Recombinant Human FSH Research Articles

Split daily recombinant human LH dose in hypogonadotrophic hypogonadism: a nonrandomized controlled pilot study

This prospective controlled nonrandomized pilot study was conducted to investigate whether split daily doses of recombinant human LH (rHLH) is more efficacious than the single daily dose in supporting follicular development and ovulation in primary hypogonadotrophic hypogonadism (HH). Twenty-seven women with HH received a 150 IU fixed daily subcutaneous dose of recombinant human FSH, supplemented by 75 IU daily dose of rHLH given either as a single dose (n=9; single-dose group) or four equally divided doses (n=18; split-dose group). Ovulation was defined by three efficacy end points: at least one follicle ⩾17mm in diameter, pre-ovulatory serum oestradiol ⩾400pmol/l and a midluteal progesterone ⩾25nmol/l. Although lacking statistical significance, the proportion of women in the rHLH split-dose group who fulfilled all three end points was higher than the single-dose group (72.2% versus 55.6%). Women in the split-dose group achieved higher serum oestradiol concentrations per follicle, endometrial thickness measurements and numbers of follicles than in the single-dose group (not statistically significant). The odds ratio for ovulation rate was 2.08 (not statistically significant). There were no serious untoward side effects. Administering rHLH in split daily doses could provide superior results compared with the traditional single daily dose.We conducted this clinical study to investigate whether a split daily dose protocol of recombinant human LH (rHLH) is more efficacious than the single daily dose in supporting follicular development and ovulation in primary hypogonadotrophic hypogonadism (HH). HH is an uncommon entity that can lead to very low or undetectable serum gonadotrophin concentrations. It manifests in anovulation, amenorrhoea and subsequent infertility. Twenty-seven women with HH received a 150 IU fixed daily subcutaneous dose of recombinant human FSH, supplemented by a 75 IU daily dose of rHLH given either as a single dose (n=9; single-dose group) or four equally divided doses (n=18; split-dose group). Ovulation was defined by these three efficacy end points: at least one follicle ⩾17mm in mean diameter, pre-ovulatory serum oestradiol concentration ⩾400pmol/l and a midluteal progesterone concentration ⩾25nmol/l. The proportion of women in the rHLH split-dose group who fulfilled all three end points was higher than the single-dose group (72.2% versus 55.6%). Women in the split-dose group achieved higher serum oestradiol concentrations per follicle, endometrial thickness measurements and numbers of follicles than in the single-dose group, without statistical significance. Women who received the split-dose regimen were more likely to have ovulation than the other group. We had no serious problematic side effects. Our results suggest that administering rHLH in split daily doses could provide superior results compared to the traditional single daily dose.

Photoperiod-gonadotropin mismatches induced by treatment with acyline or FSH in Siberian hamsters: impacts on ovarian structure and function

Many seasonal breeders time their reproductive efforts to specific times of the year to ensure adequate resources for the production and care of young. For long-day (LD) breeders, females born before the summer solstice (LDs) reach sexual maturity quickly and often breed that same year, whereas females born after the summer solstice (short days (SDs)) may delay reproductive development to the following spring when environmental conditions are favorable for reproduction. In Siberian hamsters, development in SD is associated with structural and functional differences in the ovary compared with females held in LD, including a greater number of primordial follicles and an abundance of hypertrophied granulosa cells (HGCs), which are immunoreactive for anti-Müllerian hormone. The goal of this study was to determine whether SD-induced gonadotropin suppression is responsible for these phenotypic differences. Gonadotropin levels were suppressed in LD hamsters using the GNRH antagonist acyline. Conversely, to determine whether the SD ovarian phenotype is completely reversed by gonadotropin stimulation, recombinant human FSH (rhFSH) was administered. Our treatments were successful in mimicking FSH concentrations of the opposite photoperiod, but they did not produce a comparable change in the ovarian phenotype. Most notable was the lack of HGCs in the ovaries of acyline-treated LD females. Similarly, HGCs were maintained in the ovaries of SD females treated with rhFSH. Our data suggest that gonadotropins alone do not account for the SD ovarian phenotype. Future studies will determine whether SD-induced changes in other factors underlie these phenotypic changes.

