration.