Recombinant Human LH Research Articles

Randomized trial to compare the effect of recombinant human FSH (follitropin alfa) with or without recombinant human LH in women undergoing assisted reproduction treatment

Women undergoing intracytoplasmic sperm injection (ICSI) for male factor infertility were randomly assigned to receive ovarian stimulation in a long agonist protocol with a combination of recombinant human FSH (r-hFSH; Gonal-F®) and recombinant human LH (r-hLH; Luveris®) ( n = 212) starting on day 6 of FSH stimulation until human chorionic gonadotrophin (HCG) at a daily fixed dose of 150 IU r-hLH, or with r-hFSH alone ( n = 219). There was no significant difference in the number of metaphase II oocytes retrieved (10.3 versus 10.4) in patients treated with r-hFSH and r-hLH versus r-hFSH alone; however, more embryos were transferred in the LH-supplemented group (2.9 versus 2.8, P = 0.037). Overall, the implantation rates were 22.9 versus 27.0% in patients treated with r-hFSH and r-hLH versus with r-hFSH alone respectively (NS). The respective numbers of MII oocytes retrieved in patients <35 or ≥35 years were 11 versus 8.3 ( P = 0.010) for patients treated with r-hFSH alone, and 10.7 versus 9.3 (NS) for those given supplemental r-hLH (150 IU) from day 6. Implantation rates in patients <35 years treated with r-hFSH were higher (30.7%) than those receiving r-hFSH and r-hLH, (23.5%) ( P = 0.068). In patients ≥35 years, the implantation rates were 21.7% for those patients supplemented with 150 IU r-hLH from day 6 of stimulation versus 15.7% when treated with FSH alone (NS). Younger patients therefore do not seem to benefit from an LH-supplemented ovarian stimulation protocol, but women ≥35 years undergoing assisted reproduction may benefit from using r-hLH in addition to r-hFSH.

Comparative efficacy and safety of cetrorelix with or without mid-cycle recombinant LH and leuprolide acetate for inhibition of premature LH surges in assisted reproduction

An open label, randomized, multi-centre study was performed to compare cetrorelix and leuprolide acetate for prevention of premature LH surge and to assess whether patients treated with cetrorelix benefit from addition of recombinant human (r-h)LH. Normo-ovulatory women ( n = 74) undergoing ovarian stimulation prior to intracytoplasmic sperm injection were treated with leuprolide acetate ( n = 25) before ovarian stimulation with recombinant human FSH (r-hFSH) or with cetrorelix 3 mg on stimulation day 7 (with ( n = 25) or without ( n = 24) r-hLH 150 IU on days 7–10). The main outcome measures were the number of metaphase II (MII) oocytes retrieved; secondary efficacy end-points; adverse events (AE) and other safety measures. There were no significant differences between groups for MII oocytes retrieved, duration of stimulation, total r-hFSH dose and pregnancy rates. The group treated with cetrorelix alone had a significantly lower concentration of oestradiol per follicle compared with the other groups. The majority of AE were mild to moderate in severity. Cetrorelix and leuprolide acetate appear to have comparable efficacy and safety, although cetrorelix has the advantage of typically requiring only one injection.

Effects of recombinant LH supplementation in women undergoing assisted reproduction with GnRH agonist down-regulation and stimulation with recombinant FSH: an opening study