Thyrotropic activity of recombinant human glycoprotein hormone analogs and pituitary mammalian gonadotropins in goldfish (Carassius auratus): Insights into the evolution of thyrotropin receptor specificity

Thyrotropin (TSH) is a pituitary glycoprotein hormone heterodimer that binds to its G-protein coupled receptor (TSH-R) at the thyroid to promote the synthesis and secretion of thyroid hormone. Very little is known about TSH–TSH-R interactions in teleost fish. Mammalian gonadotropins have been reported to have an intrinsic ability to activate teleost fish TSH-Rs, suggesting the TSH-R in teleost fish is more promiscuous than in other vertebrates. In this study we utilized the goldfish T4-release response and recombinant human TSH analogs as in vivo tools to evaluate the structural constraints on hormone–receptor interactions. We found that four positively charged lysines substituted for neutral or negatively charged amino acids within positions 11–20 of the glycoprotein hormone subunit α (GSUα) significantly increased biological activity of hTSH in fish, as it does in mammals. We further found that bovine follicle stimulating hormone but not luteinizing hormone, whose GSUα subunits also contain four lysine or arginine amino acid residues in the N-terminal portion of GSUα, was thyrotropic in goldfish, suggesting gonadotropin β subunit contributes to the heterothyrotropic activity. Though recombinant human FSH did not produce a dose-dependent increase in T4, thyrotropic activity could be acquired with the addition of positively charged amino acids at the N-terminal portion of its GSUα, confirming the importance of the charge on those amino acids for activation of the goldfish TSH-R. These studies demonstrate that mammalian glycoprotein hormone analogs can be utilized to evaluate the conservation of receptor binding and activation mechanisms between fish and mammals.

The Available Time Window for Embryo Transfer in the Rhesus Monkey (Macaca mulatta)

Much effort has been focused on improving assisted reproductive technology procedures in humans and nonhuman primates (NHPs). However, the pregnancy rate after embryo transfer (ET) has not been satisfactory, indicating that some barriers still need to be overcome in this important procedure. One of the key factors is embryo–uterine synchronicity, which is little known in NHPs. The objective of this study was to investigate the available ET time window in rhesus monkey (Macaca mulatta). Eighty-two adult female rhesus monkeys were superovulated with recombinant human FSH. Ovarian phases were identified according to estrogen (E2) and progesterone (P4) levels as well as ovarian examination by ultrasonography and laparoscopy. A total of 259 embryos were transferred by the laparoscopic approach into the oviducts of 63 adult female monkeys. Ovarian phases were divided into late follicular and early luteal phases. Similar pregnancy rates (30–36.4%) were obtained from recipients receiving ET either in their late follicular or early luteal phases, regardless of embryo developmental stages. This study indicates that the available time window for ET in rhesus monkeys is from the late follicular to early luteal phases.

PDB42 Economic Evaluation of Recombinant Human FSH in Comparison with Urinary HMG in Assisted Reproduction in the Greek Setting

PDB42 Economic Evaluation of Recombinant Human FSH in Comparison with Urinary HMG in Assisted Reproduction in the Greek Setting

Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin

An alginate-based matrix supports the three-dimensional (3D) architecture of non-human primate follicles and, in the presence of FSH, permits the in vitro development of pre-antral follicles to the small antral stage, including the production of ovarian steroids and paracrine factors. The current study investigated the ability of gonadotrophins, fetuin and oxygen (O₂) to improve primate follicle growth and oocyte maturation in vitro. Macaque secondary follicles were isolated from the early follicular phase ovaries, encapsulated in a sodium alginate matrix and cultured individually for 40 days in supplemented medium. The effects of recombinant human (rh) FSH (15, 3 and 0.3 ng/ml for high, medium and low FSH, respectively), bovine fetuin (1 or 0 mg/ml) and O₂ (5 or 20% v/v) were examined. Half of the follicles in each culture condition received rhLH on Day 30-40. Follicles that reached antral stage were treated with rh chorionic gonadotrophin for 34 h to initiate oocyte meiotic maturation. Media were analyzed for ovarian steroids and anti-müllerian hormone (AMH). Improved culture conditions supported non-human primate, secondary follicle growth to the antral stage and, for the first time, promoted oocyte maturation to the MII stage. In the presence of fetuin at 5% O₂, follicles had the highest survival rate if cultured with high or medium FSH, whereas follicles grew to larger diameters at Week 5 in low FSH. Oocyte health and maturation were promoted under 5% O₂. High FSH stimulated steroid production by growing follicles, and steroidogenesis by follicles cultured with low FSH was promoted by LH. AMH biosynthesis was elevated with high compared with low FSH and for longer under 5% O₂ than under 20% O₂. This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

Suboptimal response to GnRHa long protocol is associated with a common LH polymorphism

The aim of this observational preliminary trial was to estimate the association between the most common polymorphism of LH (LH-β variant: v-βLH), with different profiles of ovarian response to recombinant human FSH (rhFSH). A total of 60 normogonadotrophic patients undergoing a gonadotrophin-releasing hormone analogue long down-regulation protocol followed by stimulation with recombinant human FSH (rhFSH) for IVF/intracytoplasmic sperm injection, and in whom at least five oocytes were retrieved were retrospectively included. On the basis of the total rhFSH consumption, patients were divided into three groups: Group A: 22 women requiring a cumulative dose of rhFSH >3500 IU; Group B: 15 patients requiring 2000-3500 IU; Group C (control): 23 women requiring <2000 IU. The presence of v-βLH was evaluated using specific immunoassays. Peak oestradiol concentrations were significantly lower in Group A when compared with both groups B ( P < 0.05) and C ( P < 0.001). Group A had a significantly lower ( P < 0.05) number of oocytes retrieved (7.3 ± 1.5, 11.7 ± 2.4 and 14.7 ± 4.1 in the three groups, respectively). Seven carriers (31.8%) of v-βLH were found in Group A, whereas only one variant (6.7%) was observed in Group B; no variant was detected in Group C. These preliminary results suggest that v-βLH is more frequent in women with ovarian resistance to rhFSH.

Mid-follicular LH supplementation in women aged 35–39 years undergoing ICSI cycles: a randomized controlled study

This single-centre, randomized, parallel group, comparative study aimed to identify potential benefits of mid-follicular recombinant human LH (r-HLH) supplementation in women aged 35-39 years undergoing ovarian stimulation for intracytoplasmic sperm injection (ICSI). The main endpoint was the number of metaphase II oocytes retrieved. After pituitary suppression with a gonadotrophin-releasing hormone agonist, ovarian stimulation was initiated with recombinant human FSH (r-HFSH; 300-450 IU/day). On stimulation day 6, patients were randomized to receive r-HFSH alone or r-HFSH + r-HLH (r-HLH 150 IU/day) for the remainder of the stimulation period. Final follicular maturation was triggered with 250 μg of recombinant human chorionic gonadotrophin. After assessing oocyte nuclear maturity, oocyte were fertilized by ICSI and afterwards embryo quality was analyzed. Of the 131 women enrolled, 68 were allocated to r-HFSH alone and 63 to r-HFSH + r-HLH. No significant differences were observed in markers of either oocyte or embryo quality or quantity. However, higher rates of implantation and live birth per started cycle were observed with r-HLH supplementation than with r-HFSH alone. Although additional large studies are required to further investigate these findings, r-HLH supplementation for women aged 35-39 years undergoing ICSI is recommended as it may have a beneficial action on implantation.

