In silico insights into intra- and inter-species interactions of piscine gonadotropin hormones and receptor crosstalk.

In mammals, the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) are macromolecules secreted during specific reproductive phases and display strict specificity towards their cognate receptors. However, fish gonadotropins (GTH) and their receptors (GTHR) display diverse species-specific expression patterns, secretion patterns, and intra- and interspecies cross-activation. To uncover the molecular basis of this diversity, we generated and analyzed 29 in-silico models of intra- and inter-species combinations of sturgeon, carp, tilapia, and human gonadotropins with piscine receptors and analyzed the resulting receptor activation and signal transduction of these GTHR-GTH complexes in-vitro. Our results suggest that unlike humans, the surface charge on piscine FSH/LH ß-seatbelt and N107huLHCGR/K104hFSHR homologs does not necessarily determine binding specificity. Instead, sequence and structural variations allow piscine GTHs significant conformational flexibility when binding to the receptor extracellular domain, thereby enabling cross-activation. The resulting diversity may support various reproductive strategies in different environmental niches.

A single-domain intrabody targeting the follicle-stimulating hormone receptor impacts FSH-induced G protein-dependent signalling.

Intracellular variable fragments of heavy-chain antibody from camelids (intra-VHH) have been successfully used as chaperones to solve the 3D structure of active G protein-coupled receptors bound to their transducers. However, their effect on signalling has been poorly explored, although they may provide a better understanding of the relationships between receptor conformation and activity. Here, we isolated and characterized iPRC1, the first intra-VHH recognizing a member of the large glycoprotein hormone receptor family, the follicle-stimulating hormone receptor (FSHR). This intra-VHH recognizes the FSHR third intracellular loop and decreases cAMP production in response to FSH, without altering Gαs recruitment. Hence, iPRC1 behaves as an allosteric modulator and provides a new tool to complete structure/activity studies performed thus far on this receptor.

FSHR activation through small molecule modulators: Mechanistic insights from MD simulations.

Follicle-stimulating hormone receptor (FSHR) is a glycoprotein hormone receptor that plays a vital role in reproduction, cancer progression and osteoporosis. Owing to its therapeutic importance, several small molecule modulators have been identified by researchers through high throughput studies that usually include virtual screening of chemical libraries followed by in vitro validation through radio-ligand binding assays, cAMP accumulation and luciferase-based luminescence assays. The binding site of these modulators and structural changes that accompany modulator binding remains elusive. Here, we address these aspects through molecular docking and MD simulations on well-studied FSHR modulators and comparing the domain motions between agonist/FSH bound and antagonist bound FSHR structures. It was observed that agonist and antagonist modulators bind to the same site, but interact with distinct residues in transmembrane domain(TMD). FSHR(TMD) residues Ile522, Ala595, Ile602 and Val604 were found to interact only with agonist. Notably, these residues are conserved in the close homolog luteinizing hormone/choriogonadotropin receptor (LHCGR) and participate in interaction with its agonist Org43553. We observed distinctly prominent domain motions and conformational changes in TM helices 3, 4 and 6 for agonist bound FSHR structure. These structural changes have also been reported for LHCGR, and few GPCR members suggesting an important and well conserved mechanism of GPHR activation that could be exploited for design of novel modulators.

The effect of Nateglinide and Octreotide on follicular morphology and free radical scavenging system in letrazole-induced rat model of PCOS.

OBJECTIVE: To investigate the effects of octreotide and nateglinide on ovarian follicle count, ovarian tissue damage, biochemical parameters and free radical scavenging system in letrazole-induced rat model of PCOS. MATERIALS AND METHODS: Forty-two female Sprague-Dawley rats were divided into six groups. Group 1 (Control Group): after localizing the ovaries and the uterine horns, the abdominal wall was closed without any surgical procedure. Group 2 (PCOS Group): PCOS was induced by administrating Letrozole orally for 21 successive days. At the end of 21 days, rats underwent ovarian biopsies. The experimental PCOS model was considered successful in the presence of atretic follicles without granulosa cell stratification. Group 3 (PCOS + Nateglinide Group): Nateglinide was administered by oral dropper for 30 days to the rats in which PCOS model was created. Group 4 (Nateglinid only Group): 30 days of NG was applied to the rats without PCOS. Group 5 (PCOS+Octreotide Group): 0.1 mg/kg/day Octreotide was given intraperitoneally for 4 weeks to the rats in which PCOS model was created. Group 6 (Octreotide only Group): animals without PCOS given 0.1 mg/kg/day Octreotide at the end of the treatment, bilateral oophorectomy was performed and blood samples were collected from all groups. Ovarian tissue was stained immunohistochemically with TLR-4 in addition to conventional staining. In addition to follicle classification, ovarian damage was graded. Serum insulin, FSH and LH, TNF-α, IL-6, SHBG, SOD, IGF-1, MDA and GSH levels were also measured. RESULTS: The cystic and degenerated follicle density of PCOS group was high compared with the other groups. Both cystic and degenerated follicles were significantly reduced in PCOS+NG and PCOS+OC groups compared to PCOS group. There was no difference between the groups in terms of serum LH, FSH and insulin levels (p>0.05). Serum testosterone level was significantly higher in the PCOS group compared to the other groups (p<0.01). Adding OC or NG to PCOS groups did not cause significant changes in testosterone levels. TNF-α and IL-6 levels were high in PCOS group (p<0.03). IGF-1 and MDA levels were higher in PCOS than in other groups (p<0.03, p<0.01 respectively). Adding OC or NG to the treatment normalized IGF-1 and MDA levels. Serum GSH levels were significantly lower in the PCOS group (p<0.05). Adding NG to the treatment increased GSH levels. CONCLUSIONS: Both NG and OCT reverses atretic and degenerate follicle damage due to PCOS through TLR-4, antioxidant and anti-inflammatory pathways.

