IU rhFSH Research Articles

P-238 Non-interventional study comparing the effectiveness and safety of r-hFSH:r-hLH versus r-hFSH in women aged 35-42 years with normal ovarian reserve undergoing GnRH antagonist ART treatment

Abstract Study question Is ovarian stimulation (OS) with r-hFSH:r-hLH more effective than OS with r-hFSH alone in women aged 35-42 with normal ovarian reserve treated with ART? Summary answer Compared to r-hFSH alone, OS with r-hFSH:r-hLH for ART treatment was associated with a significant increase in clinical pregnancy and live birth per initiated cycle. What is known already Meta-analyses and secondary analyses of clinical data provide evidence that OS during ART treatment with r-hFSH:r-hLH is associated with improved reproductive outcomes when compared to r-hFSH alone in various patient groups with: a) advanced maternal age , b) low ovarian reserve or , c) hypo response to OS. Other subgroups of patients might also benefit of the use of r-hFSH:r-hLH during OS for ART treatment. We aimed to determine if, in a real-world setting, OS with r-hFSH:r-hLH is associated with improved clinical benefit when compared to r-hFSH alone in patients aged 35-42 years with normal ovarian reserve. Study design, size, duration A non-interventional study including women aged 35-42 years with normal ovarian reserve biomarkers (menstrual cycle day 2 or 3 FSH<12IU/L or 9<AFC<20 with a 2<diameter<10 mm, or AMH ≥1.2ng/ml) undergoing OS for ART treatment (fresh transfer only; GnRH antagonist protocol) in 12 French centers between 1/1/2008 and 31/12/2016, with a follow-up period up to 31/12/2017. The analysis included two cohorts: a) 4,323 OS cycles with r-hFSH alone, and b) 1,070 OS cycles with r-hFSH:r-hLH. Participants/materials, setting, methods Anonymized data were extracted from the Retrospective Multicenter Statistical database, including 12 French centers. A generalized linear model was used to address lack of randomization. The fixed effect part included age, an ovarian reserve estimator and stimulation; the random effect included age, AMH, AFC, FSH, cause of infertility and number of attempts, center and patient effect. Clusters (matched subsets) were built based on the propensity score by using a two-stage algorithm. Main results and the role of chance We included in our analysis 4,472 women receiving ART treatment after OS with r-hFSH (mean age: 37.8 years; 4,323 cycles) or with r-hFSH:r-hLH (mean age: 38.1 years; 1,070 cycles). In the r-hFSH cohort the mean AMH level was 2.81 ng/ml and mean AFC was 12.0, in the r-hFSH:r-hLH cohort they were 2.14 ng/ml and 10.0, respectively. The mean total r-hFSH dose were 2,275 IU (r-hFSH cohort) and 3,002 IU (r-hFSH:r-hLH cohort). Mean ± SD number of oocytes were 8.1 ± 5.7 (r-hFSH cohort) and 6.1 ± 4.3 (r-hFSH:r-hLH cohort); mean ± SD number of embryos were 4.0 ± 3.4 (r-hFSH cohort) and 3.3 ± 3.2 (r-hFSH:r-hLH cohort). The unadjusted ongoing pregnancy rate reached 17.1% in the r-hFSH cohort and 20.4% in the r-hFSH:r-hLH cohort, whilst the unadjusted live birth rate reached 17.0% and 20.2%, respectively. Unadjusted pregnancy failure rate was 6.6% and 3.7%, respectively. After adjusting for confounders, when compared to OS with r-hFSH, OS with rhFSH:rhLH was associated with a higher clinical pregnancy rate (Risk ratio,RR 1.232; 95% CI 1.058 to 1.436; p-value = 0.007) and a higher live birth rate (RR 1.452; 95% CI 1.227-1.1719, p-value <0.001) per initiated cycle. Limitations, reasons for caution Since this was a real-world data study, no random assignment of stimulation was performed, and some residual confounding is possible due to unmeasured confounders. Live births resulting from further transfers of frozen embryos were not included in the analysis. Wider implications of the findings This study demonstrated that OS with r-hFSH:r-hLH improves live birth rate in women > 35 years old with a normal ovarian reserve, compared to r-hFSH only. Sound RWE studies can be combined with RCTs to guide clinical and payer’s decision making, paving the way to precision medicine. Trial registration number Not applicable

Comparison of two different starting dose of rhFSH in GnRH antagonist protocol for patients with normal ovarian reserve.

To evaluate different starting doses of recombinant human follicle-stimulating hormone (rhFSH) on pregnancy outcomes for patients with normal ovarian reserve during gonadotropin- releasing hormone antagonist (GnRH-ant) protocol-controlled ovarian stimulation of in vitro fertilization (IVF) cycles. In this retrospective study, a total of 1138 patients undergoing IVF cycles following the GnRH-ant protocol were enrolled. Patients were divided into two groups according to the starting dose of rhFSH. 617 patients received a starting dose of rhFSH of 150 IU, and 521 patients received a starting dose of rhFSH of 225 IU. We compared demographic characteristics, ovarian stimulation and embryological characteristics, and pregnancy and birth outcomes between the two groups. Multivariate logistic regression analysis was performed to examine the possible effects of the known potential confounding factors on pregnancy outcomes. The number of oocytes retrieved in the 150 IU rhFSH group was significantly lower than those in the 225 IU rhFSH group. There was no significant difference between the two groups referring to embryological characteristics. The proportion of fresh embryo transfer in the 150 IU rhFSH group was significantly higher than that in the 225 IU rhFSH group (48.30% vs. 40.90%), and there was no difference in the risk of ovarian hyperstimulation syndrome and pregnancy outcomes between the two groups. In conclusion, the starting dose of rhFSH of 150 IU for ovarian stimulation has a similar pregnancy outcome as starting dose of rhFSH of 225 IU in GnRH-ant protocol for patients with normal ovarian reserve. Considering the potential cost-effectiveness and shorter time to live birth, the starting dose of rhFSH of 150 IU may be more suitable than 225 IU.