Individualizing FSH dose for assisted reproduction using a novel algorithm: the CONSORT study

The CONSORT dosing algorithm individualizes recombinant human FSH (r-hFSH) doses for assisted reproduction technologies, assigning 37.5 IU increments according to patient characteristics: basal FSH, body mass index, age and antral follicle count. A prospective, uncontrolled, international, 18-centre, pilot study of normo-ovulatory women aged 18-34 years inclusive undergoing a long agonist treatment protocol was performed. Follitropin alfa filled-by-mass (GONAL-f®) dose was assigned by the algorithm and was intended to be altered only for risk of ovarian hyperstimulation syndrome (OHSS). Primary end-point was number of oocytes retrieved. Dose groups containing ≥5 patients were analysed: 75 IU (n = 48), 112.5 IU in = 45), 150 IU (n = 34), 187.5 IU (n = 24), 225 IU (n = 10). Cancellations due to inadequate response were higher than expected in the 75 IU group (12/48). Overall, a median of 9.0 oocytes were retrieved (8.5, 8.0, 10.0, 12.0 and 8.0 in the 75, 112.5, 150, 187.5 and 225 IU groups respectively). Clinical pregnancy rates/cycle started were 31.3, 31.1, 35.3, 50.0 and 20.0%, respectively (overall, 34.2%). Two patients had severe OHSS. Use of the CONSORT algorithm achieved an adequate oocyte yield and good pregnancy rates in this preliminary study. Adjustment of the algorithm could reduce cancellation rates.

Abdominal mesotherapy injection extended the absorption of follicle-stimulating hormone

Abdominal mesotherapy injection of recombinant human FSH (rhFSH) was well tolerated with increased net absorption (AUC0-∞ 4,655.3 IU·h/L and t1/2 247.6 h) up to 360 hours compared with those of 120 hours (AUC0-∞ 1,915.7 IU·h/L and t1/2 101.8 h). The extended absorption of rhFSH suggests that abdominal mesotherapy injection mode be considered for future administration of rhFSH in controlled ovarian hyperstimulation.

GnRH antagonist multiple dose protocol with oral contraceptive pill pretreatment in poor responders undergoing IVF/ICSI

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

Effect of levothyroxine treatment on in vitro fertilization and pregnancy outcome in infertile women with subclinical hypothyroidism undergoing in vitro fertilization/intracytoplasmic sperm injection

To investigate whether levothyroxine (LT4) treatment has beneficial effects on IVF results and pregnancy outcome in infertile patients with subclinical hypothyroidism undergoing IVF/intracytoplasmic sperm injection (ICSI). Prospective, randomized trial. University-affiliated infertility clinic. A total of 64 infertile patients with subclinical hypothyroidism, defined as an elevated serum TSH level associated with a normal free T4 level and without frank symptoms of hypothyroidism. Patients were randomized into an LT4 treatment group or control group. For the LT4 treatment group, 50 μg LT4 was administered from the first day of controlled ovarian stimulation for IVF/ICSI. Results of IVF and pregnancy outcome. There were no differences in patient characteristics between the two groups. Total dose and days of recombinant human FSH used for controlled ovarian stimulation were also similar. The number of grade I or II embryos was significantly higher in the LT4 treatment group than in the control group. There was no significant difference in the clinical pregnancy rate per cycle between the two groups. However, the miscarriage rate was significantly lower in the LT4 treatment group than in the control group. Embryo implantation rate and live birth rate were significantly higher in the LT4 treatment group. In the control group, both thyroid peroxidase antibody and thyroglobulin antibody levels were significantly higher in the miscarried subgroup than in the delivered subgroup. LT4 treatment can improve embryo quality and pregnancy outcome in subclinical hypothyroid women undergoing IVF/ICSI.