A single blood sample for stimulated LH assayed by ICMA is useful for monitoring the treatment efficacy of triptorelin depot in girls.

BACKGROUND: There is still no consensus on the optimal monitoring method to evaluate the hypothalamic-pituitary-gonadal axis (HPGA) inhibition. METHODS: There were 124 girls treated with triptorelin depot due to puberty disorders, including 77 central precocious puberty and 47 early puberty. After treatment, triptorelin stimulation tests were performed, and blood samples were collected at 0, 20, 40 and 60 min. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured by immunochemiluminometric assay (ICMA). RESULTS: Peak LH (PLH), peak FSH and estradiol in 124 girls were significantly decreased after treatment, while 2 cases had inadequate treatment efficacy. Areas under the receiver operating characteristic curves (AUC) of PLH and peak FSH after stimulation for the diagnosis of HPGA suppression were 0.984 and 0.121. When the cut-off value of PLH was ≤ 2.25 IU/L, the sensitivity was 96.7% and specificity was 100.0%. There was no difference in AUC between PLH and a single LH at 20, 40, or 60 min (p > 0.05). When LH were ≤ 2.34 IU/L, ≤ 2.21 IU/L and ≤ 2.00 IU/L at 20, 40 and 60 min, respectively, the sensitivity were 99.1%, 96.7% and 98.4%, and the specificity were all 100.0%. The correlation coefficients between PLH and LH at 20, 40 or 60 min were 0.947, 0.975 and 0.961. CONCLUSION: A single blood sample for stimulated LH at 20 min, 40 min, or 60 min assayed by ICMA during triptorelin stimulation test is useful for monitoring the treatment efficacy of triptorelin depot in girls with puberty disorders.

Integrative model of the FSH receptor reveals the structural role of the flexible hinge region.

The follicle-stimulating hormone receptor (FSHR) belongs to the glycoprotein hormone receptors, a subfamily of G-protein-coupled receptors (GPCRs). FSHR is involved in reproductive processes such as gonadal development and maturation. Structurally, the extensive extracellular domain, which contains the hormone-binding site and is linked to the transmembrane domain by the hinge region (HR), is characteristic for these receptors. How this HR is involved in hormone binding and signal transduction is still an open question. We combined in vitro and in situ chemical crosslinking, disulfide pattern analysis, and mutation data with molecular modeling to generate experimentally driven full-length models. These models provide insights into the interface, important side-chain interactions, and activation mechanism. The interface indicates a strong involvement of the connecting loop. A major rearrangement of the HR seems implausible due to the tight arrangement and fixation by disulfide bonds. The models are expected to allow for testable hypotheses about signal transduction and drug development for GPHRs.

Overlapping synthetic peptides as a tool to map protein-protein interactions ̶ FSH as a model system of nonadditive interactions.

In earlier work, we used partially overlapped synthetic peptides as a tool to find regions of interaction between the human FSH hormone and its receptor, aiming to find possible antagonists or agonists. Years later, the FSH and FSH receptor 3D structures were reported by other laboratories. The 3D results were in close agreement with the interacting regions predicted by using synthetic peptides. These earlier studies are reviewed here, and the predicted regions of interaction compared to the FSH and FSH receptor 3D structures to illustrate the usefulness of the synthetic peptide strategy to find binding regions. Different contact regions contribute multiplicatively to the high affinity of the entire ligand; thus, peptides covering a fraction of the anchor sites and with low free energy density cannot reach the affinity of the entire molecule. The earlier use of multiple linear regression to find the relevant predictors for effective binding, and a new way to estimate ΔG° and nonadditive interactions for the synthetic peptides in solution, by using the buried surface area (BSA), will be discussed.

