Recombinant hormones: In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin--HP

In this study the in-vitro biopotency and glycoform distributionof human recombinant follicle stimulating hormone (FSH, Org32489) has been assessed. The biopotency of recombinant FSHwas studied using animal (rat Sertoli) and human (granulosa—lutein)cell models. Recombinant FSH, as measured in the rat Sertolicell assay, was more potent than the urinary preparations Metrodin,Metrodin—HP and IS 70/45 with half maximal stimulation(ED₅₀; mean ± SEM, n > 3) occurring at 2.2 ±0.5 IU/I (recombinant FSH), 4.7 ± 1.1 IU/I (Metrodin),13.2 ± 0.7 IU/I (Metrodin—HP) and 6.4 ±0.3 IU/I (IS 70/45); the pituitary preparation IRP 83/575 hadan ED₅₀ of 10.4 ± 0.1 IU/I. Using human granulosa—luteincells, cultured for up to 4 days in the absence of exogenoussteroid precursors, recombinant FSH was either without effect(three out of five patients) or inhibited both oestradiol andprogesterone secretion. FSH (83/575) was without effect on oestradiolwith preparations from any of the patients but slightly stimulated(134 ± 8%; mean ± SEM, P < 0.05) progesteroneproduction at the highest dose (80 IU/I). The distribution ofFSH isoforms, assessed by polyclonal radioimmunoassay, followingchromatofocusing over the ranges pH < 3.5 and pH 3.5–7.0respectively was recombinant FSH, 12.4 and 87.6%; Metrodin,19.8 and 80.2%; Metrodin—HP, 50.2 and 49.8%; IS 70/45,15.0 and 85.0%; IS 83/575, 70.9 and 29.1%. All glycoforms werepl <7.0 for the five preparations. In conclusion: (i) thepotency of FSH as measured in the rat Sertoli cell assay increasesin the order Metrodin—HP < pituitary IRP 83/575 <<Metrodin < IS 70/45 < recombinant FSH; (ii) in contrastto 83/575, recombinant FSH inhibits steroidogenesis in humangranulosa—lutein cells isolated from some patients; (iii)the glycoform distribution of recombinant FSH resembles Metrodinmore closely than Metrodin—HP which is far more acidicin nature.     

Ovarian stimulation during assisted reproduction treatment: a comparison of recombinant and highly purified urinary human FSH. On behalf of The Feronia and Apis study group

This randomized, single-blind, multicentre, multinational study compared recombinant human FSH (rhFSH, Gonal-F) with highly purified urinary human FSH (uhFSH, Metrodin HP) in women undergoing ovarian stimulation for IVF/intracytoplasmic sperm injection (ICSI). Following desensitization in a long gonadotrophin-releasing hormone (GnRH) agonist protocol, patients received s.c. Gonal-F or Metrodin HP, at a fixed dose of 150 IU, until there was adequate follicular development. Of 496 women randomized, 232 and 231 in the Gonal-F and Metrodin HP groups respectively received human chorionic gonadotrophin (HCG). The duration of FSH treatment was significantly shorter with Gonal-F than with Metrodin HP (11.6 +/- 1.9 days versus 12. 4 +/- 2.7 days; P < 0.0001) and significantly fewer ampoules were required (mean 22.6 +/- 5.0 versus 24.3 +/- 5.1, P < 0.0002). There were, however, significantly more follicles > or =10 mm in diameter with Gonal-F (15.6 +/- 8.2 versus 13.6 +/- 7.1, P < 0.01) and oocytes retrieved (13.1 +/- 7.7 versus 11.4 +/- 7.6, P < 0.002). Although no statistical difference in pregnancy rate was recorded, patients receiving Gonal-F had a higher pregnancy rate per cycle than patients given Metrodin HP (25.1 versus 20.1%). Moderate to severe ovarian hyperstimulation syndrome occurred in 2.8 and 1.2% of Gonal-F and Metrodin HP patients respectively (not significant). In conclusion, FSH stimulation in combination with a long GnRH agonist protocol is effective in inducing multiple follicular development and embryos with a high implantation potential. However, Gonal-F is clearly more effective than Metrodin HP in inducing multifollicular development.     