269 EXPRESSION PATTERN OF STRESS MARKER GENES IN BOVINE OOCYTES MATURED IN VITRO IN MEDIA WITH DIFFERENT HORMONE COMPOSITION

Evaluation and prediction of oocyte quality are 2 critical issues in assisted reproductive technologies. The lack of reliable and objective predictors of oocyte developmental competence, and subsequent embryonic development, negatively affects the efficiency of assisted reproductive technologies. The aim of this work was to study the effect of different bovine oocyte maturation protocols on the relative transcript abundance from genes involved in cellular stress (70-kDa heat shock proteins), endoplasmic reticulum (ER) stress (Bip: immunoglobulin heavy chain binding protein and proteasome subunit β-5 protease), and apoptosis (Bax and Caspase-3). Oocytes were matured in 5 different media that varied in the composition of hormones and cysteamine: 1) porcine FSH (Folltropin®, Bioniche Animal Health, Belleville, Ontario, Canada) and cysteamine, 2) epidermal growth factor (EGF), 3) EGF and cysteamine, 4) recombinant human FSH (rhFSH), and 5) rhFSH and cysteamine. Three groups of 10 matured oocytes in each of the 5 different maturation media were analysed by real-time RT-PCR. The results were normalized to the expression of the endogenous control (glyceraldehyde-3-phosphate dehydrogenase). Messenger RNA abundance data were analysed using the InfoStat software (Universidad Nacional de Córdoba, Argentina). One-way ANOVA, followed by Tukey multiple comparison test, was used for the analysis of differences in mRNA abundance. The abundance of the β-5 subunit of the proteasome mRNA increased 70% (P &lt; 0.05) in oocytes matured in medium supplemented with porcine FSH and cysteamine compared with that in the oocytes matured in the other maturation media supplemented with rhFSH and EGF with or without cysteamine. The expression of the ER chaperone, Bip, increased 120% (P &lt; 0.05) in oocytes that were matured in the same medium, compared with that in those matured in medium supplemented with rhFSH and cysteamine. The results suggest that maturation of oocytes in medium supplemented with porcine FSH and cysteamine induced ER stress, which is reflected by the increased abundance of the chaperone Bip and β-5 protease. Given that the abundance of the cytoplasmic chaperone Hsp70 was not altered in any of the treatments, there was no evidence of widespread cellular stress. However, it seemed that these conditions were not stressful enough to induce apoptosis because Bax and Caspase-3 markers were not modified. This study could complement morphological analysis to help develop an effective and accurate technique to diagnose oocyte quality during assisted reproductive technologies.

