Follicle-stimulating Hormone Analogs Research Articles

Production of Recombinant Single-Chain Eel Luteinizing Hormone and Follicle-Stimulating Hormone Analogs in Chinese Hamster Ovary Suspension Cell Culture.

We produced rec-single chain eel luteinizing (rec-eel LH) and follicle-stimulating (rec- eel FSH) hormones displaying high biological activity in Chinese hamster ovary suspension (CHO-S) cells. We constructed several mutants, in which a linker, including an O-linked glycosylated carboxyl-terminal peptide (CTP) of an equine chorionic gonadotropin (eCG) β-subunit, was attached between the β- and α-subunit (LH-M and FSH-M) or in the N-terminal (C-LH and C-FSH) or C-terminal (LH-C and FSH-C) regions. The plasmids were transfected into CHO-S cells, and culture supernatants were collected. The secretion of mutants from the CHO-S cells was faster than that of eel LHβ/α-wt and FSHβ/α-wt proteins. The molecular weight of eel LHβ/α-wt and eel FSHβ/α-wt was 32-34 and 34-36 kDa, respectively, and that of LH-M and FSH-M was 40-43 and 42-45 kDa, respectively. Peptide-N-glycanase F-treatment markedly decreased the molecular weight by approximately 8-10 kDa. The EC50 value and the maximal responsiveness of the eel LH-M and eel FSH-M increased compared with the wild-type proteins. These results show that the CTP region plays a pivotal role in early secretion and signal transduction. We suggest that novel rec-eel LH and FSH proteins, exhibiting potent activity, could be produced in large quantities using a stable CHO cell system.

Rational design of hyper-glycosylated human follicle-stimulating hormone analogs (a bioinformatics approach)

N-glycosylation is a complex mechanism in which the carbohydrate molecules bind to the Asn amino acid in the N-glycan consensus sequence (AsnXxxThr/Ser sequon, where Xxx is any residue, excluding Pro). Introduction of additional N-linked glycosylation site into proposed location in the protein causes to its hyper-glycosylation and can enhance the protein characteristics to provide promising prospects in treatment. Glycoengineering is a favorably used strategy to design and generate hyper-glycosylated variants. In this research, human follicle-stimulating hormone (HuFSH) was considered to identify appropriate positions for adding novel N-glycan sites. A rational computational strategy was applied to predict functional/structural variations induced through changes in polypeptide chain. We analyzed the amino acid chain of FSH to find out the proper locations to introduce asparagine and/or threonine for creating novel N-glycan positions. This analysis resulted in the recognition of 40 possible N-glycosylation positions, and then the eight adequate ones were chosen for additional investigation. The model validation techniques were used to examine 3-dimensional structures of the chosen mutant proteins. Finally, 2 mutants with a further glycan site were recommended as eligible FSH hyper-glycosylated analogs, which may be regarded for subsequent experimental studies. Our in silico approach may decrease tedious and time-wasting laboratory researches of the mutants. Communicated by Ramaswamy H. Sharma

Evaluation of gonadotropin-replacement therapy in male patients with hypogonadotropic hypogonadism.

Hypogonadotropic hypogonadism (HH) is a rare disease in which medical treatment has a high success rate to achieve fertility. This study aimed to analyze the efficacy of hormone replacement therapy and determine predictive factors for successful spermatogenesis and spontaneous pregnancy in patients with idiopathic HH. A total of 112 patients with low testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and normal prolactin levels were diagnosed with HH and administered LH and FSH analogs as hormone replacement therapy. During treatment, 96 (85.7%) patients had sperm present in ejaculate samples. Among these patients, 72 were married and wanted a child. Of these 72 patients, 48 (66.7%) of couples had pregnancies from natural conception. After initiation of treatment, the mean time for the appearance of sperm in semen was 9.48 months. There were no significant differences between baseline FSH, T, and LH levels; however, older age, larger testicular size, and low rate of undescended testes were favorable factors for successful spermatogenesis. Larger testicular size and older age were also the main predictive factors for natural conception. We found that patients with undescended testes had a younger age, smaller testes, and lower T levels compared with patients exhibiting descended testes. The rate of sperm found in the ejaculate was not significantly decreased in patients with undescended compared with descended testis (73.7% vs 87.6%, P = 0.261). The medical approach for males with HH and azoospermia provides a successful treatment modality in regard to successful spermatogenesis and achievement of pregnancy.