Subcutaneous low dose leuprolide acetate depot versus leuprolide acetate for women undergoing ovarian stimulation for in-vitro fertilization

A total of 100 women undergoing ovarian stimulation with gonadotrophin-releasinghormone agonist (GnRHa) and a human menopausal gonadotrophin(HMG) for in-vitro fertilization (IVF) participated in thisrandomized comparative study. Leuprolide acetate at a dose of0.5 mg/day s.c. (n = 52, group I), or low-dose leuprolide acetatedepot at a dose of 1.88 nig s.c. (n = 48, group II), was startedon days 21–23 of the cycle. Stimulation with 225 IU/dayHMG was started after pituitary desensitization had been achieved.The luteal phase was supported by human chorionic gonadotrophin(HCG) i.m. injection. There were nostatistical differences inbaseline oestradiol (24.5 ± 4.8 versus 21.9 ±4.5 pg/ml) and follicle stimulating hormone (FSH) concentrations(3.9 ± 1.9 versus 3.2 $ 1.8 mlU/ml), and concentrationson the day of HCG administration of oestradiol (1657 ±245 versus 1512$165 pg/ml), luteinizing hormone (LH; 6.2 ±4.8 versus 5.6 ± 4.3 mlU/ml) and FSH (10.6 ± 2.8versus 10.8 ± 3.6 mIU/ml). There were also no statisticaldifferences in the HMG dosage (26.8 ± 1.8 versus 28.5± 1.5), the number of oocytes retrieved (7.6 ±3.0 versus 8.1 ± 4.3), the number of oocytes fertilized(5.3 ± 2.1 versus 5.6 ± 3.0) and the number ofembryos transferred (3.5 ± 1.3 versus 3.4 ± 1.6).There was no evidence of a premature LH surge in either group,but two patients appeared to have a poor response in the leuprolideacetate group (group I). There were 11 pregnancies (21.2%) afterthe use of leuprolide acetate and 12 pregnancies (25.0%) inthose given leuprolide acetate depot; no statistical differenceexisted between these two groups. Thus, an s.c. low-dose leuprolideacetate depot injection may offer a useful alternative for pituitarysuppression in ovarian stimulation for IVF.     

A double-blind, randomized study to compare recombinant human follicle stimulating hormone (FSH; Gonal-F) with highly purified urinary FSH (Metrodin) HP) in women undergoing assisted reproductive techniques including intracytoplasmic sperm injection. The French Multicentre Trialists

This prospective, double-blind, randomized, multicentre study compared the efficacy and safety of recombinant human follicle stimulating hormone (r-hFSH; Gonal-F((R))) versus highly purified urinary FSH (u-hFSH HP; Metrodin((R)) HP) in women undergoing ovarian stimulation for in-vitro fertilization, including intracytoplasmic sperm injection. A total of 278 patients began a long gonadotrophin-releasing hormone agonist protocol, then 139 received r-hFSH and 139 u-hFSH HP, 150 IU/day administered s.c., for the first 6 days of treatment. On day 7, the dose was adjusted, if necessary, according to ovarian response. Human chorionic gonadotrophin (HCG, 10 000 IU, s.c.) was administered once there was more than one follicle 18 mm in diameter and two others >/=16 mm. Oocyte retrieval was performed 36-38 h after HCG injection: 128 patients (92%) receiving r-hFSH and 113 (81%) receiving u-hFSH HP had at least one oocyte retrieved. Among patients receiving r-hFSH, there was a significantly higher mean (+/- SD) number of oocytes retrieved (11.0 +/- 5.9 versus 8.8 +/- 4.8 with u-hFSH HP; P = 0. 002) and mean number of embryos obtained (5.1 +/- 3.7 versus 3.5 +/- 2.9 with u-hFSH HP; P = 0.0001). With r-hFSH, significantly fewer FSH treatment days (11.7 +/- 1.9 versus 14.5 +/- 3.3) and 75 IU ampoules (27.6 +/- 10.2 versus 40.7 +/- 13.6) were required than with u-hFSH HP (P = 0.0001). Embryo replacement on day 2-3 after oocyte retrieval resulted in 36 liveborn children in the Gonal-F((R)) group and 33 in the Metrodin HP((R)) group (not significant). There were seven cases (5.0%) of ovarian hyperstimulation syndrome in the r-hFSH group and three (2.2%), in the u-hFSH HP group (not significant). It is concluded that r-hFSH is more effective than u-hFSH in inducing multiple follicular development.     

Randomized controlled trial of follicle stimulating hormone versus human menopausal gonadotrophin in in-vitro fertilization*

The adverse effect of raised luteinizing hormone (LH) concentrationson reproductive outcome suggests that exogenous LH administrationfor ovarian stimulation may not be desirable. The aim of thisstudy was to compare the clinical pregnancy rates between folliclestimulating hormone (FSH) and human menopausal gonadotrophin(HMG) used in in-vitro fertilization (IVF) cycles. A total of232 infertile patients, with a mean duration of infertilityof 67.1 ± 32.9 months, were selected for IVF (femaleage <38 years, FSH <15 IU/1, and total motile sperm count>5x10⁶). A short (flare-up) protocol with daily leuprolideacetate was followed randomly from day 3 with FSH (n = 115)or human menopausal gonadotrophin (HMG; n = 117), at an initialdose of two ampoules per day. A maximum of three embryos wastransferred, and the luteal phase was supported with four dosesof HCG (2500 IU). No differences were observed between the twogroups in any of the cycle response variables except fertilizationrates per oocyte and per patient, both of which were significantlyhigher with FSH. Clinical pregnancy rates per cycle initiated,per oocyte retrieval and per embryo transfer were 19.1, 21.0and 22.7% respectively for FSH, and 12.0, 12.8 and 15.4% respectivelyfor HMG. Whilst these differences were not statistically significant,the results of this interim analysis suggest that HMG may beassociated with a lower clinical pregnancy rate than FSH.     