Intraovarian Injection of Recombinant Human Follicle-Stimulating Hormone for Luteal-Phase Ovarian Stimulation during Oocyte Retrieval Is Effective in Women with Impending Ovarian Failure and Diminished Ovarian Reserve

It is a challenge to obtain sufficient eggs during in vitro fertilization (IVF) in women with impending ovarian failure (IOF)/diminished ovarian reserve (DOR). Although studies have suggested that more than one wave of follicle growth exists, the efficacy of controlled ovulation stimulation (COS) in both follicular and luteal phases of the same ovarian cycle (DuoStim) is not established in women with IOF/DOR. We investigated the efficacy of DuoStim using the intraovarian injection of recombinant human follicle-stimulating hormone (rhFSH) during oocyte retrieval in women with DOR. For luteal-phase stimulation, intraovarian (Group A, N = 28) or superficial subcutaneous (Group B, N = 18) injection of 300 IU rhFSH immediately after oocyte retrieval was administered as the first dose, and intermittent superficial subcutaneous addition of gonadotropins was employed accordingly for further COS in both groups. In Group A, significantly lower Gn doses, a shorter duration of COS, a greater number of antral follicle counts, and an increased number of retrieved mature and total oocytes were noted. Compared with the clinical outcomes of luteal-phase COS, the average daily doses of rhFSH used in Group A were significantly lower. In summary, the novel approach using intraovarian rhFSH injection provides an efficient treatment regimen in women with IOF/DOR.

Extended Injection Intervals of Gonadotropins by Intradermal Administration in IVF Treatment

Gonadotropins can be administered every 5 days under intradermal injection in in vitro fertilization (IVF) treatment. To explore the effectiveness of intradermal injection of recombinant human FSH (rhFSH) for women undergoing IVF. Women who received their first IVF treatment enrolled in this prospective intervention in 2018. All women received a bolus of 900 IU rhFSH intradermally at day 2 of the treatment cycle followed by additional dosage of rhFSH at day 7 and/or day 10. The main outcome measures included the total dose of rhFSH and number of injections required, sequential serum FSH level detected, and number of mature oocytes retrieved. Seventy women completed the study. On average, 2.31 ± 0.73 injections and 1662 ± 397 IU of rhFSH were administered. While the baseline FSH level was 5.6 ± 2.2 IU/L, the serum concentrations of FSH after rhFSH administration were 35.3 ± 7.0 on the first day (24 hours) and 10.7 ± 3.7 IU/L on the fifth day (120 hours). A total of 10.5 ± 6.6 mature oocytes were retrieved, resulting in 7.3 ± 5.1 pronuclear embryos; 1.8 ± 0.6 embryos were transferred to the uterus. Our findings resulted in 72% fertilization, 91% cleavage, 31% implantation, and 36% live birth rates. Although fewer larger follicles were found, noninferiority results were noted in the mature oocytes retrieved, good embryos available, and clinical pregnancy rate compared with those received conventional daily subcutaneous rhFSH administration. Intradermal administration of rhFSH, with a smaller dose of rhFSH and fewer injections, may achieve the goal of a cost-effective and more patient-friendly regimen.

Treatment of idiopathic oligozoospermia with recombinant human follicle-stimulating hormone: a prospective, randomized, double-blind, placebo-controlled clinical study in Chinese population.

Follicle-stimulating hormone plays a crucial role in spermatogenesis. The aim of this study was to evaluate the efficacy of treatment with FSH in Chinese infertility population. Prospective, randomized, double-blind, placebo-controlled clinical study. A total of 354 men affected by idiopathic oligozoospermia from three medical centres. This study contained three parts: (i) treatment with different doses of rhFSH (50 IU, 100 IU, 200 IU and 300 IU); (ii) the efficacy of rhFSH at different periods (2, 3, 4, 5 months); (iii) FSH treatment in patients with different levels of inhibin B (normal-level group, low-level group and high-level group). Semen parameters were evaluated in all subjects. The patients who had not reached spontaneous pregnancy underwent assisted reproductive techniques. Sperm number was significantly increased after treatment with FSH at doses of at least 200 IU, and the improvement was observed beginning at the third month. The significant improvement in both morphology and forward motility was observed beginning at the fifth month. Moreover, 300 IU rhFSH administration for 5 months could significantly improve the spontaneous pregnancy rate (12/40) and ART pregnancy rate (14/28), while the rates for placebo group were two of twenty-nine and five of twenty-seven, respectively. The seminal parameters (total sperm count, sperm concentration, forward motility and morphology) were significantly improved in the normal- and low-level inhibin B groups, but no significant variation was observed in the high-level group at the end of the study. The efficacy of FSH treatment was associated with the dose of FSH and duration of treatment, and FSH therapy was more effective in patients with normal level and low level of inhibin B.

