Internal carbohydrate complexity of the oligosaccharide chains of recombinant human follicle stimulating hormone (Puregon, Org 32489): a comparison with Metrodin and Metrodin-HP

Glycoforms of recombinant human follicle stimulating hormone (rhFSH) (Org 32489, Puregon) were characterized using concanavalin A lectin affinity chromatography to reveal information about the internal carbohydrate complexity (extent of carbohydrate side-chain branching) of the preparations. The rhFSH glycoforms were measured by radioimmunoassay and a two-site immunoradiometric assay and compared with those in two urinary preparations (Metrodin and Metrodin-HP) used in assisted reproduction programmes and a urinary FSH international standard 70/45 (uFSH IS 70/45). Similar data were obtained with both assays; rhFSH had 6% complex internal carbohydrate structures compared with 22-27% for Metrodin, Metrodin-HP and uFSH. The proportion of simple carbohydrate structures was also different, with rhFSH having 18.5 compared with 4.5-9.3% for Metrodin, Metrodin-HP and uFSH. A linear relationship was observed between the percentage glycoforms with an isoelectric point (pl) < 4 and the log percentage simple forms (logarithmic regression; r = 0.93) indicating a direct relationship between carbohydrate complexity and charge heterogeneity. In summary, rhFSH contains fewer complex forms and an increased proportion of simple carbohydrate structures in comparison with Metrodin, Metrodin-HP and IS 70/45.     

The effect of recombinant follicle stimulating hormone (Gonal-F) on endogenous luteinizing hormone secretion in women

Parenteral administration of follicle stimulating hormone (FSH) has been shown to lower luteinizing hormone (LH) concentrations in women undergoing ovulation induction. This study was designed to explore the physiological mechanism of this effect. Seven healthy women were recruited into a double-blind placebo-controlled study. LH secretion, after the administration of variable i.v. boluses (37.5, 75 and 150 IU) of recombinant FSH (Gonal-F), was evaluated. LH was measured at 10 min intervals for 2 h before and 4 h after the FSH/placebo infusion. LH pulse frequency and amplitude were evaluated and there was no significant difference between control and trial cycles for each subject. A linear regression analysis revealed that in the group receiving 150 IU FSH, the mean plasma LH concentration decreased significantly due to a reduction tonic LH secretion. This could be a result of the suppression of secretion or an alteration of clearance. This decrease was not seen in the other dosage groups, revealing that above a dosage threshold, FSH reduced non-pulsatile LH secretion. Therefore the effect of FSH in this study exposed the likely presence of two components of LH concentration: an FSH-sensitive, non-pulsatile tonic secretion and a gonadotrophin-releasing hormone-stimulated, pulsatile release that is unaffected by FSH. Although an indirect effect involving ovarian regulation is not excluded, the rapidity of the effect suggests that FSH acts directly on the pituitary gland.     

General pharmacology studies on recombinant human follicle stimulating hormone

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein secreted by anterior pituitary cells. Its main actions are to lead ovarian follicles to maturation and to maintain spermatogenesis. Up to now the FSH preparations used in clinical practice (CAS 9002-68-0 and CAS 97048-13-0) have been purified from human postmenopausal urine. Only recently, a product was successfully obtained by recombinant-DNA technology, r-hFSH (Gonal-FTM). This recombinant protein is highly pure and has a very high specific activity. In view of its clinical use, this hormone has been submitted to an extensive panel of general pharmacology studies with the aim of defining its pharmacological profile and determine possible side effects not related to the main therapeutic action. Subcutaneous or intravenous doses of 5 to 500 IU/kg were assayed in several tests for their effects in vivo on various systems in different animal species. The substance under study was also tested in vitro on isolated guinea-pig ileum preparations at final concentrations of 0.05 to 2 IU/ml of bath. The results of this study showed that r-hFSH does not influence the general activity and behaviour of mice, as measured by the multidimensional Irwin's test. Similarly, the drug was not found to affect the normal body temperature in rats nor the locomotor activity in mice for as long as 7 h post-injection; in addition, it was not found to induce pharmacologically significant alterations of the cardiovascular and respiratory parameters in rats and dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant follicle stimulating hormone (rFSH; Puregon) in assisted reproduction: more oocytes, more pregnancies. Results from five comparative studies