Circulating endogenous concentrations of LH are reduced in women undergoing down-regulation with gonadotrophin-releasing hormone agonists (GnRHa) and ovarian stimulation with recombinant human FSH (r-hFSH). The effect of recombinant human LH (r-hLH) supplementation on ovarian response and pregnancy outcome was evaluated in a prospective randomized study (sealed envelopes) including 231 cycles. Normogonadotrophic women were stimulated with either r-hFSH or a combination of r-hFSH and r-hLH in a ratio of 2:1. LH supplementation was started from day 8 of the cycle. Blood samples for oestradiol, LH and androstendione were prospectively collected on days 1, 8 and on the day of aspiration and analysed retrospectively. Overall, the two groups did not differ with respect to pregnancy rate. In contrast, women aged ≥35 years responded to exogenous LH supplementation with significantly increased implantation rates and significantly reduced total FSH consumption as compared with the non-supplemented group. In addition, the implantation rate for a subgroup of patients with the highest endogenous LH concentrations (i.e. ≥1.99 IU/l) on day 8 was significantly increased by LH supplementation. Exogenous LH supplementation from day 8 has no detrimental effect on ovarian response and pregnancy outcome. On the contrary supplementation with r-hLH seems to benefit treatment outcome for women above 35 years of age and for the subgroup of women exhibiting LH concentrations above 1.99 IU/l on stimulation day 8.

Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability

The objective of this work was to study the effect of a preparation of human recombinant gonadotrophins (r-FSH and r-LH) on the in vitro maturation (IVM) and development of sheep oocytes. In addition, the viability of fresh and vitrified blastocysts obtained after transfer was tested. Oocytes collected from slaughtered animals were divided into five different maturation groups. All groups were matured in a medium containing TCM199 with 4 mg/ml BSA, 100 μM cysteamine and 1 μg/ml estradiol-17β. Each group was also treated with one of the following: 0.1 UI/ml r-FSH (r-FSH group), 0.1 UI/ml r-LH (r-LH group), 0.1 UI/ml r-FSH and 0.1 UI/ml r-LH (r-FSH/r-LH group), 5 μg/ml FSH and 5 μg/ml LH hypophysial gonadotrophins (h-G group) as a control, or no gonadotrophins (no-G group). After in vitro fertilization with fresh ram semen, presumptive zygotes were cultured in vitro for 6–7 days and a total of 109 blastocysts were then transferred in pairs into synchronized ewes. To determine the viability of embryos after vitrification, 36 blastocysts from the r-FSH/r-LH group and 30 from the h-G group were vitrified in 10% ethylene glycol (EG) and 10% dimethylsulphoxide (DMSO) for 5 min, followed by 20% EG, 20% DMSO and 0.5 M Sucrose (S) for <45 s. They were loaded into open pulled straws (OPS) and plunged into LN 2. After warming, the blastocysts were transferred in pairs into synchronized ewes. The highest maturation rate was reached in the r-FSH/r-LH group (91.9%). However, no statistical difference was found when this group was compared with the h-G group (84.0%). Likewise, the cleavage rate of the r-FSH/r-LH group (81.4%) was not significantly different from that of the h-G group (82.3%). The cleavage rates of all other groups, however, were significantly lower than the r-FSH/r-LH and h-G groups. The blastocyst rate was highest in the h-G group (53.6%), and it was statistically higher than in the r-FSH/r-LH group (41.5%). The blastocyst rate was very similar between groups r-FSH and r-FSH/r-LH (42.0 and 41.5%, respectively). The lowest lambing rate (31.8%) was in the no-G group. The highest lambing rate was achieved in the r-FSH/r-LH group (66.6%). The vitrified embryos of h-G and r-FSH/r-LH groups had a very similar lambing rate (16.6% and 19.4%). In conclusion, these data provide support for the hypothesis that sheep oocytes respond to human recombinant gonadotrophins used for in vitro embryo production.

Increased 17-hydroxyprogesterone (17P) responsiveness to gonadotropin stimulation in hyperthecosis (HT) compared to polycystic ovary syndrome (PCOS)

Objective: Both PCOS and HT are syndromes defined by ovarian hyperandrogenism and chronic anovulation. In PCOS, increased LH secretion together with increased theca cell responsiveness to LH is responsible for excess androgen production. In contrast, serum LH levels in HT are normal and circulating androgen levels are generaly higher than those found in PCOS. These observations suggest that theca cell sensitivity to LH in HT is greater than that of PCOS. To begin to address this issue, we compared 17P responses to recombinant human LH (r-hLH) in HT and PCOS.