253 OOCYTE PREMATURATION IN THE PRESENCE OF MILRINONE IMPROVES NUCLEAR BUT NOT CYTOPLASMIC MATURATION OF MACAQUE OOCYTES

During oocyte growth chromatin configuration of the germinal vesicle (GV) oocyte undergoes modification in relation to changes in transcriptional activity crucial for conferring meiotic as well as developmental competence on the oocyte. In the macaque oocyte, there are 3 distinct GV states: GV1, noncondensed chromatin; GV2, an intermediate state; and GV3, condensed chromatin. The aim of this study was to test the effects of a prematuration culture (PMC) system, using the phosphodiesterase type 3 inhibitor milrinone (MIL), on the synchronization of GV chromatin to the GV3 stage and assess metaphase II (MII) oocyte reduced glutathione (GSH) content as a measure of cytoplasmic maturation. Reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. To assess the effect of PMC on GV chromatin status, immature oocytes retrieved from unstimulated ovaries were either fixed (2% paraformaldehyde+0.1% Triton-X100) immediately after follicular aspiration (t = 0) or after culture in a humidified atmosphere of 6% CO2 in air at 37°C for 24 h in modified Connaught Medical Research Laboratories medium (mCMRL) supplemented with 10% FCS (Hyclone, Logan, UT, USA) and 12.5 μM MIL in the absence (MILNil) or presence of 1.0 IU of FSH (MILFSH). For chromatin assessment, fixed GV oocytes were stained with 5 μg mL–1 of 4′,6-diamidino-2-phenylindole (Molecular Probes, Leiden, the Netherlands) and imaged using confocal microscopy. Following PMC, MILFSH oocytes were transferred to fresh mCMRL+FCS supplemented with 1.0 IU of recombinant human FSH and 1.0 IU of hLH and cultured for a further 30 h. Control and MILFSH oocytes were denuded of cumulus cells and assessed for maturation. The MII oocytes were prepared for GSH analysis, and total GSH content was determined using a commercial 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB)-GSH reductase recycling assay kit (North-West Life Science). The MII rates were compared using chi-square. Differences in oocyte GSH content were compared using t-test. Significant differences were determined at P &lt; 0.05. There was no significant difference in the proportion of oocytes remaining at the GV stage following 24 h of PMC in MILNil or MILFSH (42/44, 96% v. 32/35, 91%, respectively). However, there was a significant reduction in GV1 chromatin (15/49, 31% v. 28/54, 52% and 22/58, 38%) and a significant increase in GV3 chromatin (23/49, 47% v. 14/54, 26% and 16/58, 28%) observed in MILFSH oocytes compared with both MILNil and t = 0 oocytes, respectively. The MII rate of MILFSH oocytes following in vitro maturation was significantly higher compared with the MII rate of control in vitro matured oocytes (91/167, 55% v. 83/243, 34%). There was no significant difference in the GSH content of GV oocytes from the time of oocyte collection (t = 0) or GV oocytes following PMC in MILFSH (3.69 ± 0.16 and 4.14 ± 0.28 pmol/oocyte, n = 39–49 oocytes). The GSH content of control in vitro matured MII oocytes was significantly greater than that of MILFSH-treated MII oocytes (3.13 ± 0.16 v. 2.02 ± 0.04 pmol/oocyte, n =53–54 oocytes). The PMC supported high rates of nuclear maturation, but cytoplasmic maturation, assessed by GSH content, was negatively affected. Further assessment following fertilization and development is required to determine the practical utility of PMC in a primate in vitro maturation setting.

Dynamic endocrine test of inhibin B and anti-Müllerian hormone in men with non-obstructive azoospermia

Although both serum inhibin B (Inh-B) and anti-Müllerian hormone (AMH) concentrations have been proposed as markers of spermatogenesis in men with subfertility, there are wide overlaps with fertile controls. The main aim of this study was to evaluate stimulated serum Inh-B and AMH concentrations in men with non-obstructive azoospermia (NOA). Thirty-seven men with NOA confirmed by testicular fine-needle aspiration and 17 fertile controls participated at this prospective, case–control study. All subjects underwent the Exogenous FSH SErtoli Reserve Test (EFSERT): estimation of serum Inh-B and AMH concentrations before, 24 and 48 hours after administration of 300 IU human recombinant FSH (hrFSH). Basal serum Inh-B and AMH concentrations, as well as Inh-B concentrations at 24 and 48 h were lower in men with NOA as compared to controls. No changes in Inh-B or AMH concentrations were recorded throughout the EFSERT in either men with NOA or controls nor when men with NOA were classified according to their clinical, hormonal and cytological diagnosis. Thus, stimulated serum concentrations of Inh-B and AMH, as obtained by an EFSERT, do not contribute to the diagnostic evaluation of the men with NOA, as the same information can be acquired by the basal serum concentrations of these hormones.