Molecular regulation of follicle-stimulating hormone synthesis, secretion and action.

Follicle-stimulating hormone (FSH) plays fundamental roles in male and female fertility. FSH is a heterodimeric glycoprotein expressed by gonadotrophs in the anterior pituitary. The hormone-specific FSHβ-subunit is non-covalently associated with the common α-subunit that is also present in the luteinizing hormone (LH), another gonadotrophic hormone secreted by gonadotrophs and thyroid-stimulating hormone (TSH) secreted by thyrotrophs. Several decades of research led to the purification, structural characterization and physiological regulation of FSH in a variety of species including humans. With the advent of molecular tools, availability of immortalized gonadotroph cell lines and genetically modified mouse models, our knowledge on molecular mechanisms of FSH regulation has tremendously expanded. Several key players that regulate FSH synthesis, sorting, secretion and action in gonads and extragonadal tissues have been identified in a physiological setting. Novel post-transcriptional and post-translational regulatory mechanisms have also been identified that provide additional layers of regulation mediating FSH homeostasis. Recombinant human FSH analogs hold promise for a variety of clinical applications, whereas blocking antibodies against FSH may prove efficacious for preventing age-dependent bone loss and adiposity. It is anticipated that several exciting new discoveries uncovering all aspects of FSH biology will soon be forthcoming.

Preparation, characterization and application of long-acting FSH analogs for assisted reproduction

Assisted reproduction technologies are widely used in humans and domestic animals and often include follicle stimulating hormone (FSH) in the protocol. One limitation with most of the available FSH preparations is the relative short half-life in the circulation that dictates multiple daily injections for the desired follicle development and superovulation. The development of bioactive long-acting structurally modified FSH analogs is desirable for human and veterinary use. In addition, optimal preparations and/or formulations are expected to improve the regimen and efficiency of the treatment. This review briefly describes the approaches that have been explored to extend the half-life of FSH in the circulation. These include strategies to increase the mass and/or charge of FSH and to prevent the dissociation of the hormone to inactive subunits components. Most of these strategies, except one that led to a registered drug (Elonva) indicated for controlled ovarian stimulation protocols in humans, are still in experimental stage.

LAPS-FSH: a new and effective long-acting follicle-stimulating hormone analogue for the treatment of infertility

Although several long-acting follicle-stimulating hormone (FSH) therapies have been developed to enhance the ovarian response, a disadvantage of FSH therapy is its relatively short half-life, which requires women to receive one to two injections per day for almost 2 weeks. In the present study, we developed a novel FSH analogue by conjugating recombinant human FSH (rhFSH) and the constant region of the human immunoglobulin G4 fragment via non-peptidyl linkers. The efficacy of the FSH analogue was evaluated in vitro by cAMP level assessments, pharmacokinetic studies and a determination of ovarian weight and by comparing these findings with the results from other FSH analogues. In addition, the total number of antral and Graafian follicles was determined after 7 days of treatment with control, 6µgkg(-1) follitropin β, 6, 12 or 42µgkg(-1) corifollitropin α or 3, 6 or 12µgkg(-1) long acting protein/peptide discovery-follicle-stimulating hormone (LAPS-FSH). As a result, the animals treated with 12µgkg(-1) LAPS-FSH produced additional and larger healthy follicles. These data demonstrate that LAPS-FSH promotes growth and inhibits atresia of the ovarian follicle compared with other available drugs, suggesting that our new drug enhances the efficacy and duration of treatment. It is expected that our new FSH analogue will result in a higher chance of pregnancy in patients who are unresponsive to other drugs.

Drug delivery to the testis: current status and potential pathways for the development of novel therapeutics.