The 112.5IU r-hFSH starting dose is enough for successful IVF in AMH ≥3 ng/ml

Controlled ovarian hyperstimulation(COH) is needed in order to improve the pregnancy rate in IVF cycles. Recently, there has been an interest in using a much lower dosage of FSH for mild stimulation in IVF protocols. The purpose of this study was to assess the mild stimulation with starting dose of recombinant human FSH (r-hFSH) 112.5IU could be enough for successful IVF outcomes in patients with AMH ≥3ng/ml in IVF cycles. Retrospective. We retrospectively reviewed 657 consecutive cycles of 594 normo-ovulatroy women(AMH ≥3ng/ml) who underwent IVF treatment over a 3-year period (2010 –2012) at our institution. Our study population was divided into two groups according to r-hFSH starting dose for COH-IVF cycles. Group A: r-hFSH(Gonal-F®) starting dose 112.5IU (n=257), Group B: starting dose 150IU (n=400) in women with AMH ≥3ng/ml. Outcomes were compared the number of oocytes retrieved, total dose of r-hFSH, total r-hFSH stimulation days, clinical pregnancy and abortion rate between the two groups.. The total dose of r-hFSH administered was significantly different between the group A and the group B (1055.1 IU and 1365.5 IU, p<0.01). The total number of r-hFSH administration days (9.6days (5–17) and 9.1days (9–16), p=0.08) and embryo quality were comparable in both groups. Although the number of retrieved oocytes (14.9 ± 6.8 vs. 13.3 ± 6.1, P<0.01) and fertilized oocytes(9.1 ± 4.9 vs. 8.8 ± 4.3, P=0.02) were significantly higher in group A than group B. However, the pregnancy rate (63.4% vs 58.3%, p=0.139) and abortion rate (12.9% vs13.3%) were not significantly different between both groups. In our results, a total mean r-hFSH dose of 1055.1IU with the 112.5IU starting dose in COH cycle was sufficient for the number of retrieved oocytes and the pregnancy rates in patients with AMH≥3ng/ml. Therefore, These seems clear that a subset of patients with AMH ≥3ng/ml undergoing ovarian stimulation can be adequately treated with a 112.5 IU starting dose of r-hFSH.

Dextran vitrification media prevents mucin coat and zona pellucida damage in rabbit embryo

Vitrification of embryos is being increasingly important for cryopreservation in mammals. However, damage and toxicity has to be reduced even more. The composition of cryoprotective medium used to immerse the embryos affects viability and developmental potential. The aim of this work was to assess the effect of the Polyvinylalcohol-PVA- and Dextran addition to vitrification media on the in vitro development of rabbit embryos from superovulated and non-superovulated females. Superovulation group were treated intramuscularly with 25 IU rhFSH. The vitrification media contained the same permeable cryoprotectans (Ethylene Glycol-ET- and Dimethyl Sulfoxide-Me 2SO-) and different macromolecules (PVA and Dextran) in different combinations. There was a significantly higher proportion of embryos without damages in mucin coat or zona pellucida after warming (undamaged embryos) in the control than in the superovulation group (95.8% vs. 83.2%, respectively). The proportion of undamaged embryos was significantly affected by the vitrification solution composition. The rate of undamaged embryos after warming in media containing 20% Me 2SO was significantly lower in media supplemented with PVA than in media with dextran (67.3 vs. 93.8, respectively). However, the proportion of undamaged embryos for the medium supplemented with dextran was similar for media with 15 or 20% Me 2SO. In conclusion, the addition of dextran to the vitrification media improve the preservation of rabbit embryos and permits to reduce the amount of Me 2SO for vitrification. Additionally, in vitro developmental ability of undamaged embryos were not affected by superovulation treatment nor vitrification media.

Recombinant follicle-stimulating hormone (follitropin alfa) for ovulation induction in Japanese patients with anti-estrogen-ineffective oligo- or anovulatory infertility: results of a phase II dose-response study.

To determine the optimal regimen of recombinant human follicle-stimulating hormone (r-hFSH) for ovulation induction (OI) in Japanese women with amenorrhea I or anovulatory infertility. In this randomized, double-blind, dose-finding study, women aged 20-39 years were enrolled. Patients underwent a chronic low-dose step-up regimen with starting doses of r-hFSH of 37.5 IU (group L; n = 62), 75 IU (group M; n = 62) or 150 IU (group H; n = 60). Primary endpoint was time to achieve a dominant follicle with mean diameter ≥18 mm. Secondary endpoints included percentage of patients producing a dominant follicle ≥18 mm, achieving ovulation, and biochemical pregnancy. Median time to achieve a dominant follicle ≥18 mm was 14 days (group L; 87.7%), 10 days (group M; 98.4%), and 8 days (group H; 94.5%). In group M, ovulation occurred in 95.1% of patients, with pregnancy in 18.0%, compared with 86.0% and 15.8% in group L, and 50.9% and 9.1% in group H, respectively. Twelve patients developed ovarian hyperstimulation syndrome (nine in group H). Most adverse events in groups L and M were mild in severity. A starting dose of 75 IU r-hFSH was associated with a favorable efficacy and safety profile for OI in Japanese women.