The lutropin/choriogonadotropin receptor-induced phosphorylation of the extracellular signal-regulated kinases in leydig cells is mediated by a protein kinase a-dependent activation of ras.

The pathways involved in activation of the ERK1/2 cascade in Leydig cells were examined in MA-10 cells expressing the recombinant human LH receptor (hLHR) and in primary cultures of rat Leydig cell precursors. In MA-10 cells expressing the recombinant hLHR, human choriogonadotropin-induced activation of ERK1/2 is effectively inhibited by overexpression of a cAMP phosphodiesterase (a manipulation that blunts the human choriogonadotropin-induced cAMP response), by addition of H89 (a selective inhibitor of protein kinase A), or by overexpression of the heat-stable protein kinase A inhibitor, but not by overexpression of an inactive mutant of this inhibitor. Stimulation of hLHR did not activate Rap1, but activated Ras in an H89-sensitive fashion. Addition of H89 to MA-10 cells that had been cotransfected with a guanosine triphosphatase-deficient mutant of Ras almost completely inhibited the hLHR-mediated activation of ERK1/2. We also show that 8-bromo-cAMP activates Ras and ERK1/2 in MA-10 cells and in primary cultures of rat Leydig cells precursors in an H89-sensitive fashion, whereas a cAMP analog 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP (8CPT-2Me-cAMP) that is selective for cAMP-dependent guanine nucleotide exchange factor has no effect. Collectively, our results show that the hLHR-induced phosphorylation of ERK1/2 in Leydig cells is mediated by a protein kinase A-dependent activation of Ras.

Inhibitory and stimulatory regulation of testicular inhibin B secretion by luteinizing hormone and follicle-stimulating hormone, respectively, in the rhesus monkey (Macaca mulatta).

This study examined the relative role of FSH and LH in governing testicular inhibin B secretion in the rhesus monkey. Adult male monkeys, rendered hypogonadotropic and hypogonadal by administration of a GnRH receptor antagonist (acyline), were implanted with testosterone (T)-filled or empty capsules. Following T-induced restoration of spermatogenesis, both groups received recombinant human FSH and vehicle for 12 d. Juvenile male monkeys received an 11-d infusion of single-chain recombinant human LH and recombinant human FSH, either alone or in combination. In adults, chronic hypogonadotropism resulted in a modest reduction of circulating inhibin B levels, which was more than fully reversed by FSH. In the presence of T, which exerted a marked suppression in inhibin B secretion, FSH restored inhibin B levels only to those observed before acyline treatment. In juveniles, treatment with single-chain recombinant human LH led to a suppression of inhibin B secretion and curtailed the FSH-induced stimulation of this testicular hormone. The T-induced decrease in inhibin B secretion was associated with suppression in inhibin-beta(B) mRNA levels, but FSH stimulation of inhibin B secretion occurred in the absence of clear changes in expression of this subunit gene. These findings indicate that inhibin B secretion by the monkey testis is governed by the inhibitory and stimulatory action of LH and FSH, respectively. The action of LH is presumably indirect and likely mediated by T inhibition of inhibin-beta(B) gene expression. The molecular basis of the stimulatory action of FSH on inhibin B secretion requires further study.

Clinical evidence for an LH 'ceiling' effect induced by administration of recombinant human LH during the late follicular phase of stimulated cycles in World Health Organization type I and type II anovulation