Nanotechnology has been increasingly utilized for the targeting and delivery of novel therapeutic agents to different tissues and cell types. The current therapeutic options for testicular disorders fall short in many instances due to difficulty traversing the blood-testis barrier, systemic toxicities, and complicated dosing regiments. For testicular tissue, potential targeting can be obtained either via anatomic methods or specific ligands such as luteinizing hormone or follicle-stimulating hormone analogs. Potential novel therapeutic agents include DNA, RNA, cytokines, peptide receptor antagonists, peptide receptor agonists, hormones, and enzymes. Nanotherapeutic treatment of testicular cancer, infertility, testicular torsion, orchalgia, hypogonadism, testicular infections, and cryptorchidism within the framework of potential target cells are an emerging area of research. While there are many potential applications of nanotechnology in drug delivery to the testis, this remains a relatively unexplored field. This review highlights the current status as well as potential future of nanotechnology in the development of novel therapeutics for testicular disorders.

Efficacy of native and hyperglycosylated follicle-stimulating hormone analogs for promoting fertility in female mice

To compare the efficacy of recombinant human FSH (rhFSH) with rhFSH with four additional O-linked carbohydrates (rhFSH-CTP), rhFSH with four additional N-linked carbohydrates (rhFSH-N4), and the current gold standard for rodent ovarian stimulation, pregnant mare serum gonadotropin (PMSG), on fertility parameters in mice. Animal study. Academic research center. Adult C57Bl/6J female mice. Ovarian stimulation with 5 IU of rhFSH, rhFSH-CTP, rhFSH-N4, or PMSG. Forty-six hours later, 5 IU of hCG was injected to promote ovulation and females were mated overnight. Eggs retrieved after ovulation, in vitro embryo development, delivery rate, and litter size. The hyperglycosylated FSH analogs, rhFSH-CTP and rhFSH-N4, enhanced ovulation and embryo maturation significantly better than rhFSH. RhFSH-N4 produced more eggs (28.5 +/- 1.9 per mouse) and embryos (17.8 +/- 1.6) compared with rhFSH-CTP (18.3 +/- 1.2 and 9.0 +/- 1.0, respectively). Treatment with rhFSH, rhFSH-N4, and PMSG produced statistically equivalent delivery rates and litter sizes. The delivery rate was surprisingly lower with rhFSH-CTP (14%) compared with PMSG (33%). Compared with rhFSH, treatment with hyperglycosylated rhFSH-CTP and rhFSH-N4 led to superior rates of ovulated eggs and subsequent in vitro embryo development. RhFSH-N4 was equivalent to PMSG, while all of the fertility parameters studied were lower with rhFSH-CTP than with PMSG therapy.

Effects of long-acting recombinant human follicle-stimulating hormone analogs containing N-linked glycosylation on murine folliculogenesis

To evaluate the efficacy of two novel long-acting rhFSH analogs, rhFSH-N2 and rhFSH-N4, in stimulating murine folliculogenesis. Experimental study. Academic research environment. Immature female mice. Recombinant hFSH-N2 and -N4 were administered via single IP injection to 3-week-old female mice (n = 10) who were killed 48 hours later for dissection and histologic examination of reproductive organs and serum inhibin A. Results were compared with other groups of mice who received either single or q 12 hour injections for 48 hours of commercial rhFSH, or a single injection of pregnant mare serum gonadotropin (PMSG). A subgroup of the mice receiving rhFSH-N4 was supplemented with daily injections of small doses of hCG to simulate LH add-back. Serum inhibin A levels, ovarian and uterine weights, and ovarian antral follicle counts. Recombinant human FSH-N2 and -N4 administration induced a statistically significant increase in ovarian weights, uterine weights, and inhibin A levels compared with single and twice-daily injection of rhFSH. PMSG induced the greatest increases in all three measured parameters. There was no statistical difference between rhFSH-N2 and rhFSH-N4 for any parameter analyzed. A single injection of rhFSH-N2 or -N4 induced a greater number of antral follicles than did either single or q 12 hour injections of rhFSH. The addition of small doses of hCG to rhFSH-N4 increased inhibin A levels and antral follicle number to reach statistical equivalence to PMSG treatment. Addition of a synthetic polypeptide containing two or four N-linked glycosylation sites to rhFSH increases in vivo bioactivity of the hormone compared to commercial rhFSH. After a single injection, both rhFSH-N2 and rhFSH-N4 effectively induced a greater follicular response in the mouse than did rhFSH.