The clinical assessment of recombinant follicle stimulating hormone (rFSH; Puregon) in assisted reproduction technologies such as in-vitro fertilization (IVF) has probably been the most extensive clinical trial programme ever performed for the evaluation of a new fertility drug. It started with a pilot study to evaluate the potential of rFSH to stimulate the ovaries in the absence of luteinizing hormone (LH) using various gonadotrophin-releasing hormone (GnRH) agonists. After it became clear that FSH-induced steroidogenesis was not jeopardized even after severe pituitary suppression, comparisons between rFSH and urinary FSH or human menopausal gonadotrophins were made using different GnRH agonists or no agonists at all. In addition, the effects of the route of administration (s.c. or i.m.) were assessed. The study with the strongest statistical power to truly assess clinically relevant differences between rFSH and urinary FSH included approximately 1000 patient cycles. It indicated that after rFSH treatment, significantly more oocytes were retrieved, more embryos obtained and, as a result, more pregnancies achieved when the results of the cryoprogramme were included.     

First established pregnancy after controlled ovarian hyperstimulation with recombinant follicle stimulating hormone and the gonadotrophin-releasing hormone antagonist ganirelix (Org 37462)

This case report describes the first established pregnancy after the use of gonadotrophin-releasing hormone (GnRH) antagonist, ganirelix (Org 37462; Organon), to prevent a premature luteinizing hormone surge during ovarian hyperstimulation with recombinant human follicle stimulating hormone (rhFSH). The pregnancy progressed normally and ended with the birth of a healthy boy and a girl after an elective Caesarean section at gestational age of 37 weeks. This case illustrates, for the first time, the use of a GnRH antagonist in combination with a pure FSH preparation for ovarian stimulation.     

Luteinizing hormone response to gonadotrophin-releasing hormone in normal women undergoing ovulation induction with urinary or recombinant follicle stimulating hormone

Oestradiol enhances pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) in normal women, while in women undergoing ovulation induction the putative factor gonadotrophin surge attenuating factor (GnSAF) attenuates the response of luteinizing hormone (LH) to GnRH. To study the relationships between oestradiol and GnSAF during ovulation induction, 15 normally ovulating women were investigated in an untreated spontaneous cycle (control, first cycle), in a cycle treated with daily i.m. injections of 225 IU urinary follicle-stimulating hormone (FSH) (Metrodin HP, uFSH cycle) and in a cycle treated with daily s.c. injections of 225 IU recombinant FSH (Gonal-F, rFSH cycle). Treatment with FSH started on cycle day 2. The women during the second and third cycle were allocated to the two treatments in an alternate way. One woman who became pregnant during the first treatment cycle (rFSH) was excluded from the study. In all cycles, an i.v. injection of 10 microg GnRH was given to the women (n = 14) daily from days 2-7 as well as from the day on which the leading follicle was 14 mm in diameter (day V) until mid-cycle (n = 7). The response of LH to GnRH at 30 min (deltaLH), representing pituitary sensitivity, was calculated. In the spontaneous (control) cycles, deltaLH values increased significantly only during the late follicular phase, i.e. from day V to mid-cycle, at which time they were correlated significantly with serum oestradiol values (r = 0.554, P < 0.01). Initially during the early follicular phase in the uFSH and the rFSH cycles, deltaLH values showed a significant decline which was not related to oestradiol (increased GnSAF bioactivity). Then, deltaLH values increased significantly on cycle day 7 and further on day v with no change thereafter up to mid-cycle. On these two days, deltaLH values were correlated significantly with serum oestradiol values (r = 0.587 and r = 0.652 respectively, P < 0.05). During the pre-ovulatory period, deltaLH values in the FSH cycles were significantly lower than in the spontaneous cycles. Significantly higher serum FSH values were achieved during treatment with uFSH than rFSH. However, serum values of oestradiol, immunoreactive inhibin, and deltaLH as well as the number of follicles > or = 12 mm in diameter did not differ significantly between the two FSH preparations. These results suggest that in women undergoing ovulation induction with FSH, oestradiol enhances pituitary sensitivity to GnRH, while GnSAF exerts antagonistic effects. The rFSH used in this study (Gonal-F) was at least as effective as the uFSH preparation (Metrodin-HP) in inducing multiple follicular maturation in normally cycling women.     