The objective of these studies was to test the hypothesis that over-dosing with recombinant human LH (rLH) during the late follicular phase would suppress the development of follicles. Two double-blind studies were conducted. In study A, WHO I anovulatory patients received treatment with rFSH and rLH. When at least one follicle reached a mean diameter of 10-13 mm, patients were randomized using a computer-generated randomization list (stratified by centre) to rFSH and rLH (225 IU/day) (n = 8) or rLH alone (n = 6) or rFSH alone (n = 6). In study B, WHO II anovulatory patients with a hyper-responsive FSH were randomized to rLH (225 IU/day) (n = 4) or rLH 450 IU/day (n = 8) or placebo (n = 5). Study A: the mean number of follicles >/=11 mm was 4.2 +/- 0.3 in the rFSH group, 1.5 +/- 0.7 in the rLH group and 6.0 +/- 2.3 in the rFSH/rLH group (P = 0.07). 0/8 patients presented follicular growth arrest in the rFSH group, but 4/6 in the rLH group and 1/6 in the rFSH/rLH did. Study B: 5/12 patients presented follicular growth arrest in the rLH groups, but none in the placebo group. The mean number of follicles >/=11 mm was 4.6 +/- 1.8 for the placebo group, 2.5 +/- 1.9 for the rLH 225 IU group and 4.2 +/- 1.4 in the rLH 450 IU group (not significant). Results of this pilot study suggest that rLH alone can trigger follicular growth arrest in a significant number of patients, suggesting the existence of an 'LH ceiling' during late follicular maturation.

Pregnancy after administration of high dose recombinant human LH alone to support final stages of follicular maturation in a woman with long-standing hypogonadotrophic hypogonadism

Traditionally, the roles of LH in folliculogenesis have been considered to be limited to stimulating theca cells androgen production, triggering ovulation and supporting the corpus luteum. However, in the late stages of follicle development, granulosa cells become receptive to LH stimulation and LH becomes capable of exerting its actions on both theca cells and granulosa cells. Thus, it has been postulated that once an appropriate (i.e. LH-responsive) stage of follicular development has been achieved in response to treatment with FSH, there are theoretical grounds for reducing or completely withdrawing FSH and maintaining tonic stimulation of the dominant follicle with exogenous LH. This hypothesis was tested in a woman with long-standing hypogonadotrophic hypogonadism, which is the best and only true model to investigate correctly any LH hypothesis. Ovulation induction treatment was carried out with daily s.c. injections of 150 IU recombinant human FSH (rhFSH) (increased to 225 IU daily on stimulation day 15) and 375 IU recombinant human LH (rhLH). When a 14-mm follicle was identified on stimulation day 26, rhFSH was discontinued and from treatment days 26 to 29 the patient was given only rhLH at the above-mentioned dose of 375 IU/day. On treatment day 30, the single dominant follicle measured 22 mm in diameter and oestradiol serum concentration was 148 pg/ml. Thus, an injection of 10,000 IU i.m. human chorionic gonadotrophin was given and sexual intercourse was advised. The patient conceived and a viable singleton intrauterine pregnancy was obtained.

The clinical therapeutic window for luteinizing hormone in controlled ovarian stimulation

Objective: To discuss the clinical therapeutic window for LH during the follicular phase. Design: Review of selected papers that were retrieved through a Medline search and a review of clinical trials, the results of which are in the process of publication. Patient(s): Women undergoing infertility treatment. Intervention(s): Recombinant human LH (r-hLH) was administered SC as a supplement to FSH during controlled ovarian hyperstimulation. Main Outcome Measure(s): Follicular development, E 2 production, and endometrial thickness. Result(s): Optimal follicular maturation is the result of both FSH and LH stimulation. In patients with hypogonadotropic hypogonadism, 75 IU of r-hLH and 150 IU of FSH per day resulted in more follicles and provided sufficient E 2 for optimal endometrial proliferation. Additional r-hLH (>250 IU/day), in patients with either hypogonadotropic hypogonadism or polycystic ovary disease, may precipitate a series of deleterious physiological actions leading to atresia of developing follicles. Adding r-hLH to FSH in women treated with GnRH agonist showed no benefits in terms of number of mature oocytes, fertilization, and cleavage. However, those who experience profound pituitary desensitization may benefit from adding LH to the stimulation protocol. No obvious clinical criteria have been established to define this group of patients. Conclusion(s): A “threshold” and “ceiling” level for LH (therapeutic window) is proposed, below which E 2 production is not adequate and above which LH may be detrimental to follicular development.