Endocrinology: Variability in the immunoreactive and bioactive follicle stimulating hormone content of human urinary menopausal gonadotrophin preparations

The within- and between-batch variation in the immuno-reactiveand in-vitro bioactive FSH content of Pergonal, Metrodin andMetrodin-HP was investigated. Three batches of Pergonal andMetrodin, consisting of three ampoules in each batch, and threebatches of Metrodin-HP, consisting of between one and threeampoules per batch, were selected at random. The follicle stimulatinghormone (FSH) content of Pergonal, Metrodin and Metrodin-HPwas determined by radioimmunoassay (R-FSH) and the in-vitrorat Sertoli cell bioassay (B-FSH) using the international urinarystandard 70/45. The variability in the FSH content of the preparationswas evaluated within and between batches by analysis of variance.Within-batch variability of B-FSH was not observed in Pergonalor Metrodin-HP but was seen in two batches of Metrodin in whichthe potency varied by up to 2.4 fold (P = 0.03). The between-batchR-FSH potencies of Pergonal (P1–P3) varied, with P2 (59.8± 0.6) and P3 (61.7 ± 0.9) being higher than P1(47.1 ± 1.5 mean ± SEM IU/ampoule, P < 0.01).A similar pattern of variability was observed for B-FSH. ForMetrodin, each of the batch R-FSH potencies was dissimilar (P< 0.02), with estimates ranging from 34.9 ± 1.2 to64.3 ± 1.8 IU/ampoule. Furthermore, the extensive within-batchB-FSH variation from two batches confounded any meaningful comparisonof between-batch variability. For Metrodin-HP, there was nobetween-batch B-FSH variation (29.0 ± 6.1 to 33.0 ±0.3 IU/ampoule) and the R-FSH content was also well controlled(41.2 ± 0.5 to 46.9 ± 1.1 IU/ampoule). The B:I(bioassay: immunoassay) FSH ratios of Pergonal (0.9 ±0.1, n = 9), Metrodin (2.2 ± 0.3, n = 9) and Metrodin-HP(0.7 ± 0.04, n = 5) were all dissimilar (P < 0.03).Within- and between-batch variations were observed to varyingdegrees in three human urinary gonadotrophin preparations. Thevariations in B-FSH content between the preparations appearedmore extensive than for R-FSH. Of the preparations studied,Metrodin-HP exhibited the least variability.     

Endocrinology: Inhibition of ovarian-derived prorenin to angiotensin cascade in the treatment of ovarian hyperstimulation syndrome

The purpose of this experiment was to determine whether useof the angiotensin-converting enzyme (ACE) inhibitor, enalapril,would prevent the occurrence of ovarian hyperstimulation syndrome(OHSS) in the rabbit model. A total of 20 adult female New Zealandwhite rabbits were studied. All rabbits received 75 IU of humanmenopausal gonadotrophin s.c. each day for 7 days. On day 8,all rabbits received 2500 IU of human chorionic gonadotrophin(HCG). Ten rabbits were randomly chosen to receive enalaprilorally. Five received 1 mg/kg of enalapril and five received2 mg/kg of enalapril twice daily. The remainder received placeboorally twice daily. On day 10, all rabbits underwent surgicalexploration. Total body weight was found to increase significantlyin the placebo group (by 293 g, P < 0.001) but not in eithergroup receiving enalapril. Haematocrit also increased significantlyin the placebo group (by 3%, P < 0.013) but not in the enalaprilgroups. Ovarian weights were highest for the 2 mg/kg enalaprilgroup (5.80 ± 0.52 g), followed by the 1 mg/kg enalaprilgroup (3.64 ± 0.45), and least for the placebo group(2.69 ± 0.17). All 10 placebo rabbits met criteria forsevere OHSS whereas only six in the enalapril groups did. Weconcluded that angiotensin II may play a significant role inthe development of weight gain, third space fluid accumulationand intravascular fluid depletion in OHSS. ACE inhibition resultedin a 40% decrease in the incidence of OHSS in the rabbit model.     