Effect of recombinant gonadotropins on embryo quality in superovulated rabbit does and immune response after repeated treatments

This study aimed first to evaluate the effect of recombinant human FSH (rhFSH) with and without recombinant human LH (rhLH) on fresh and frozen-thawed embryo development and also to analyze the immune response of rabbit does (Oryctolagus cuniculus) subjected to repeated rhFSH treatments. Nulliparous New Zealand White does were used. In Experiment 1, 120 does were superovulated with 25 IU rhFSH alone or in combination with 5% or 10% rhLH (1.25 IU or 2.50 IU rhLH). A total of 1116 embryos at the compacted morula stage were cultured at 38.5 °C, 5% CO 2, and saturated humidity for 48 h. The embryo development to hatching blastocyst was significantly lower for the group with 10% rhLH versus that of the control group (65.6 vs. 79.5 for rhFSH + 10% rhLH vs. control, respectively). However, no significant difference was found in development to hatching blastocyst for the control, rhFSH alone, and rhFSH + 5% rhLH groups. The developmental potential of frozen-thawed embryos obtained from all groups was similar, with an 83.5% in vitro development rate until the expanded blastocyst stage. To detect anti-FSH antibodies, in Experiment 2, does were subject to four superovulation treatments. The hormone administration had a significant effect on immune response in the superovulation group after two treatments (0.14 ± 0.074 and 0.15 ± 0.076 vs. 0.46 ± 0.078 and 0.50 ± 0.078 optical density for the first, second, third, and forth cycles, respectively). Nevertheless, none of the treated does had an immune response in both the first and second treatments; on the contrary, a significant increase in the antibody levels was observed in these females at the moment of the third and fourth superovulation treatments. In conclusion, rhFSH superovulation treatments increase the reproductive potential of rabbit does.

Ovarian response to gonadotropin stimulation in juvenile rhesus monkeys

The objective of this study was to investigate juvenile rhesus monkeys responding to various gonadotropin regimen stimulations. Thirty-two prepubertal rhesus monkeys were randomly allocated into five groups for ovarian stimulation as follows: Groups I, II, and III were given 35, 18, and 9 IU recombinant human follicle-stimulating hormone (rhFSH), respectively, twice daily for 8 d; Group IV was given 18 IU rhFSH twice daily until the appearance of maximal increase in sex skin during the breeding season; and Group V was treated identically to Group II but during the nonbreeding season. In addition, nine menarchial monkeys (Group VI) were treated identically to Group II. Menarchial monkeys yielded two- to fivefold the numbers of MII oocytes (24.1) and almost twice the development potential of in vitro–fertilized oocytes (blastocyst rate: 50.0%) compared with those of the other groups. Moreover, prepubertal monkeys in Group V had approximately double the numbers of MII oocytes and in Groups IV and V twice the development potential compared with those of Groups I and II, whereas Group III did not respond to stimulation. The most prominent sex skin swelling was in association with peak serum estradiol concentrations, and good responses to stimulation were associated with reduced body temperatures. All stimulated monkeys had normal reproductive performance at adulthood, except those in Group I. In conclusion, gonadotropin stimulation of menarchial monkeys could be appropriate for addressing the high cost and limited availability of rhesus monkeys in studying reproductive biology in primates.

Correlation between body mass index (BMI) and serum beta hCG levels post hCG with ART cycle outcomes