In vitro maturation (IVM) and parthenogenesis of adult murine primary ovarian follicles is ameliorated in the absence of anti-mÜllerian hormone (AMH)

OBJECTIVE: To improve the IVM outcome of adult primary ovarian follicles in a murine model with the emphasis on the role of AMH in folliculogenesis.DESIGN: Primary(1°) and secondary(2°) ovarian follicles were isolated from adult mice and cultured in different serum and AMH conditions. The competence of the oocytes grown was assessed through parthenogenesis followed by cleavage and blastocyst formation.MATERIALS AND METHODS: 1° and 2° follicles were isolated from CBA/BL6 F1 mice at 9 weeks. 1° follicles were grown in droplets of aMEM with 1% ITS, 10 mIU/mL of LH, 100 mIU/mL of recombinant human (rh) FSH, with the following conditions 1)FBS containing AMH from male fetuses, 2)female FBS, and 3)female FBS with 5ug/mL of rhAMH. 2° follicles were grown in aMEM with 1% ITS, 100 mIU/mL of rhFSH in the same serum and AMH conditions as 1° follicles. Maturation was performed with hCG and EGF. Parthenogenesis was performed in Ca2+-free KSOM with SrCl2 and CCB and cultured in CZB medium up to 5 days for blastocyst development.RESULTS: 1° follicle growth into 2° follicles was enhanced with female FBS compared to unsexed FBS(86.4% : 64.9% p<0.01) and blocked by AMH. MII yield, in the female FBS group was improved in comparison with those cultured in unsexed FBS for both 1° and 2° follicles(69.5% : 50.9% p<0.01; 80.9% : 63.5% p<0.01). For 2° follicles, AMH significantly compromised MII oocytes yield(80.9% : 33.3 p<0.01) but the complete blockage observed in 1° follicles did not occur. The yield of competent oocytes via parthenogenesis was improved in both the 1° and 2° follicles(26.6% : 15.1% p<0.05; 57.2% : 30.4% p<0.05) when cultured in female FBS and adversely affected by AMH. 2 blastocysts were derived from 85 2° follicles in female FBS and 1 blastocyst out of 89 2° follicles in unsexed FBS.CONCLUSION: AMH plays a vital role in suppressing adult 1° to 2° follicle development in vitro. The yield of primary follicle growth, maturation, and developmental competence of oocytes is significantly improved in conditions of absent AMH. OBJECTIVE: To improve the IVM outcome of adult primary ovarian follicles in a murine model with the emphasis on the role of AMH in folliculogenesis. DESIGN: Primary(1°) and secondary(2°) ovarian follicles were isolated from adult mice and cultured in different serum and AMH conditions. The competence of the oocytes grown was assessed through parthenogenesis followed by cleavage and blastocyst formation. MATERIALS AND METHODS: 1° and 2° follicles were isolated from CBA/BL6 F1 mice at 9 weeks. 1° follicles were grown in droplets of aMEM with 1% ITS, 10 mIU/mL of LH, 100 mIU/mL of recombinant human (rh) FSH, with the following conditions 1)FBS containing AMH from male fetuses, 2)female FBS, and 3)female FBS with 5ug/mL of rhAMH. 2° follicles were grown in aMEM with 1% ITS, 100 mIU/mL of rhFSH in the same serum and AMH conditions as 1° follicles. Maturation was performed with hCG and EGF. Parthenogenesis was performed in Ca2+-free KSOM with SrCl2 and CCB and cultured in CZB medium up to 5 days for blastocyst development. RESULTS: 1° follicle growth into 2° follicles was enhanced with female FBS compared to unsexed FBS(86.4% : 64.9% p<0.01) and blocked by AMH. MII yield, in the female FBS group was improved in comparison with those cultured in unsexed FBS for both 1° and 2° follicles(69.5% : 50.9% p<0.01; 80.9% : 63.5% p<0.01). For 2° follicles, AMH significantly compromised MII oocytes yield(80.9% : 33.3 p<0.01) but the complete blockage observed in 1° follicles did not occur. The yield of competent oocytes via parthenogenesis was improved in both the 1° and 2° follicles(26.6% : 15.1% p<0.05; 57.2% : 30.4% p<0.05) when cultured in female FBS and adversely affected by AMH. 2 blastocysts were derived from 85 2° follicles in female FBS and 1 blastocyst out of 89 2° follicles in unsexed FBS. CONCLUSION: AMH plays a vital role in suppressing adult 1° to 2° follicle development in vitro. The yield of primary follicle growth, maturation, and developmental competence of oocytes is significantly improved in conditions of absent AMH.