In-vitro biopotency and glycoform distribution of recombinant human follicle stimulating hormone (Org 32489), Metrodin and Metrodin-HP

In this study the in-vitro biopotency and glycoform distributionof human recombinant follicle stimulating hormone (FSH, Org32489) has been assessed. The biopotency of recombinant FSHwas studied using animal (rat Sertoli) and human (granulosa-lutein)cell models. Recombinant FSH, as measured in the rat Sertolicell assay, was more potent than the urinary preparations Metrodin,Metrodin-HP and IS 70/45 with half maximal stimulation (ED₅₀;mean ± SEM, n 3) occurring at 2.2 ± 0.5 IU/I (recombinantFSH), 4.7 ± 1.1 IU/I (Metrodin), 13.2 ± 0.7 IU/I(Metrodin-HP) and 6.4 ± 0.3 IU/I (IS 70/45); the pituitarypreparation IRP 83/575 had an EDM of 10.4 ± 0.1 IU/I.Using human granulosa-lutein cells, cultured for up to 4 daysin the absence of exogenous steroid precursors, recombinantFSH was either without effect (three out of five patients) orinhibited both oestradiol and progesterone secretion. FSH (83/575)was without effect on oestradiol with preparations from anyof the patients but slightly stimulated (134 ± 8%; mean± SEM, P < 0.05) progesterone production at the highestdose (80 IU/I). The distribution of FSH isoforms, assessed bypolyclonal radioimmunoassay, following chromatofocusing overthe ranges pH < 3.5 and pH 3.5–7.0 respectively wasrecombinant FSH, 12.4 and 87.6%; Metrodin, 19.8 and 80.2%; Metrodin-HP,50.2 and 49.8%; IS 70/45, 15.0 and 85.0%; IS 83/575, 70.9 and29.1%. All glycoforms were pi <7.0 for the five preparations.In conclusion: (i) the potency of FSH as measured in the ratSertoli cell assay increases in the order Metrodin-HP < pituitaryIRP 83/575 < < Metrodin < IS 70/45 < recombinantFSH; (ii) in contrast to 83/575, recombinant FSH inhibits steroidogenesisin human granulosa-lutein cells isolated from some patients;(iii) the glycoform distribution of recombinant FSH resemblesMetrodin more closely than Metrodin-HP which is far more acidicin nature. biopotency/glycoform/isoform/Metrodin/recombinant FSH     

The effect of gonadotrophins with differing LH/FSH ratios on the secretion of the various species of inhibin in women receiving IVF

We have measured secretory patterns of inhibin A, B, total alpha inhibin, pro-alphaC inhibin and oestradiol in women following pituitary suppression who were randomised into two groups to receive either urinary gonadotrophin (25:75 IU/ampoule of luteinizing hormone (LH) and follicle stimulating hormone (FSH; Normegon; n = 11) or recombinant (r)FSH (75 IU/ampoule of FSH alone, n = 16). The women were of similar age (approximately 33 years) and length of infertility (approximately 4 years) and had a normal endocrine evaluation. Plasma FSH, LH, oestradiol, inhibin A, B, pro-alphaC and total alpha inhibin were measured by immunoassay prior to and following gonadotrophin stimulation. Immunoactive FSH, LH and oestradiol blood concentrations following pituitary down regulation were similar in the two groups being <2.0, <3.6 IU/l and <82 pmol/l respectively. The units of FSH given (2230 versus 2764 IU; Normegon versus rFSH), duration of treatment (9.1 versus 9.4 days) and number of follicles of > or =14mm on the day of human chorionic gonadotrophin (HCG) administration (17 versus 14) were also similar. Inhibin A or B concentrations rose similarly during Normegon or rFSH administration, peaking at days 9-11. Total alpha and pro-alphaC inhibin concentrations were lower (P < 0.05) in the rFSH group during days 10 and 11 of treatment being 18.9 +/- 15.9 ng/ml (Normegon) and 4.6 +/- 2.8 ng/ml (rFSH) for total alpha inhibin and 8.5 +/- 6.8 ng/ml (Normegon) and 2.8 +/- 1.6 ng/ml (rFSH) for pro-alphaC inhibin on day 10. Overall, higher total alpha inhibin concentrations were associated with more mature follicles and oocytes, greater fertilization rates and better quality embryos. We conclude that inhibin A and B secretion was similar in both groups and is primarily controlled by FSH, whereas total alpha inhibin and pro-alphaC increased preferentially in the Normegon group over the rFSH group, indicating that they are, in part, stimulated by LH.     