Pulsatile iv Infusion of Recombinant Human LH in Leuprolide-Suppressed Men Unmasks Impoverished Leydig-Cell Secretory Responsiveness to Midphysiological LH Drive in the Aging Male

The present investigation tests the clinical hypothesis that Leydig-cell responsiveness to pulsatile and midphysiological LH drive is impaired in older men. To this end, we implemented a novel clinical investigative paradigm consisting of preadministration of an LH-down-regulating dose (3.75 mg) of leuprolide acetate followed, 3–4 wk later, by controlled challenge of the testis, with pulsatile iv infusions of saline vs. recombinant human (rh) LH. Based on a preliminary dose-finding experiment, we evaluated LH action in 8 young (ages, 18–25 yr) and 7 older (ages, 60–85 yr) volunteers by infusing eight consecutive 6-min squarewave pulses of saline or 50 IU rhLH iv every 2 h. Analyses were carried out 48 or 72 h apart in a prospective, randomly assigned, double-blind, within-subject cross-over design. Serum concentrations of T (RIA) and LH (immunoradiometric assay) were measured in blood sampled every 10 min concurrently. Leuprolide injection suppressed pre-LH-infusion (0800 h baseline) serum T concentrations (pooled mean ± sem) markedly in both age groups (P < 10−3); namely, to 40 ± 20 ng/dl (young) and 12± 3.1 ng/dl (older; P < 0.05 vs. young) (to convert to nm, multiply by 0.0347). Successive iv pulses of rhLH stimulated T output, over time, to an asymptotic maximum of 166 ± 42 ng/dl in young men (P = 0.0008 vs. saline) and 57 ± 9.8 ng/dl in older subjects (P = NS vs. saline, and P < 0.05 vs. young). Further regression analyses identified significant reductions of both the initial rate and maximum of the time-dependent incremental rise in LH-driven serum T concentrations in older men. In contrast, infused serum LH concentrations, distribution volumes, and calculated LH half-lives were comparable in the two age cohorts. We conclude that older men manifest both a delayed initial and reduced maximal serum T concentration rise compared with young men exposed to identical controlled midphysiological pulsatile LH drive.

Differential regulation of inhibin A and inhibin B by luteinizing hormone, follicle-stimulating hormone, and stage of follicle development.

Inhibin B and inhibin A exhibit unique patterns of secretion across the follicular phase of the menstrual cycle. To test the hypothesis that the distinct patterns of inhibin B and inhibin A secretion result from differential regulation by LH and FSH, a series of controlled experiments was designed to dissect the specific effects of LH and FSH at distinct stages of follicle development. After GnRH agonist desensitization, women with small antral follicles were treated with recombinant human LH (rhLH), rhFSH, or rhFSH and estradiol (E(2)). rhLH or rhFSH was also administered when follicles reached the preovulatory stage in gonadotropin-stimulated or spontaneous cycles. At the small antral stage of development, rhFSH, but not rhLH, administration increased inhibin B (17.4 +/- 4.6 to 321.0 +/- 97.0 pg/mL; P < 0.05), inhibin A (0.6 +/- 0.1 to 2.6 +/- 0.6 IU/mL; P < 0.05), and E(2) [15.8 +/- 3.6 to 95.3 +/- 26.9 pg/mL (58.0 +/- 13.2 to 349.8 +/- 98.7 pmol/L); P < 0.05]. The inhibin B increase preceded inhibin A by 48 h. Addition of E(2) to FSH resulted in a greater increase in inhibin B (23.2 +/- 6.4 to 865.2 +/- 294.5 pg/mL; P < 0.05) than FSH alone (P < 0.05). At the preovulatory stage, rhLH administration increased inhibin A (15.9 +/- 10.3 to 21.5 +/- 13.7 IU/mL; P < 0.05) and E(2) [669.4 +/- 285.5 to 943.6 +/- 388.1 pg/mL (2457.4 +/- 1048.1 to 3464.0 +/- 1424.7 pmol/L); P < 0.05], but not inhibin B, as did rhFSH administration in spontaneous cycles [E(2): 226.4 +/- 102.7 to 264.7 +/- 121.0 pg/mL (831.1 +/- 377.0 to 971.7 +/- 444.2 pmol/L); P < 0.05; inhibin A: 2.6 +/- 1.3 to 3.7 +/- 1.9 IU/mL; P < 0.05; and inhibin B: 76.3 +/- 32.2 to 77.6 +/- 32.8 pg/mL; P = NS]. These findings suggest that increases in both FSH and E(2) in the early follicular phase result in increased inhibin B secretion at early stages of follicle development, whereas the selective LH rise in the late follicular phase favors inhibin A secretion from more mature follicles. Thus, both differential secretion of LH and FSH and the stage of follicle development determine the patterns of inhibin A and inhibin B secretion in the normal menstrual cycle.