Improved oocyte quality is obtained with follicle stimulating hormone alone than with follicle stimulating hormone/human menopausal gonadotrophin combination

The aim of this study was to compare the efficacy of pure follicle stimulating hormone (FSH) with that of FSH/human menopausal gonadotrophin (HMG) combination in downregulated cycles. A total of 357 patients was evaluated retrospectively. Sixty percent of patients in the FSH group and 55% in the FSH/HMG group were new; the others were repeat patients. Ovulation was suppressed with leuprolide acetate in all patients, followed by either FSH (n = 218) or FSH/HMG (n = 119). There was no difference in patients' age, infertility factors, number of ampoules used, length of stimulation, oestradiol levels on day of human chorionic gonadotrophin (HCG) administration, number of oocytes recovered or the number of embryos transferred. Also, nuclear maturity at aspiration and fertilization rates were not different between the two groups. FSH stimulation resulted in a significantly higher percentage of mature oocytes that showed the typical 'mature' morphological characteristics (P < 0.0001). The clinical pregnancy rates per transfer were 40 and 28% in patients stimulated with pure FSH and FSH/HMG respectively (P < 0.05). The significantly higher number of immature oocytes matured in vitro in the FSH/HMG group (P = 0.001) suggests a possible effect on in-vitro maturation, due to luteinizing hormone present in HMG. The difference in mature oocyte quality may be an important determinant in the higher pregnancy rates for the FSH-stimulated patients.     

Structure-function relationship of follicle-stimulating hormone and its receptor

Follicle stimulating hormone (FSH) is one of the two pituitary gonadotrophins involved in the regulation of gonadal function. Structurally, this gonadotrophin is a heterodimer composed of two non-covalently associated subunits containing several heterogenous oligosaccharide residues which play an important role in both the in-vivo and in-vitro bioactivity of the hormone. Its cognate receptor, which belongs to the superfamily of the G protein-linked cell surface receptors, also displays a high degree of functional and molecular complexity. Studies employing monoclonal antibodies, synthetic peptides and/or site directed mutagenesis, have unveiled some of the multiple structural determinants involved in FSH and FSH receptor function and interaction. Despite their structural complexity, both molecules exhibit a high degree of plasticity and diversity that allows formation of distinct ligand-receptor complexes capable of selectively activating or deactivating a variety of signalling pathways. Knowledge and mapping of the structural determinants and functional epitopes for intra- and extracellular hormone action are of paramount importance not only for a better and more detailed understanding of the molecular basis of FSH action and FSH receptor function but also for the rational design of analogues with predicted properties and effects.     

Radioimmunologic method for determining serum follicle stimulating hormone in humans

Three methods of treatment of acute transverse myelitis are presented. The first group of 16 patients were treated with antibiotics (average age 32.1). Three patients died, 11 remained unrecovered, while in two cases remarkable improvement was recorded. The second group of 15 patients, whose average age was 32.4, were treated with corticosteroid per os or instrumuscularly. Four of the patients died (three of them with ascendent course of disease), there was no change in five cases, while marked improvement ("cured" and "walking with assistance") was recorded in the remaining six cases. The third group of ten patients, eight women two men, ranging from 15 to 47 years of age (average age 26.9) was treated with methylprednisolone acetate intrathecally. Five patients were practically cured after 3 to 4 weeks. Two patients were cured to an extent that they could walk with assistance. In two cases no improvement was recorded, while one of the patients, who in addition suffered from serum myelitis, died of bilateral bronchopneumonia. In all these cases the drug was administered comparatively late, on the 6th or 7th day ofthe disease.     