Andrology: Inhibin and steroid responses to testicular stimulation in normal men

Static measurements of immunoreactive inhibin have proved tobe of little relevance in the diagnosis of testicular disorders.To explore whether a dynamic evaluation of inhibin secretionmight yield a more useful parameter of testicular function wecompared the responses of inhibin with steroids to i.v. injectionsof pure follicle-stimulating hormone (FSH; 300 IU) or humanchorionic gonadotrophin (HCG; 1500 IU) and oral administrationof the antioestrogen Tamoxifen (20 mg/day for 7 days) in fournormal fertile men. Blood was aspirated between 1 and 72 h afterthe injections and daily during Tamoxifen intake. Four controlswere injected with physiological saline solution. An additionalfour men were injected with pure FSH, and blood was taken after24, 48 and 72 h. Injection of FSH was accompanied by nycthemeralvariations of testosterone comparable with those observed inthe controls. The concentration of inhibin showed similar nycthemeralvariations but a significant increase was observed in all eightcases at 12 noon on days 2 and 3 after FSH injection. HCG injectionresulted in the expected biphasic response of testosterone.Inhibin displayed a pronounced increase 18 h after injectionbut the delayed response after 48 and 72 h was not observed.Tamoxifen intake increased testosterone but not inhibin, andcaused a moderate and temporary increase of luteinizing hormoneand FSH. It was concluded that primary stimulation both of Leydigcells by HCG and Sertoli cells by FSH increase circulating inhibin.Comparison with the testosterone response suggests that theinhibin peak 18 h after HCG administration may reflect Leydigcell function, and that the delayed response 48 and 72 h afterFSH administration can be used as a parameter of Sertoli cellfunction.     

Infertility: Composition of commercial gonadotrophin preparations extracted from human post-menopausal urine: characterization of non-gonadotrophin proteins

Gonadotrophin preparations extracted from post-menopausal urineare of low purity and the major protein components are not gonadotrophins.A study was undertaken to identify some of these non-gonadotrophinproteins present in the extracted human urinary gonadotrophinpreparations that are commercially available, i.e. Humegon (Organon),HMG Massone (Massone), Metrodin (Serono), Metrodin HP (Serono),Pergonal (Serono) and Progonadyl (Elea). As revealed by sodiumdodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)with Coomassie blue staining and Western blotting analysis,these products had electrophoretic protein profiles which differedin the amounts and species of proteins present. With the exceptionof Metrodin HP, all the other preparations tested containedtumour necrosis factor binding protein-I, transferrin, and immunoglobulin-relatedproteins. Some of the products contained in addition: urokinase,Tamm-Horsfall glyco-protein and epidermal growth factor. Recently,a highly purified human urinary follicle stimulating hormone(FSH) preparation (Metrodin HP) became available. In this preparationhuman FSH represents >95% of the total proteins ( 10 000IU of FSH/mg of protein). Metrodin HP was demonstrated to bethe purest preparation tested, with none of the above-mentionedcontaminants detected.     

Recombinant human follicle stimulating hormone (r-hFSH; Gonal-F) versus highly purified urinary FSH (Metrodin HP): results of a randomized comparative study in women undergoing assisted reproductive techniques

A prospective, randomized, comparative, assessor-blind study was carried out in two centres to compare the efficacy and safety of recombinant human follicle stimulating hormone (r-hFSH; Gonal-F) versus highly purified urinary FSH (u-hFSH HP; Metrodin HP), both administered s.c. in women undergoing ovarian stimulation for in-vitro fertilization including intracytoplasmic sperm injection (ICSI). A total of 235 patients started a long gonadotrophin-releasing hormone agonist protocol: 119 received r-hFSH and 114 received u-hFSH HP (150 IU/day) for the first 6 days. Two patients were excluded from the study because they mistakenly received the incorrect treatment combination. Human chorionic gonadotrophin (HCG; 10000 IU, s.c.) was administered once there was at least one follicle 18 mm in diameter and two others > or = 16 mm. In all, 119 (100%) and 102 (89%) of the patients respectively in the r-hFSH and u-hFSH HP groups achieved the criteria for HCG. The mean numbers (+/- SD) of oocytes recovered (the primary endpoint) were 12.2 +/- 5.5 and 7.6 +/- 4.4 in the r-hFSH and u-hFSH HP groups respectively (P < 0.0001). However, the number of FSH treatment days (11.0 +/- 1.6 versus 13.5 +/- 3.7) and the number of 75 IU ampoules (21.9 +/- 5.1 versus 31.9 +/- 13.4) used were significantly less (P < 0.0001) in the r-hFSH group than in the u-hFSH HP group. In patients treated using ICSI (63 patients in each group), no difference in oocyte maturation was observed. The mean numbers of embryos obtained were 8.1 +/- 4.2 and 4.7 +/- 3.5 (P < 0.0001), in favour of the r-hFSH group. In the majority of patients (96 and 99% respectively) only one or two embryos were replaced (mean 2.0 +/- 0.2 and 1.9 +/- 0.1 respectively) in the r- hFSH and u-hFSH HP groups. The clinical pregnancy rates per started cycle and per embryo transfer were 45 and 36%, and 48 and 47%, respectively in the r-hFSH and u-hFSH HP groups (not significant). There were six (5.1%) and two (1.7%) cases of ovarian hyperstimulation syndrome respectively. In conclusion, it was found that r-hFSH was more effective than u-hFSH at inducing multiple follicular development. However, the high rate of low ovarian response in the u-hFSH group compared with our general experience was unexpected. The availability of a gonadotrophin with less inter-batch variation would be beneficial for clinicians. r-hFSH seems to fulfil such a requirement.      