Reactivity of different LH and FSH standards and preparations in the world health organization matched reagents for enzyme-linked immunoassays of gonadotrophins.

The immunoreactivity of various LH and FSH calibration standards and recombinant preparations in the enzyme-linked immunoassay (EIA) systems for gonadotrophins developed for the Special Programme of Research in Human Reproduction of the World Health Organization (WHO) were compared. The preparations tested included three LH and two FSH pituitary standards (calibrated against LH 80/552 and 68/40 and FSH 78/549 respectively) provided with the EIA or radioimmunoassay WHO matched reagent kits, the pituitary preparation LER-907, and recombinant human LH (rhLH) and FSH (rhFSH). Simultaneous curve fitting of the EIA dose-response curves revealed no significant differences among the slopes generated by the WHO LH standards and LER-907; in contrast, no parallelism was found between the curves of rhLH and the pituitary-derived LH standards. No significant differences were found among the slopes of the curves elicited by the pituitary and recombinant FSH preparations. Each LH preparation exhibited a high degree of charge heterogeneity. Considerable variations in charge isoform distribution among the WHO LH standards, rhLH and LER-907 were also evident. In contrast, the FSH preparations were less heterogeneous and exhibited minor differences in charge distribution. Despite the existing differences in charge isoform distribution, all the pituitary-derived preparations as well as rhFSH seem appropriate for using as calibration standards in this particular EIA system.

Titrating luteinizing hormone replacement to sustain the structure and function of the corpus luteum after gonadotropin-releasing hormone antagonist treatment in rhesus monkeys.

These studies were designed to identify 1) a regimen of a third generation GnRH antagonist that abolishes primate luteal function, and 2) the amount of LH replacement required to maintain the structure and functional life span of the corpus luteum of the menstrual cycle after GnRH antagonist treatment. A single injection of antide at 3 or 5 mg/kg BW on day 6 of the luteal phase suppressed serum progesterone levels within 1 day of treatment, but levels recovered within 4 days. Administration of antide (3 mg/kg) for 3 days (luteal days 6-8) reduced (P < 0.05) serum progesterone below 1 ng/mL and maintained these low levels for the entire sampling period; in subsequent experiments, all monkeys received this antide regimen. Fixed doses (5, 10, or 20 IU) of recombinant human LH administered at 8-h intervals during and after antide treatment stimulated progesterone production in a dose-dependent manner; these monkeys menstruated earlier than controls regardless of treatment group. Replacement with an escalating dose regimen (5-20 IU) of LH resulted in typical serum progesterone and relaxin levels throughout a luteal phase of normal length. Corpora lutea removed on day 10 from monkeys treated with antide alone had decreased wet weight (P < 0.05) and few large luteal cells; coadministration of the escalating dose regimen of LH maintained luteal structure similar to that seen in time-matched controls. Antide-only treatment increased progesterone receptor (PR) messenger ribonucleic acid, but decreased PR immunostaining in luteal tissue; the escalating dose regimen of LH maintained PR messenger ribonucleic acid and immunostaining similar to those in controls. This study indicates that during GnRH antagonist administration, an escalating dose regimen of LH replacement is optimal for maintenance of the structure and functional life span of the primate corpus luteum.