OBJECTIVE: To evaluate if 250 mcg rhCG in women with different BMIs achieved similar serum beta hCG levels for follicular maturation and successful ART outcomes.DESIGN: Retrospective study.MATERIALS AND METHODS: 84 consecutive ART cycles between January - March 2008 were included. Women were treated with GnRH agonist/antagnosists, stimulation began with 150-450 IU rhFSH and 75-150 IU rhLH or hMG added day 6 or 7. Response was assessed by ultrasound after 5 days of treatment; the dose of rhFSH ± rhLH/hMG adjusted as needed. Final follicular maturation was with 250 mcg r-hCG; serum beta hCG levels were measured 12-14 hrs later. Retrieval was done 36-37 hrs post hCG injection. ET was on day 5 or 6. Patients were stratified according to their BMI (kg/m2): 16-20, 21-25, 26-28, and 29-36. Statistical analysis: ANOVA test for continuous data and X2 for categorical data; significance: p<0.05.Table 1Body Mass Index (BMI (kg/m2)16-2021-2526-2829-36P valueN cycles13461411Age (yrs)32.1±4.733.5±4.637.0±3.4∗34.1±4.0∗0.02BMI19.3±1.222.8±1.327.0±0.931.1±2.2<0.001days of stim9.2±3.49.3±1.49.5±1.29.9±1.50.78Total 75 IU FSH24.0±13.832.4±13.034.6±15.341.8±17.2∗∗0.04Total 75 IU LH5.6±1.95.9±3.55.5±3.75.1±2.90.91#foll > 15 mm11.6±7.09.0±4.98.1±4.58.2±3.40.27Peak E2pg/ml2195±14402495±12952186±8321922±7860.48E2 post hCG3219±21662968±14242707±12552583±13850.74serum hCG (IU/L)135±51102±6389±4270±560.05# eggs17.4±11.613.6±6.113.6±6.711.4±4.50.21# mature oocytes15.0±11.111.3±5.712.0±6.77.8±4.30.09MII/total eggs0.810.810.840.660.05# 2PN8.3±8.06.8±4.36.5±2.54.0±2.80.77# blasts transferred1.6±1.01.2±0.81.7±0.71.1±0.80.19# blasts frozen∗∗4.6±1.63.0±2.34.0±1.71.6±0.50.24Clinical pregnancy rate (%)46.1 (6/13)43.8 (20/46)42.8 (6/14)45.4 (5/11)0.61∗ Frozen on day 6. Open table in a new tab CONCLUSIONS: Women with lower BMI (16-20 kg/m2) achieved higher serum levels of beta hCG, had more MII oocytes and blastocysts than those in the highest BMI group. Pregnancy rates were similar for all groups. Patients with a BMI >29 kg/m2 may require a higher dose of rhCG to achieve similar higher serum beta hCG levels which are associated, in women with lower BMIs, with more mature oocytes and higher blastulation rate. OBJECTIVE: To evaluate if 250 mcg rhCG in women with different BMIs achieved similar serum beta hCG levels for follicular maturation and successful ART outcomes. DESIGN: Retrospective study. MATERIALS AND METHODS: 84 consecutive ART cycles between January - March 2008 were included. Women were treated with GnRH agonist/antagnosists, stimulation began with 150-450 IU rhFSH and 75-150 IU rhLH or hMG added day 6 or 7. Response was assessed by ultrasound after 5 days of treatment; the dose of rhFSH ± rhLH/hMG adjusted as needed. Final follicular maturation was with 250 mcg r-hCG; serum beta hCG levels were measured 12-14 hrs later. Retrieval was done 36-37 hrs post hCG injection. ET was on day 5 or 6. Patients were stratified according to their BMI (kg/m2): 16-20, 21-25, 26-28, and 29-36. Statistical analysis: ANOVA test for continuous data and X2 for categorical data; significance: p<0.05. ∗ Frozen on day 6. CONCLUSIONS: Women with lower BMI (16-20 kg/m2) achieved higher serum levels of beta hCG, had more MII oocytes and blastocysts than those in the highest BMI group. Pregnancy rates were similar for all groups. Patients with a BMI >29 kg/m2 may require a higher dose of rhCG to achieve similar higher serum beta hCG levels which are associated, in women with lower BMIs, with more mature oocytes and higher blastulation rate.

Effects of rhFSH regimen and time interval on ovarian responses to repeated stimulation cycles in rhesus monkeys during a physiologic breeding season

We studied the effects of repeated stimulation by recombinant human FSH (rhFSH) at various time intervals during a physiologic breeding season in rhesus monkeys. Ovarian recovery and responses were assessed by ultrasonography, serum steroid concentrations, number of oocytes retrieved, and in vitro blastocyst development following IVF. One group underwent a single stimulation regimen with 18 IU rhFSH i.m., followed by 1000 IU hCG, and serum steroid concentrations and ovarian status were determined in the following three menses. Another group was stimulated as before and then allocated into three subgroups; each subgroup was re-stimulated once at the beginning of the ensuing first, second, or third menses. In the final experiment, one group was stimulated with 37.5 IU rhFSH, whereas another group received 18 IU rhFSH. In subsequent cycles, all were re-stimulated twice with 18 IU rhFSH at time intervals of two menstrual cycles (MCs). At the first menses after stimulation, serum progesterone concentrations were significantly higher and the ovaries larger than before stimulation. Monkeys that were re-stimulated at the first menses responded poorly; at the second menses, progesterone concentrations and ovarian size recovered, but the number of oocytes retrieved from re-stimulated monkeys was still significantly reduced. However, animals that were re-stimulated in two MCs later responded well (i.e., percentage of the animals responding, oocytes recovered, and potential for fertilization and blastocyst formation). In conclusion, rhesus monkeys were likely to have similar ovarian responses to repeated stimulation with the same regimen spaced at least two MCs apart.

Src-Family Protein Tyrosine Kinases Are Required for Meiotic Maturation in the Mouse.

Oocyte maturation involves both cytoplasmic maturation processes as well as meiotic divisions that prepare the oocyte for fertilization and progressive development. Three members of the Src-family of protein tyrosine kinases (PTKs) are expressed in mammalian oocytes: Fyn, Yes and Src. These PTKs have been implicated in both the first and second meiotic maturation processes. The objective of the current studies was to examine the specific biochemical and cellular events requiring Src-family PTKs during oocyte maturation. We have used a combination of chemical inhibitors and siRNA knock-down to impair Src PTKs in order to examine Src PTK involvement in meiotic maturation. Oocytes were collected from mature CF1 mice with or without 300 μM dbcAMP to maintain GV arrest. Maturation media were supplemented with 0.2 IU rhFSH and 10 ng/ml EGF. To test the requirement for Src PTKs, oocytes were matured in the presence of Src-specific inhibitors PP2 (10, 20 or 50 μM) or SKI606 (1, 5 or 10 μM). Both inhibitors had no effect on GVBD, but inhibited meiotic progression from MI to MII. This inhibition was dose dependant. At lower doses, some oocytes progressed to metaphase-II but displayed abnormalities in spindle and chromatin organization; the percentage of abnormal oocytes was also dose dependant. Continuous 17-h exposure of COC to 10 μM SKI606 arrested all oocytes at metaphase of MI. Wash-out experiments in which oocytes were held for 1, 2.5 or 5 h with or without cAMP and/or SKI606 and were returned to control medium demonstrated that the effects of SKI606 were reversible. As an alternative approach, we have used siRNA for a specific knock-down of Fyn PTK in GV stage oocytes, since our previous studies implicated Fyn kinase in the completion of meiosis following fertilization. The siRNA injected GV oocytes were maintained in cAMP media for 5 h to allow time for the siRNA actions prior to cAMP removal thus allowing meiotic resumption. Following 17 h of maturation, 61% of the Fyn knock-down eggs arrested at the MI stage (39% matured to MII, n=96), compared to scrambled control siRNA injected and non-injected control eggs which matured to MII (86% n=112 and 93% n=27, respectively). These studies demonstrate that Src PTK activity is required during the early stages of meiotic maturation and further demonstrates the involvement of Src PTKs in meiotic spindle morphogenesis and cell cycle progression at the first metaphase anaphase transition. Fyn PTK specifically appears critical for healthy meiotic maturation and early developmental progression of the mammalian egg.