Endocrine composition of follicular fluid comparing human chorionic gonadotrophin to a gonadotrophin-releasing hormone agonist for ovulation induction

Concentrations of inhibin, oestradiol and progesterone weredetermined in pre-ovulatory follicular fluid from 16 women undergoingin-vitro fertilization and embryo transfer treatment. A prospectiverandomized design was used such that ovulation was induced ineight women with human chorionic gonadotrophin (HCG) (9000 IU),and in eight women with an endogenous surge of luteinizing hormone(LH) and follicle stimulating hormone (FSH) caused by a singleinjection of gonadotrophin-releasing hormone agonist (GnRHa).Inhibin was measured by an enzyme-linked immunosorbent assay,and oestradiol and progesterone were measured by radioimmunoassay.Concentrations of inhibin and progesterone are significantlyhigher in follicular fluids collected after ovulation inductionwith HCG compared with ovulation induction with GnRHa (P <0.001, P < 0.02, respectively). Concentrations of oestradiolwere similar in the two groups. This study shows that the methodby which ovulation is triggered significantly affects the micro-environmentof the oocyte just prior to ovulation. The results indicatethat HCG causes a prolonged luteotrophic effect well beforeovulation, compared to an endogenous surge of gonadotrophinscaused by GnRHa, and suggest that follicular maturation withan endogenous surge of gonadotrophins may be closer to the naturalcycle than those cycles in which HCG is administered for ovulationinduction. In addition, this study shows that the concentrationsof inhibin and progesterone in follicular fluid may be valuableparameters in assessing the midcycle LH surge requirements forinduction of ovulation.     

Serum inhibin A, VEGF and TNFalpha levels after triggering oocyte maturation with GnRH agonist compared with HCG in women with polycystic ovaries undergoing IVF treatment: a prospective randomized trial

BACKGROUND: We aimed to examine the serum levels of inhibin A, vascular endothelial growth factor (VEGF), tumour necrosis factor alpha (TNFalpha), estradiol (E2) and progesterone levels after triggering of final oocyte maturation with GnRH agonist compared with HCG in patients with polycystic ovaries (PCO) and to investigate the relationship between these markers and ovarian hyperstimulation syndrome (OHSS). METHODS: Twenty-eight patients with PCO, undergoing controlled ovarian hyperstimulation with FSH and GnRH antagonist for IVF-embryo transfer treatment, were randomized for triggering of final oocyte maturation with GnRH agonist (GnRH agonist group, n = 15) or HCG (HCG group, n = 13). Blood samples were obtained on the day of randomization and thereafter every 2-7 days. Serum levels of inhibin A, VEGF, TNFalpha, E2 and progesterone, the incidence of OHSS, ovarian size and pelvic fluid accumulation were evaluated. RESULTS: Serum inhibin A, E2 and progesterone levels were significantly lower in the GnRH agonist group compared with the HCG group, particularly on the day of embryo transfer (P < 0.0001). Serum VEGF and TNFalpha levels were similar between the two groups. Four patients in the HCG group developed severe OHSS, whereas no patient had any symptoms or signs of OHSS in the GnRH-agonist group (P < 0.05). CONCLUSIONS: In patients with PCO treated with FSH/GnRH antagonist, final oocyte maturation with GnRH agonist instead of HCG reduces significantly inhibin A, E2 and progesterone levels during the luteal phase. This phenomenon reflects the inhibition of the corpus luteum function and may explain, at least in part, the mechanism of OHSS prevention in high-risk patients. Our results do not support a crucial role for VEGF or TNFalpha in OHSS.     