Long-acting follicle-stimulating hormone analogs containing N-linked glycosylation exhibited increased bioactivity compared with o-linked analogs in female rats.

The effects of altering the number and type of additional carbohydrate moieties on the pharmacokinetic and pharmacodynamic properties of FSH were examined in this report. A series of single-chain follitropins, containing variable numbers of additional N- (or O-) linked carbohydrates, were designed and expressed in Chinese hamster ovary cells. Proper folding, efficient receptor binding, and signal transduction were confirmed by in vitro assays. Pharmacokinetic and pharmacodynamic parameters were evaluated in immature female Sprague Dawley rats. Increasing the number of glycosylation sites with either N- (or O-) linked moieties extended the elimination half-life as much as 2-fold compared with recombinant human FSH (rhFSH). However, there was a maximum elimination half-life such that further glycosylation provided no additional lengthening of the half-life. Conversely, biopotency, as assessed by inhibin A levels 74 h post injection, and follicle production were significantly higher for the N-linked analogs. Rats stimulated with the longest acting analogs (either N- or O-linked) showed significantly higher ovarian weights than rats receiving a single injection of rhFSH. The analog containing four additional N-linked sites (rhFSH-N4) had the greatest number of large, preovulatory follicles. Although the half-life of rhFSH-N4 displayed no further enhancement beyond the other longest acting analogs, this analog exhibited significantly increased biopotency in rats. This work provides the basis for the generation of a series of reagents potentially useful for therapeutic applications.

Novel long-acting human follicle stimulating hormone (FSH) analogue requires fewer injections than standard recombinant FSH during non-human primate ovarian hyperstimulation

Current recombinant gonadotropin preparations are administered as daily injections due to their relatively short half-life. Decreasing the frequency of administration would facilitate therapy and be more tolerable for the patient. We have developed a single chain recombinant human FSH (rhFSH) analogue with four additional N-linked glycosylation sites (rhFSH-N4) which in the murine model, has a prolonged half-life and greater in vivo bioactivity than commercially available rhFSH. In a previously reported controlled ovarian hyperstimulation (COH) trial in the baboon, 75 IU of rhFSH-N4 was shown to have a prolonged half-life of 48 hours, but failed to induce adequate follicular growth. The objective of this study was to induce folliculogenesis in the baboon using a single high-dose injection of rhFSH-N4 and to compare its effectiveness to daily rhFSH. Experimental study. COH was initiated in a multiparous, regularly cycling female baboon by administering a single 200 IU injection of rhFSH-N4 subcutaneously (SC) on the second day of her menstrual cycle. Concurrently, a second female baboon began daily SC injections of 75IU rhFSH also on day 2 of her menstrual cycle. Both baboons were monitored daily with serum estradiol (E2), luteinizing hormone (LH) and FSH levels. Pelvic ultrasound was performed under anesthesia (ketamine/xylazine) every other day throughout the cycle to monitor number and size of follicles. Once E2 levels plateaued in the baboon receiving rhFSH-N4, daily SC injections of 75 IU rhFSH were begun to maintain follicular growth during the COH cycle. Once E2 levels had peaked in both baboons, daily progesterone levels were added to detect spontaneous ovulation. Gonadotropin stimulation was terminated on cycle day 12, when no further increases in E2 levels or follicular growth could be detected. After a single high-dose injection of rhFSH-N4, serum FSH levels rose rapidly to a peak level of 17.9 mIU/mL, and gradually declined to a level of 7.2 mIU/mL by cycle day 7, at which point daily injections of 75 IU rhFSH were initiated (Fig. 1.) Estradiol levels mimicked the FSH levels, in that a single injection of rhFSH-N4 maintained elevated estradiol levels for six days before requiring additional daily rhFSH (Fig. 2.) Multiple follicular growth (4–7 follicles of >5mm diameter) was documented by ultrasound on cycle day 8 for both baboons. Neither baboon demonstrated an endogenous LH surge during the stimulation protocol, indicating the need for hCG to induce ovulation in future COH cycles. The use of a 200 IU loading dose of a novel, long-acting hFSH analogue, rhFSH-N4, in baboons as part of a modified human ovarian stimulation protocol was successful in recruitment of multiple follicles while requiring significantly fewer injections than commercially available rhFSH. Decreasing the number of daily injections is particularly advantageous for baboon COH protocols as it minimizes the amount of daily stress on the animal. Ultimately, use of a long-acting hFSH analogue could reduce the number of daily injections required of women undergoing ovarian hyperstimulation during in-vitro fertilization cycles.