Superovulatory response to a low dose single‐daily treatment of rhFSH dissolved in polyvinylpyrrolidone in rhesus monkeys

To simplify the procedure for superovulation in the rhesus monkey, this study was designed using polyvinylpyrrolidone (PVP) solution as a solvent for gonadotropins. Thirty-five cycling females (aged 5-8 years old) were divided into six groups during the breeding season (November- February). The groups were as follows: Group I, animals received twice-daily 35 IU recombinant human follicle-stimulating hormone (rhFSH) dissolved in 0.5 ml saline for 8 days as the control; Groups II and III, animals received single-daily 35 IU and 17 IU rhFSH in 0.5 saline, respectively, for 9 days; Groups IV, V and VI, received single-daily injection of 35 IU rhFSH, 17 IU rhFSH and 8.5 IU rhFSH dissolved in 0.5 ml 30% PVP (w/v) solution, respectively, for 9 days. After human chorionic gonadotropin was administered to induce the nuclear maturation of oocytes, oocytes were retrieved and the development competence of recovered oocytes treated with in vitro fertilization were observed. The plasma concentrations of follicle-stimulating hormone and ovarian responses were monitored during the treatment. The results showed that the number of recovered oocytes and the in vitro developmental competence of mature oocytes was equivalent among monkeys when treated with a single-daily treatment of 17 and 35 IU rhFSH with PVP preparation in Groups IV and V compared with the twice-daily 35 IU rhFSH treatments received by Group I. However, almost all animals in Groups II, III and VI responded poorly to corresponding stimulations. These findings indicate that a single-daily low dose of rhFSH dissolved in PVP solution can induce the satisfactory ovarian responses in rhesus monkeys. This has the potential to reduce treatment distress, stress to the animals, the labor of the operator as well as the amount of rhFSH used in ovarian stimulation, compared with traditional superovulation methods.

P-748: Post marketing surveillance analysis of the routine use of of follitropin alfa in a prefilled ready to use injection pen in ART

This report presents data from an observational study conducted in Germany on the use of the Gonal-f®1 (follitropin alfa) pre-filled pen in routine ART procedures. It focuses on the patient’s evaluation of the pen ease-of-use and acceptability. Observational study, patient questionnaire. Between April 2004 and December 2005, 40 German IVF centers participated in the study. Routine data for stimulated IVF and ICSI cycles were prospectively entered into an electronic database, and patients completed a questionnaire regarding: (1) the ease of use of the pen, and (2) the patients′ preference for the Gonal-f® pre-filled pen versus their prior experience with ampoules or vials, which have to be reconstituted, or a reusable pen with loadable cartridges. 3,522 of 35,724 prospective ART-cycles in Germany (IVF-register report 2004), one cycle per patient, were documented. Mean days of stimulation was 10.8+/-2.6 days, at a mean dose of 2,138+/-983 IU r-hFSH per patient. 2052 patients with previous gonadotropin treatment experience rated that the prefilled pen needed less training for 63.5% (n=1303), equal for 29.3% (n=602) and more for 7.2% (n=147) patients compared to the monodose or multidose vials with follitropin alfa (reconstitution required). 383 patients with prior experience using both pens judged that compared to the follitropin beta reusable pen with cartridges, the follitropin alfa ready-to-use solution in a prefilled pen device was easier to use for 59.8% (n=229), equal for 32.4% (n=124) and less easy for 7.8% (n=30) patients. From 2686 patients, 83.2% (n=2234) said they would prefer the GONAL-f® prefilled pen versus 8.9% (n=238) for the follitropin beta pen, 7.2% (n=194) for the multidose vials and 0.7% (n=20) for ampoules respectively, if future treatment was required. The routine use of follitropin alfa as a ready-to-use solution in a prefilled injection pen in ART was well accepted by patients. Patients′required less training compared to vials or ampoules and preferred the prefilled pen compared to the loadable device.