Pharmacodynamics and pharmacokinetics after repeated subcutaneous administration of three gonadotrophin preparations

Recently, several new urinary gonadotrophin preparations have been developed, containing less luteinizing hormone (LH) activity than human menopausal gonadotrophin. Normegon is a gonadotrophin preparation with a follicle stimulating hormone (FSH)/LH ratio of 3:1; Follegon and Metrodin-HP are purified FSH preparations. The aim of the present randomized study was to compare pharmaco-dynamics, -kinetics and local tolerance of these preparations after repeated s.c. administration. Thirty-six healthy female subjects were treated with Lyndiol contraceptive pills for 5 weeks to suppress endogenous gonadotrophin concentrations. After 3 weeks of Lyndiol treatment, 150 IU of Normegon, Follegon or Metrodin HP were administered once daily, s.c. for 7 days. Blood samples were collected once daily during the fourth and fifth weeks of the study and assayed for FSH and oestradiol. After the last gonadotrophin injection, blood samples were collected more frequently to determine pharmacokinetic parameters of FSH. During the fourth and fifth study weeks, daily ultrasound measurements of follicular growth were performed. Endogenous FSH and LH values were extremely suppressed during Lyndiol treatment. Serum FSH values showed similar patterns in the three groups. The maximum FSH concentration was reached 9-11 h post- injection, the terminal half-life was 43-47 h. The preparations were bioequivalent with respect to FSH immunoreactivity. The number of follicles tended to be larger after Normegon than after Follegon and Metrodin HP treatment, though this was not statistically significant. Serum oestradiol concentrations were significantly higher after Normegon treatment. In general, s.c injections were well tolerated. In conclusion, the three preparations were bioequivalent with respect to FSH immunoreactivity. Nevertheless, the biological activity of Normegon tended to be higher than that of Follegon and Metrodin HP in Lyndiol- suppressed women.      

Human prolactin release induced by follicle stimulating hormone, luteinizing hormone and human chorionic gonadotrophin

Plasma prolactin levels rise in stimulated cycles. To clarifythe effects of gonadotrophin on the lactotrophs, three studieswere performed. First, plasma concentrations of prolactin duringclomiphene citrate (CC)-human menopausal gonadotrophin (HMG)-humanchononic gonadotrophin (HCG) treatment of women enrolled forin-vitro fertilization (IYF) were compared with those duringHMG-HCG administration while under pituitary suppression witha gonadotrophin releasing hormone (GnRH) analogue (buserelin).Women suppressed with buserelin had higher basal levels of PRLin plasma (14.4 ± 4.3 nglml versus 6.9 ± 1.4 ng/ml,P<0.001). Only buserelin-suppressed women showed a significantrise in plasma prolactin before HCG administration, while bothpatient groups had marked prolactin peaks after HCG injection.This peak was higher in the buserelin group (71.9 ± 50.7ng/ml versus 52.6 ± 29.7 ng/ml). The second study showedthat plasma levels of prolactin of 6 post- menopausal womenwere significantly increased 48 h after an injection of 5000IU HCG, i.m. (24.9 ± 17.4 ng/ml versus 12.4 ±6.2 ng/ml P<0.05). Third, plasma prolactin was studied in5 women over 30 days after surgical castration. An upward trendwas observed similar to that of endogenous gonadotrophin, withthe change in prolactin values closely correlating with thechange in concentrations of follicule stimulating hormone (P<0.005).All these findings suggest that human gonadotrophins stimulatelactotrophs.     

The benefits of mid-luteal addition of human chorionic gonadotrophin in in-vitro fertilization using a down-regulation protocol and luteal support with progesterone

Luteal support is essential in in-vitro fertilization (IVF)when long-acting gonadotrophin-releasing hormone agonist (GnRHa)is used. Because progesterone lacks luteotrophic stimulation,it seems to be the drug of choice in cases with an increasedrisk of ovarian hyperstimulation syndrome (OHSS). The aim ofthis study was to assess the beneficial effect of the mid-lutealaddition of human choriomc gonadotrophin (HCG) in IVF, usinga down-regulation protocol and luteal support with progesterone,in a prospective randomized study. The study included 170 IVFcycles down-regulated with long-acting GnRHa which were supportedwith 50 mg/day progesterone i.m. during the luteal phase. Patientswere evaluated in the mid-luteal period. Those without clinicalsigns of OHSS, oestradiol concentrations <1000 pg/ml andprogesterone concentrations <50 mg/ml were randomly allocatedto either the addition of 2500 IU HCG (HCG+ group) or no HCG(HCG– group). End luteal phase progesterone concentrationsamong non-pregnant patients were used to assess the contributionof exogenous progesterone and to categorize pregnancies accordingto their corpus luteum function. Similar low OHSS (2.7 and 1.8%)and pregnancy (30 and 29%) rates were observed in the HCG+ andHCG– groups respectively. Of the 26 pregnancies in theHCG+ cases, there was only one case with reduced corpus luteumfunction, compared with 12 of the 25 pregnancies among HCG–patients. Cases with reduced corpus luteum function requiredcontinuous progesterone support and presented lower HCG concentrationsand a higher rate of adverse pregnancy outcome. We concludethat mid-luteal HCG addition does not affect pregnancy rate,but in fact helps to preserve corpus luteum function and avoidsthe need for further supplementation during early pregnancy.     