A bolus of recombinant human follicle stimulating hormone at midcycle induces periovulatory events following multiple follicular development in macaques

The efficacy of follicle stimulating hormone (FSH) as an alternative to luteinizing hormone (LH)/human chorionic gonadotrophin (HCG) for the initiation of periovulatory events in primate follicles is unknown. A single bolus of 2500 IU recombinant (r)-hFSH was compared to 1000 IU r-HCG for its ability to promote oocyte nuclear maturation and fertilization, granulosa cell luteinization and corpus luteum function following r-hFSH (60 IU/day) induction of multiple follicular development in rhesus monkeys. Following the r-hFSH bolus, bioactive luteinizing hormone concentrations were <3 ng/ml. Peak concentrations of serum FSH (1455+/-314 mIU/ml; mean+/-SEM) were attained 2-8 h after r-hFSH, and declined by 96 h. Bioactive HCG concentrations peaked between 2-8 h after r-HCG and remained > or = 100 ng/ml for >48 h, while immunoreactive FSH concentrations were at baseline. The proportion of oocytes resuming meiosis and undergoing in-vitro fertilization (IVF) were comparable for r-hFSH (89%; 47+/-19%) and r-HCG (88%; 50+/-17%). In-vitro progesterone production and expression of progesterone receptors in granulosa cells did not differ between groups. Peak concentrations of serum progesterone in the luteal phase were similar, but were lower 6-9 days post-FSH relative to HCG. Thus, a bolus of r-hFSH was equivalent to r-HCG for the reinitiation of oocyte meiosis, fertilization and granulosa cell luteinization, but a midcycle FSH surge did not sustain normal luteal function in primates.     

Prader-Willi syndrome with elevated follicle stimulating hormone levels and diabetes mellitus

A 21 -year-old man with Prader-Willi syndrome (PWS) was hospitalized due to hyperglycemia. After diet therapy and transient insulin administration, his blood glucose levels improved. Based on the fact that his urinary C-peptide levels increased, the diabetes mellitus may have been due to insulin resistance with obesity. In addition, his testes had become atrophied. Testosterone levels remained low even after human chorionic gonadotropin (HCG) administration. Luteinizing hormone (LH) levels were also low after LH releasing hormone (LHRH) administration. The LH response increased slightly after daily LHRH administration, indicating hypothalamic hypogonadism. Follicle stimulating hormone (FSH) levels were, however, high and increased after LHRH administration. The selective FSH elevation may have been due to the accompanying idiopathic oligospermia.     

Developmental potential of embryos produced by in-vitro fertilization from gonadotrophin-releasing hormone antagonist-treated macaques stimulated with recombinant human follicle stimulating hormone alone or in combination with luteinizing hormone

We previously demonstrated, in luteinizing hormone (LH)-deficient macaques, that follicular growth and maturation occurred with administration of exogenous (recombinant human) follicle stimulating hormone (r-hFSH) alone, and that the oocytes recovered fertilized at a notably higher rate than their counterparts from animals receiving both r-hFSH and r-hLH (Zelinski-Wooten et al., 1995). Here, the developmental potential of embryos produced from animals treated with r-hFSH alone or in combination with r-hLH was evaluated. Embryos (n = 127) were cryopreserved, thawed and either co-cultured on buffalo rat liver cells until the hatched blastocyst stage or transferred to synchronized recipients. Although embryos from each treatment group demonstrated a similar ability to develop to hatched blastocysts with a definitive inner cell mass, a significant difference was seen in cryosurvival (56 versus 78%) and in developmental rate to the hatched blastocyst (12 versus 10 days) between embryos from the r-hFSH alone and the combination group respectively. Pregnancies resulted following oviductal embryo transfers in both groups, with corpus luteum rescue occurring on days 12-16 of the luteal phase. In summary, r-hFSH alone during the pre-ovulatory interval is adequate for the gametogenic events required to produce embryos that develop either in vitro or in vivo; however, exposure to r-hLH may improve embryo viability and the rate of development.     