Survival, growth, and maturation of secondary follicles from prepubertal, young, and older adult rhesus monkeys during encapsulated three-dimensional culture: effects of gonadotropins and insulin

A three-dimensional culture system supports the development of primate preantral follicles to the antral stage with appreciable steroid production. This study assessed i) whether in vitro developmental competence of follicles is age dependent, ii) the role of gonadotropins and insulin in supporting folliculogenesis, and iii) anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) production by growing follicles. Ovaries were obtained from prepubertal, young, and older adult rhesus macaques. Secondary follicles were encapsulated into alginate beads and cultured individually for 40 days in media containing 0.05 or 5 μg/ml insulin, with or without recombinant human (rh) FSH (500 mIU/ml). No follicles survived in the culture without rhFSH. In the presence of rhFSH, survival was lower for follicles from older animals, whereas growth, i.e. follicle diameter, was less by day 40 for follicles from prepubertal animals. The surviving follicles were categorized as no-grow (NG; ≤ 250 μm), slow-grow (SG; 250-500 μm), and fast-grow (FG; ≥ 500 μm) according to their diameters. SG follicles cultured with 5 μg/ml insulin produced more ovarian steroids than those cultured with 0.05 μg/ml insulin by week 5. SG and FG follicles produced more AMH and VEGF than the NG, and levels peaked at weeks 2 and 5 respectively. After 100 ng/ml rh chorionic gonadotropin treatment for 34 h, more healthy oocytes were retrieved from young adults whose follicles were cultured with 5 μg/ml insulin. This culture system offers an opportunity to characterize the endocrine and paracrine function of primate follicles that influence follicle growth and oocyte maturation.

HP-HMG versus rFSH in treatments combining fresh and frozen IVF cycles: success rates and economic evaluation

The economic implications of the choice of gonadotrophin influence decision making but their cost-effectiveness in frozen-embryo transfer cycles has not been adequately studied. An economic evaluation was performed comparing highly purified human menopausal gonadotrophin (HP-HMG) and recombinant FSH (rFSH) using individual patient data (n=986) from two large randomized controlled trials using a long agonist IVF protocol. The simulation model incorporated live birth data and published UK costs of IVF-related medical resources. After treatment for up-to-three cycles (one fresh and up to two subsequent fresh or frozen cycles conditional on availability of cryopreserved embryos), the cumulative live birth rate was 53.7% (95% CI 49.3-58.1%) for HP-HMG and 44.6% (40.2-49.0%) for rFSH (OR 1.44, 95% CI 1.12-1.85; P<0.005). The mean costs per IVF treatment for HP-HMG and rFSH were pound5393 ( pound5341-5449) and pound6269 ( pound6210-6324), respectively (number needed to treat to fund one additional treatment was seven; P<0.001). With maternal and neonatal costs applied, the median cost per IVF baby delivered with HP-HMG was pound11,157 ( pound11,089-11,129) and pound14,227 ( pound14,183-14,222) with rFSH (P<0.001). The cost saving using HP-HMG remained after varying model parameters in a probabilistic sensitivity analysis.

Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo

Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG +/- FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs.

Corifollitropin alfa in a long GnRH agonist protocol: proof of concept trial

Fifty healthy women, aged 18-39 years with no known risk of ovarian hyperstimulation were treated with 100 or 150 μg corifollitropin alfa (dependent on body weight) in a long GnRH agonist protocol. At these doses, corifollitropin alfa initiated and supported growth of a large cohort of follicles during the first week of ovarian stimulation.