P-950: Correlation between day 3 embryo morphology and aneuploidy is not affected by type of pituitary suppression: An interim analysis

The purpose of this study was to compare the effects of pituitary suppression (agonist or antagonist), on embryo morphology as it correlates to aneuploidy status. Prospective randomized study, interim analysis. Patients aged 35-40 years with day 3 FSH <12 ml/L undergoing ART were recruited for the study. Patients were randomized to COH and IVF using GnRH antagonist (Cetrorelix acetate, Serono, Inc.), group 1 or GnRH agonist (leuprolide acetate, Tap Pharmaceuticals), group 2 for pituitary suppression. Both groups were pretreated with oral contraceptive. All patients received 250 IU r-hFSH (follitropin alpha, Serono, Inc.) and 75 IU hMG (menotropins, Serono, Inc.) daily for fives days, after which dose adjustments according to ovarian response were allowed. Group 1 began r-hFSH + hMG daily starting day 2-3 after the last OC. Cetrorelix acetate 0.25 mg was given daily once the lead follicle reached 14 mm. Group 2 began leuprolide acetate 0.5 mg daily, decreasing dose to 0.25 mg daily following pituitary desensitization. r-hFSH + hMG began day 2-3 of menses. 250 mcg r-hCG (Ovidrel®, Serono Inc.) was used to induce final oocyte maturation. Progesterone gel 8% (Serono, Inc.) was administered twice daily for luteal phase support. Day 3 embryos suitable for preimplantation genetic screening (PGS) were categorized into high or low quality based on cell number and degree of fragmentation. PGS was performed for chromosomes 13, 18, 21, X and Y. Wilcoxon test was used to analyze between treatment groups the mean number of oocytes, fertilized oocytes, embryos and aneuploidy rates. Spearman’s p correlation was utilized to assess relationship between embryo quality and aneuploidy outcome. Twenty-six patients under went treatment with antagonist (n=13) or agonist (n=13) protocols. Both treatment groups were comparable in terms of mean age, day 3 FSH, days of gonadotropin stimulation, total r-hFSH and total hMG usage, serum estradiol day of hCG and number of follicles ≥ 14mm. In the antagonist arm, oocytes retrieved, fertilized, and cleaved were 10.4 ± 5.7, 5.9 ± 3.9 (56.5%) and 6.5 ± 3.2, respectively. In the agonist arm a mean of 10.2 ± 4.3 oocytes were retrieved, 6.4 ± 3.1 (62.7%) fertilized, resulting in 6.6 ± 3.2 embryos (p>0.05). For both treatment groups, 138 embryos were assessed for morphology, 79 (56.8%) embryos were of high quality and 59 (42.8%) poor quality. Aneuploidy rate was similar, 40.7% and 42.9% for antagonist and agonist treatment groups, respectively (p>0.05). Independent of the type of pituitary suppression, high quality embryos significantly correlated with embryos normal for aneuploidy screening (p<0.0004) while poor quality embryos significantly correlated with embryos positive for aneuploidy (p<0.02). An interim analysis comparing antagonist and agonist treatment groups have not revealed significant differences between ovarian response, laboratory outcomes and aneuploidy rates. Independent of treatment regimens, high quality embryos positively correlated with normal aneuploidy outcome while poor quality embryos correlated with embryos positive for aneuploidy using a five-probe panel for chromosomes 13, 18, 21, X and Y. Our findings confirm that further evaluation of a larger powered study currently in progress should continue.

The Effect of Androgen Blockade on Granulosa Cell Estradiol Production after Follicle-Stimulating Hormone Stimulation in Women with Polycystic Ovary Syndrome

Previously, we have shown that women with polycystic ovary syndrome (PCOS) exhibit an exaggerated serum estradiol (E(2)) response to recombinant human FSH (rhFSH) (150 IU) compared with similarly treated normal women. This enhanced granulosa cell responsiveness is consistent with excessive follicular development after gonadotropin therapy and the corresponding risk of ovarian hyperstimulation syndrome. In vitro studies have shown that granulosa cells treated with androgens display greater FSH-induced E(2) production than untreated cells, suggesting a role for androgens in granulosa cell responsiveness. This study was conducted to determine whether blockade of androgen action in PCOS women by administration of the antiandrogen flutamide would alter E(2) responses to rhFSH. We conducted a prospective cohort study. We studied 11 women with PCOS at an institutional general clinical research center. On study d 1, each subject received 150 IU rhFSH iv. Frequent blood samples were obtained over 24 h. After completion of rhFSH stimulation, each subject was treated with flutamide, 125 mg, twice daily, for 6 wk. Thereafter, the rhFSH stimulation test was repeated. Baseline and stimulated E(2) levels before and after treatment were assayed. Mean baseline and maximally stimulated E(2), integrated E(2) response, and fold change in E(2) were not different before and after treatment. Levels of testosterone, androstenedione, progesterone, 17-hydroxyprogesterone, estrone, and SHBG before and after treatment were unchanged. Baseline dehydroepiandrosterone sulfate levels declined significantly after flutamide therapy. These findings indicate that in women with PCOS, the E(2) hyperresponsiveness to FSH may not be attributable to increased circulating androgens.