Follicle stimulating hormone alone supports follicle growth and oocyte development in gonadotrophin-releasing hormone antagonist-treated monkeys

Both follicle stimulating hormone (FSH) and luteinizing hormone (LH) are proposed requirements for follicular growth and steroidogenesis; however, the role of LH in primate folliculogenesis is unclear. Follicular stimulation by recombinant human FSH (n = 5) with and without recombinant LH (1:1; n = 6) following 90 days of gonadotrophin-releasing hormone (GnRH) antagonist (Antide) treatment in macaques was evaluated. Human chorionic gonadotrophin (HCG) was administered when six follicles > or = 4 mm were observed. Oocytes were aspirated 27 h later and inseminated in vitro. Chronic Antide reduced serum oestradiol and bioactive LH to concentrations observed in hypophysectomized rhesus monkeys. Multiple follicular growth required a longer interval following recombinant FSH (12 +/- 1 days) than recombinant FSH+recombinant LH (9 +/- 0.2 days), but the total number of follicles/animal did not differ between groups. The day prior to HCG, oestradiol concentrations were 4-fold less following recombinant FSH compared to recombinant FSH+recombinant LH. With recombinant FSH, more oocytes completed meiosis to metaphase II (51%) and fertilized (89 +/- 5%) relative to recombinant FSH+recombinant LH (12 and 52 +/- 11% respectively). Follicular growth and maturation in LH-deficient macaques occurred with FSH alone. Thus, LH is not required for folliculogenesis in primates. Higher fertilization rates following follicular stimulation with FSH alone suggest that the presence of LH with FSH (1:1) during the pre-ovulatory interval impairs gametogenic events in the periovulatory period.     

Immunocytochemical evidence that luteinizing hormone (LH) and follicle stimulating hormone (FSH) are present in the same cell type in the rhesus monkey pituitary gland

Antisera to oLHbeta subunits were used to identify the gonadotrophic cells in partes distalis and tuberalis of the rhesus monkey pituitary gland. The same cell type in pituitary glands obtained from male and female rhesus monkeys stained with both antisera. Immunocytochemical staining was observed in PAS positive cells that were randomly dispersed throughout the entire pars distalis. The gonadotrophs did not react with antisera to either hTSHbeta or oACTH. These observations indicate that only one gonadotrophic cell type is present in the primate pituitary gland.     

Plasma levels of estrogen, luteinizing hormone, and follicle stimulating hormone following castration and estradiol implant

Plasma levels of luteinizing hormone, follicle stimulating hormone, and estrogen were studied serially in 20 patients before and after hysterectomy and bilateral salpingo-oophorectomy. Ten patients received an implant of estradiol-17 beta (100 mg) at the time of operation. Ten patients who did not receive an implant acted as controls. Patients recorded the severity of vasomotor symptoms before and after hysterectomy. In those patients who did not receive an implant, plasma estrogen levels fell from a mean preoperative level of 18.1 +/- 10.4 ng/100 ml to 8.7 +/- 1.4 ng/100 ml by 24 hours after oophorectomy and they remained in this range for the 6 months of the study. No significant change in the plasma estrogen level was noted after oophorectomy in those patients who received an implant. The implant prevented the rise in gonadotropin levels and the appearance of vasomotor symptoms seen in those patients who underwent oophorectomy without an implant. Insertion of an implant into oophorectomized patients caused the plasma estrogen level to return to premenopausal levels within 2 weeks and the gonadotropin levels to premenopausal values within 6 weeks. Hot flashes were alleviated within 2 to 6 days. The usefulness of this type of therapy in preventing the appearance of vasomotor symptoms at the time of oophorectomy in premenopausal patients is confirmed.     

Effect of growth hormone administration on circulating levels of luteinizing hormone, follicle stimulating hormone and testosterone in normal healthy men

A new area of growth hormone (GH) therapy in adults is the treatment of infertility. The aim of this study was to evaluate the effects of pharmacological GH administration on the secretion of pituitary and gonadal hormones in normal men. Eight healthy men, 23-32 years of age (mean 28.1 years), with a normal body mass index were studied in a double-blind, placebo-controlled crossover design. All participants had a normal semen analysis before entering the study. Each participant was treated with placebo and GH (12/IU/day, Norditropin; Novo Nordisk, Denmark) during two different 14-day periods, separated by a 6 week washout period. Administration of GH for 14 days resulted in a significant increase in serum insulin-like growth factor I (IGF-I; P < 0.01) but no changes occurred in IGF-I values during placebo treatment. The concentrations of follicle stimulating hormone and luteinizing hormone displayed no change during the two periods and did not differ between the GH treatment period and the placebo period. The concentration of testosterone was unchanged during the placebo/GH periods and there was no difference between the GH treatment period and the placebo period. We conclude that GH treatment for 14 days in normal healthy men does not affect gonadotrophin or testosterone patterns.     