Clinical pharmacology of recombinant human luteinizing hormone: Part I. Pharmacokinetics after intravenous administration to healthy female volunteers and comparison with urinary human luteinizing hormone

Objective: To assess the pharmacokinetics after IV administration of a recombinant human LH and to compare them to those of a reference hMG preparation containing urinary human LH. Design: Prospective, dose-escalating, cross-over study. Setting: Phase I clinical research environment. Patient(s): Twelve healthy pituitary down-regulated females. Intervention(s): Subjects received single IV doses of 300, 10,000, and 40,000 IU of recombinant human LH, followed by a single IV dose of 300 IU of hMG, all separated by 1 week. Main Outcome Measure(s): Pharmacokinetic parameters. Results: For both preparations, LH serum levels were well described by similar biexponential models. The pharmacokinetics of recombinant human LH were linear over the 300 to 40,000 IU range. After a rapid distribution phase with an initial half-life of 1 hour, both recombinant human LH and urinary human LH were eliminated with a terminal half-life of 10–12 hours. Total serum clearance was 1.7 Lgn with <4% and 30% of the dose being eliminated in the urine for recombinant human LH and urinary human LH, respectively. The volume of distribution at steady-state was approximately 10 L. Irrespective of the dose, recombinant human LH was well tolerated. Conclusion(s): The pharmacokinetics of recombinant human LH are linear with dose and similar to those of urinary human LH.

Pharmacokinetic and pharmacodynamic interactions between recombinant human luteinizing hormone and recombinant human follicle-stimulating hormone

Objective: To assess the pharmacokinetics of a recombinant human LH preparation and its pharmacokinetic and pharmacodynamic interactions with recombinant human follicle-stimulating hormone (FSH). Design: Prospective, randomized cross-over study. Setting: Phase I clinical research environment. Patient(s): Twelve healthy pituitary down-regulated females. Intervention(s): Subjects received 150 IU of SC recombinant human LH and FSH, either alone or in combination, followed by recombinant human LH and FSH once daily for 7 days. Main Outcome Measure(s): Pharmacokinetic parameters, ovarian follicle development. Result(s): No pharmacokinetic interaction between recombinant human LH and FSH was observed, with no significant difference in baseline-corrected maximal observed concentration over baseline, area under the concentration-time curve from t = 0 to t = 24 hours, or time to maximal concentration after single doses alone or in combination. After daily administration, the mean accumulation ratio was 1.6 for LH and 2.9 for FSH, with absorption and terminal phase half-life estimates of 4 and 11 hours for LH and 8 and 16 hours for FSH, respectively. Combined administration of FSH and LH for 7 days was effective in stimulating ovarian follicular development and steroidogenesis, with large interindividual variability related to ovarian sensitivity. Conclusion(s): A new recombinant human LH preparation has a low accumulation ratio at steady-state and no pharmacokinetic or pharmacodynamic interactions with recombinant human FSH.