265. Gonadotrophic hormones affect the uterus, implantation and fetal development in mice

Although gonadotrophin stimulation using equine chorionic gonadotrophin (eCG) adversely influences pregnancy and fetal development, the effects of stimulation using recombinant human follicle stimulating hormone (rhFSH) are largely unknown. Evidence from human studies indicates that rhFSH may also be detrimental to the endometrium and implantation. We investigated the effect of gonadotrophin stimulation on ovarian hormones and uterine characteristics in the peri-implantation period, and pregnancy outcomes in mice. Adult female mice were stimulated with 2.5 IU or 10 IU rhFSH or 5 IU eCG, followed by 5 IU human chorionic gonadotrophin (hCG). Control mice received saline injections. On day 4 of pseudopregnancy mice either had embryos transferred to the uterus or were sacrificed and blood and uterine samples collected. Plasma progesterone and estradiol concentrations were determined by radioimmunoassay. Uterine mRNA levels of the estrogen and progesterone receptors (ERa and PR), leukaemia inhibitory factor (LIF), homeobox gene Hoxa10 and vascular endothelial growth factor (VEGF) were determined by real-time RT-PCR. Uterine protein distribution of PR was determined by immunohistochemistry. Embryo transfer recipients were sacrificed on day 15 to assess pregnancy outcomes. Gonadotrophin stimulation increased plasma progesterone concentration compared to controls, while estradiol concentrations were not affected. Stimulation also reduced total LIF mRNA and altered the spatial distribution of PR protein in the uterus on day 4. Embryo transfer recipients administered eCG or 10IU rhFSH had lower pregnancy rates compared to controls (11, 35 and 75% respectively) and fetuses from the rhFSH group had reduced mean weight (94 ± 6 v. 176 ± 8 mg), length (11 ± 0.2 v. 12 ± 0.1 mm) and maturity (14.1 ± 0.09 v. 14.6 ± 0.05 days) compared to controls. These results demonstrate that gonadotrophin stimulation induces changes to the maternal reproductive tract prior to implantation that have consequences for the establishment of pregnancy and fetal development in the mouse.

Effects of recombinant human follicle-stimulating hormone on embryo development in mice

We have developed a protocol using recombinant human follicle-stimulating hormone (rhFSH) to induce ovarian stimulation in the mouse to investigate its impact on preimplantation embryo development. Embryos were collected from adult female C57Bl/6 x CBA F1 mice treated with rhFSH (0, 2.5, 5.0, 10.0, or 20.0 IU) or 5 IU equine chorionic gonadotropin (eCG). Embryos were also recovered from nontreated control mice. Embryos were cultured in vitro for 88 h, and the stage of development was morphologically assessed. The allocation of cells to the inner cell mass or trophectoderm of blastocysts was determined by differential nuclear staining. The expression of insulin-like growth factor 2 (IGF-II), the insulin-like growth factor receptor (IGF-II receptor), and vascular endothelial growth factor (VEGF) in blastocysts was measured by real-time RT-PCR. Blastocyst development was reduced in the 10 (72.3 +/- 5.1%) and 20 (77.3 +/- 5.6%) IU rhFSH groups compared with control embryos (96.7 +/- 1.0%). The number of inner cell mass cells was reduced (P < 0.001) in the 5, 10, and 20 IU rhFSH groups and the eCG group compared with control embryos. We did not find any effect of rhFSH treatment on IGF-II, IGF-II receptor, or VEGF expression in blastocysts compared with the control group. eCG treatment, however, significantly increased the expression of IGF-II in blastocysts. These results indicate that ovarian stimulation with rhFSH impairs the in vitro development of preimplantation mouse embryos, and these results may have potential implications for clinical ovarian stimulation during infertility treatment and subsequent embryo quality.

Testicular Anti-Müllerian Hormone Secretion Is Stimulated by Recombinant Human FSH in Patients with Congenital Hypogonadotropic Hypogonadism

Serum anti-Mullerian hormone (AMH), a prepubertal Sertoli cell marker, declines during puberty as an early sign of testicular testosterone (T) production. When T synthesis or action is impaired, serum AMH is abnormally high in the first months after birth and at puberty but normal between these two periods. We postulated that FSH might be responsible for AMH up-regulation in the absence of androgen inhibition. To test this hypothesis, we administered recombinant human (rh) FSH to eight patients aged from 18-31 yr with untreated congenital hypogonadotropic hypogonadism. This situation is ideal to study the effect of FSH on AMH production because it avoids interference by endogenous gonadotropins and T. The patients received daily sc injections of 150 IU rhFSH for 1 month, followed in seven of them by a combined treatment of rhFSH plus human chorionic gonadotropin (hCG; 1500 UI im, twice a week) for 2 months. Gonadotropins, T, AMH, and inhibin B were measured in plasma before treatment every 10 d during rhFSH treatment and every month during combined rhFSH and hCG treatments. All hormones were at prepubertal levels before treatment. Although LH and T did not vary, AMH and inhibin B levels gradually increased after 20 d of FSH administration. However, in contrast to rhFSH alone, the combined rhFSH plus hCG stimulation of the testis dramatically suppresses the secretion of AMH and induced a modest but significant reduction of circulating inhibin B levels. We conclude that FSH stimulates AMH production in the testis when it is at a prepubertal stage. In addition, the decrease of serum AMH during combined rhFSH and hCG testicular stimulation is in agreement with the concept that during pubertal development and in adult life, the suppressive effect of LH-driven testicular androgens outweighs the stimulating effect of FSH on AMH production by Sertoli cells. Finally, the hCG-induced decrease in inhibin B suggests that in humans, as previously demonstrated in monkeys, testicular T is also able to inhibit inhibin B secretion.