Modulation of folliculogenesis and steroidogenesis in women by graded menotrophin administration

BACKGROUND: To test the effects of progressively decreasing dosages of exogenous LH we combined various amounts of HMG, containing FSH, LH and HCG, and highly purified (HP) FSH to treat 120 GnRH agonist-suppressed infertile female patients as candidates for controlled ovarian stimulation (COS). METHODS: Subjects were randomly assigned to four treatment groups that received the following daily i.m. gonadotrophin regimens: A, FSH 150 IU only; B, FSH 150 IU and LH activity 37.5 IU; C, FSH 150 IU and LH activity 75 IU; D, FSH 150 IU and LH activity 150 IU. FSH dose adjustments were allowed only after the 14th treatment day. Monitoring included transvaginal ultrasound at 2-day intervals and daily determinations of LH, FSH, estradiol (E(2)), progesterone, testosterone and HCG. RESULTS: Duration of COS was significantly shortened in patients receiving at least 75 IU daily of LH activity. Small (<10 mm diameter) pre-ovulatory ovarian follicle occurrence was inversely correlated with LH activity dose administered (r = -0.648, P < 0.0001) and serum HCG levels (r = -0.272, P < 0.01) but not to serum LH levels. Serum testosterone levels were positively correlated to the LH activity dose administered (r = 0.313, P < 0.001), while serum progesterone levels were positively correlated to the FSH dose administered (r = 0.447, P < 0.00001) but not to the LH activity dose administered. CONCLUSIONS: Firstly, HCG content considerably contributes to HMG activity; secondly, menotrophin LH activity content can reduce in a dose-dependent manner the occurrence of small pre-ovulatory follicles; and finally, contrary to common belief, enhanced FSH stimulation rather than LH activity appears to cause premature follicle luteinization during COS.     

Endocrinology: 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) withand without recombinant LH (1: 1; n = 6) following 90 days ofgonadotrophin-releasing hormone (GnRH) antagonist (Antide) treatmentin macaques was evaluated. Human chorionic gonadotrophin (HCG)was administered when six follicles >4 mm were observed.Oocytes were aspirated 27 h later and inseminated in vitro.Chronic Antide reduced serum oestradiol and bioactive LH toconcentrations observed in hypophysectomized rhesus monkeys.Multiple follicular growth required a longer interval followingrecombinant FSH (12 ± 1 days) than recombinant FSH +recombinant LH (9 ± 0.2 days), but the total number offollicles/animal did not differ between groups. The day priorto HCG, oestradiol concentrations were 4-fold less followingrecombinant FSH compared to recombinant FSH + recombinant LH.With recombinant FSH, more oocytes completed meiosis to metaphaseII(51%) and fertilized (89 ± 5%) relative to recombinantFSH + recombinant LH (12 and 52 ± 11% respectively).Follicular growth and maturation in LH-deficient macaques occurredwith FSH alone. Thus, LH is not required for folliculogenesisin primates. Higher fertilization rates following follicularstimulation with FSH alone suggest that the presence of LH withFSH (1: 1) during the pre-ovulatory interval impairs gametogenicevents in the periovulatory period.     

Response of serum inhibin B and pro-alphaC levels to gonadotrophic stimulation in normal men before and after steroidal contraceptive treatment

BACKGROUND: Testicular regulation of inhibin B may be influenced by the germ cell complement. METHODS: We examined the effects of gonadotrophin stimulation on serum inhibin B and pro-alphaC in 25 normal men at (i) control (stimulation test 1), (ii) after spermatogenic suppression induced by testosterone plus progestin treatment (stimulation test 2), and (iii) during spermatogenic recovery induced by FSH and/or hCG treatment (stimulation test 3). For each test, subjects received a single injection of 1200 IU FSH or 5000 IU hCG or both. RESULTS: Inhibin B and pro-alphaC fell with spermatogenic suppression (75 and 51% of pre-treatment baseline respectively, P < 0.05). Inhibin B response to FSH (130-144%) was similar in controls and after germ cell suppression. Pro-alphaC response after germ cell suppression compared with control was significantly increased (P < 0.05) with both FSH (210-229% versus 140-185%) and hCG (254-261% versus 145%). All treatments partially restored spermatogenesis with no clear relationship apparent between inhibin B and sperm count. CONCLUSIONS: We conclude that: (i) serum inhibin B and pro-alphaC are only partially gonadotrophin dependent, (ii) spermatogenic suppression does not modify inhibin B response to FSH but enhances pro-alphaC response to both FSH and hCG, and (iii) inhibin B is a poor marker of spermatogenesis in this model of gonadotrophic manipulation in normal men.