Clinical pharmacology of recombinant human luteinizing hormone: Part II. Bioavailability of recombinant human luteinizing hormone assessed with an immunoassay and an in vitro bioassay

Objective: To assess the single-dose pharmacokinetics of a recombinant human LH preparation administered by the IV, IM, and SC route. Design: Prospective, randomized cross-over study. Setting: Phase I clinical research environment. Patient(s): Twelve healthy pituitary down-regulated females. Intervention(s): Subjects received single IV, IM, and SC doses of 10,000 IU of recombinant human LH, each separated by 1 week. Main Outcome Measure(s): Pharmacokinetic parameters. Result(s): After single IV administration, the pharmacokinetics were described by a two-compartment model, after IM or SC administration, by a one-compartment model with zero order absorption and a lag time. Using the immunoassay, after IV administration initial half-life was 1 hour and terminal half-life was 10 hours (half-life was prolonged after extravascular administration, suggesting rate-limiting absorption). Total serum clearance was 2.6 L/h, and steady-state volume of distribution was 14 L. Observed C max, after IM and SC administration, was 43 IU/L with median t max of 9 hours (IM) and 5 hours (SC). Bioavailability was 0.54 (IM) and 0.56 (SC). The pharmacokinetics of LH are comparable using an in vitro bioassay. Conclusion(s): The terminal half-life of recombinant human LH is around 12 hours and is slightly prolonged after extravascular administration. The pharmacokinetics are similar after IM and SC injection, and one-half the administered dose is available systemically.

Follicle-stimulating hormone and luteinizing hormone/chorionic gonadotropin stimulation of vascular endothelial growth factor production by macaque granulosa cells from pre- and periovulatory follicles.

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 +/- 471 vs. 111 +/- 26 pg/mL, mean +/- SEM; P < 0.05). In vitro treatment with hCG increased (P < 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P < 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.

The mouse inhibin α-subunit promoter directs SV40 T-antigen to Leydig cells in transgenic mice

Testicular tumorigenesis was observed in transgenic mice expressing the 6-kb mouse inhibin α-subnit promoter/Simian virus 40 T-antigen (SV40 Tag) fusion gene. The tumors were confined to Leydig cells using immunohistochemistry with anti-Tag antibody, specific binding of biotinylated hCG and histochemistry for 3β-hydroxysteroid dehydrogenase. Leydig cell hyperplasia and presence of Tag protein in the testicular interstitial tissue were already evident at 5 and 6.5 days of age, respectively. An immortalized cell line, BLT-1, was established from one testicular tumor. These cells expressed the LH receptor and P450scc mRNAs, and displayed LH-responsive cAMP and progesterone production, and low testosterone production. The cells also specifically bound 125I-labeled recombinant human LH with high affinity (36 000 binding sites/cell), and the binding was regulated by 8Br-cAMP and hCG. This gonadal tumor model is valuable for further studies on endocrine functions of Leydig cells and their tumorigenesis in vivo and in vitro.

The luteinizing hormone surge—the final stage in ovulation induction: modern aspects of ovulation triggering

To compile updated information regarding gonadotropin secretion, specifically the physiology of the midcycle LH surge, in natural cycles and under various ovulation induction protocols. Studies that deal with the clinical aspects of LH surge manipulation or substitution were identified through literature and Medline searches. Three major regulatory factors have been identified as participants in the induction of the midcycle gonadotropin surge. These are hypothalamic GnRH secretion, ovarian and adrenal steroids, and less well-characterized ovarian peptide hormones. Gonadotropin-releasing hormone pulsatility is regulated by a complex mechanism that integrates multiple neurotransmitters and sex steroids. Estradiol plays a central role in the pituitary secretion of LH, which also is influenced by P concentrations. Gonadotropin surge attenuating factor also has been implicated in the regulation of timing and amplitude of the LH surge. Human chorionic gonadotropin is used extensively as a LH surrogate, but its use is associated with a number of disadvantages. Induction of an endogenous LH surge through use of the flare effect of GnRH analogues has been examined more recently and has been found to have several advantages. Recombinant human LH is in the final stages of clinical testing. Although much is known about the physiology of the midcycle LH surge and its variations under different clinical conditions, new approaches to the induction or substitution of the LH surge currently are being examined and learned. The introduction of recombinant gonadotropins into clinical practice is likely to influence ovulation induction and IVF practice to a significant degree in the near future.