Use of a novel long-acting human follicle-stimulating hormone analogue containing four extra n-linked carbohydrates in the ovarian hyperstimulation of a female baboon

Objective: A single chain human follicle-stimulating hormone (FSH) analogue with four additional N-linked glycosylation sites (rhFSH-N4) has recently been shown to prolong elimination half-life (T1/2) and increase biopotency as compared to commercially available recombinant human FSH (rhFSH) in the murine model. Longer-acting gonadotropins may improve patient compliance and comfort by requiring fewer injections in women undergoing controlled ovarian hyperstimulation (COH) protocols for in vitro fertilization (IVF). Superovulation with rhFSH given daily and collection of oocytes via aspiration has been examined in the baboon. Use of rhFSH-N4 in baboons for the purpose of COH and collection of multiple oocytes via uterine lavage, as well as T1/2 determination of the analogue, were examined. Design: Experimental study. Materials and Methods: A multiparous, regularly cycling female baboon was chosen to undergo COH using experimental rhFSH-N4. Ovarian stimulation was initiated on day 1 of the menstrual cycle with a subcutaneous (SC) injection of rhFSH-N4 (75 IU). A 2nd dose of the rhFSH-N4 (75 IU) was given on day 5 when serum estradiol (E2) levels had plateaued. A GnRH antagonist (250μg SC) was given daily starting on day 6 until the day of hCG administration. Serum E2, FSH, and progesterone levels were measured daily. Pelvic ultrasound was performed under anesthesia (ketamine/xylazine) every other day to monitor number and size of follicles until hCG was given. Recombinant hCG (250μg SC) was given when majority of follicles were >5mm in diameter. 7 days post-ovulation, uterus was lavaged under anesthesia with 10ml of warm, sterile, isotonic, culture medium using a 27-g double-barreled cannula through the cervical canal to collect unfertilized oocytes. Results: Serum E2 levels started to rise on day 3 and plateaued after 5 days, requiring a 2nd dose of rhFSH-N4 to be given. Serum E2 peaked and plateaued by day 6–9 of stimulation. Follicular development was not observed until day 8–9. Majority of the follicles (n=7) increased to >5mm in diameter on day 10 when hCG was given. No oocytes were collected via uterine lavage 7 days post-ovulation. Serum FSH levels increased after each dose of rhFSH-N4, and its T1/2 was 48 hours. Serum progesterone levels followed a pattern consistent with ovulation after administration of hCG. Conclusions: Prolonged T1/2 of rhFSH-N4 in a primate species was confirmed. Use of rhFSH-N4 in baboons as part of a modified human COH protocol is successful in inducing follicular development as evident by increased E2 production and growing follicular size, while requiring fewer injections as compared to commercially available rhFSH. However, no oocytes were collected via uterine lavage. Protocol needs to be modified to account for the delayed tubal transport that may accompany hyperstimulation. Tubal lavage, although more invasive, may be considered to increase yield of oocyte collection if inadequate results persist despite delaying the time of uterine lavage. Once the problem of oocyte collection is cleared, the protocol can be modified to allow animals to be bred during the COH cycle with the goal of collecting multiple embryos to isolate baboon embryonic stem (ES) cells, which may provide an in vitro model of ES cell differentiation similar to human ES cells. Ultimately, use of a long-acting hFSH analogue for COH in women undergoing IVF may be beneficial by requiring fewer injections, thereby increasing comfort and compliance.