Screening male intracytoplasmic sperm injection candidates for mutations of the follicle stimulating hormone receptor gene

Follicle stimulating hormone (FSH) is considered to be essential for spermatogenesis. Therefore, genetic abnormalities of FSH signalling on testicular Sertoli cells would be expected to affect sperm production negatively in males. Inactivating FSH receptor mutations have been reported earlier in both males and females. All affected males had elevated FSH serum concentrations and abnormal sperm parameters. We postulated that inactivating FSH receptor mutations might be a cause of oligozoospermia or azoospermia and reviewed the clinical data of 151 male intracytoplasmic sperm injection (ICSI) candidates with special attention to FSH serum concentrations. The exclusion criteria for mutation screening of the FSH receptor gene were: a history of operative sterilization or testicular malignancy, congenital abnormality other than cryptorchidism, and a chromosomal aberration or a Y-chromosome microdeletion. The inclusion criteria were: male (ICSI candidate) with azoospermia or oligoasthenoteratozoospermia (OAT) and elevated FSH serum concentrations. In total, 23 males with OAT and five males with azoospermia were tested for mutations of the coding sequences and the intron-exon boundaries of the FSH receptor gene by polymerase chain reaction (PCR) followed by single strand conformation polymorphism analysis (SSCP). Neutral polymorphisms were readily detected using this technique in both probands and controls. None of the 28 selected patients showed a pathogenic FSH receptor mutation. Mutations in the FSH receptor gene are not a common cause of infertility in ICSI candidates.     

Effect of profound suppression of luteinizing hormone during treatment with gonadotrophin-releasing hormone analogue and purified follicle stimulating hormone upon development of cryopreserved embryos

In response to previously published evidence from monkeys, this study examined the influence of the degree of luteinizing hormone (LH) suppression during the follicular phase of the stimulation cycle, upon cryopreserved embryo survival and development. The LH concentration of the mid-follicular phase was assessed in 250 in-vitro fertilization (IVF) cycles treated with gonadotrophin-releasing hormone analogue (GnRHa) and either purified follicle stimulating hormone (FSH) or human menopausal gonadotrophin (HMG), and was related to the performance of cryopreserved embryos in 351 subsequent embryo transfer cycles. Rates of embryo survival, embryo development rates, implantation rates, and pregnancy rates were examined with respect to the LH concentration recorded in the mid-follicular phase. In contrast to experimental evidence from other primates, there was no significant influence of the follicular phase LH concentration upon any of the parameters examined.     

On the dynamics between gonadotrophin surge-inhibiting factor and gonadotrophin releasing hormone (GnRH): role of self-priming and desensitization in the luteinizing hormone response to GnRH after follicle stimulating hormone treatment

The effects were studied of follicle stimulating hormone (FSH)-induced production of gonadotrophin surge-inhibiting factor (GnSIF) on three phases of the pituitary responsiveness to gonadotrophin releasing hormone (GnRH): the unprimed, primed and desensitized phases. Rats were injected with FSH on two occasions during the oestrous cycle. Spontaneous luteinizing hormone (LH) surges were measured as well as GnRH-induced LH surges on the day of pro-oestrus during infusions with 100-4000 pmol GnRH/rat/10 h, in phenobarbital blocked rats. The spontaneous LH surges were attenuated or completely inhibited by the FSH treatment. FSH suppresses and prolongs the unprimed LH response and delays GnRH self-priming, especially during infusions with low concentrations of GnRH. This treatment does not affect the total LH response (area under curve) to the highest concentrations of GnRH and after ovariectomy. On the other hand, this response is suppressed during infusions with the lower concentrations of GnRH. Hence, FSH, via GnSIF, delays maximal priming of the LH response to GnRH, whereas the suppression of LH release is a consequence of the GnRH-induced progressed state of desensitization. The inconsistent effects of FSH on the mid-cycle LH surges are explained as a result of the interaction between the relative strengths of GnRH and GnSIF.