Central residues of FSHß (89-97) peptide are not critical for FSHR binding: Implications for peptidomimetic design.

In our previous study, we had identified a 9-mer peptide (FSHß (89-97)) derived from seat belt loop of human FSHß and demonstrated its ability to function as FSHR antagonist in vivo. Structure analysis revealed that the four central residues 91STDC94 within this peptide may not be critical for receptor binding. In the present study, 91STDC94 residues were substituted with alanine to generate ΔFSHß 89-97(91STDC94/AAAA) peptide. Analogous to the parent peptide, ΔFSHß 89-97(91STDC94/AAAA) peptide inhibited binding of iodinated FSH to rat FSHR and reduced FSH-induced cAMP production. The peptide could impede granulosa cell proliferation leading to reduction in FSH-mediated ovarian weight gain in immature female rats. In these rats, peptide administration further downregulated androgen receptor and estrogen receptor-alpha expression and upregulated estrogen receptor-beta expression. The results indicate that substitution of 91STDC94 with alanine did not significantly alter FSHR antagonist activity of FSHß (89-97) peptide implying that these residues are not critical for FSH-FSHR interaction and can be replaced with non-peptidic moieties for development of more potent peptidomimetics.

First-in-class humanized FSH blocking antibody targets bone and fat.

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a KD of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH-FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHß subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

Downshifting nanoprobes with follicle stimulating hormone peptide fabrication for highly efficient NIR II fluorescent bioimaging guided ovarian tumor surgery.

Failure of intraoperative detection, early minimal lesion and microscopic residual tumor margins elimination causes metastatic diffusion and lethal recurrence. However, during surgical process, surgeons can only rely largely on palpation and visual examination. Intraoperative bioimaging with the aid of the second near-infrared fluorescent (NIR II FL) light has entered the surgical excision area to bridge the gap of preoperative bioimaging and intraoperative resection. Here, we demonstrate that the follicle-stimulating hormone peptide (FSHP) engineered NIR II downshifting nanoparticles (DSNPs@FSHP) selectively undergo efficient ovarian tumor targeting property. Owing to the special biocompatibility of nanoprobes, this strategy provided rapid body clearance and efficient tumor targeting with significantly tumor to background (T/B) ratio enhanced for surgical excision. Based on these, this strategy can successfully empower the detection and surgical removal for both ovarian tumor lesions and ovarian tumor margins by NIR II FL bioimaging.

Characterization of gonadotropin receptors Fshr and Lhr in Japanese medaka, Oryzias latipes.

Reproduction in vertebrates is controlled by the brain-pituitary-gonad axis, where the two gonadotropins follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) play vital parts by activating their cognate receptors in the gonads. The main purpose of this work was to study intra- and interspecies ligand promiscuity of teleost gonadotropin receptors, since teleost receptor specificity is unclear, in contrast to mammalian receptors. Receptor activation was investigated by transfecting COS-7 cells with either Fsh receptor (mdFshr, tiFshr) or Lh receptor (mdLhr, tiLhr), and tested for activation by recombinant homologous and heterologous ligands (mdFshßα, mdLhßα, tiFshßα, tiLhßα) from two representative fish orders, Japanese medaka (Oryzias latipes, Beloniformes) and Nile tilapia (Oreochromis niloticus, Cichliformes). Results showed that each gonadotropin preferentially activates its own cognate receptor. Cross-reactivity was detected to some extent as mdFshßα was able to activate the mdLhr, and mdLhßα the mdFshr. Medaka pituitary extract (MPE) stimulated CRE-LUC activity in COS-7 cells expressing mdlhr, but could not stimulate cells expressing mdfshr. Recombinant tiLhßα, tiFshßα and tilapia pituitary extract (TPE) could activate the mdLhr, suggesting cross-species reactivity for mdLhr. Cross-species reactivity was also detected for mdFshr due to activation by tiFshßα, tiLhßα, and TPE, as well as for tiFshr and tiLhr due to stimulation by mdFshßα, mdLhßα, and MPE. Tissue distribution analysis of gene expression revealed that medaka receptors, fshr and lhr, are highly expressed in both ovary and testis. High expression levels were found for lhr also in brain, while fshr was expressed at low levels. Both fshr and lhr mRNA levels increased significantly during testis development. Amino acid sequence alignment and three-dimensional modelling of ligands and receptors highlighted conserved beta sheet domains of both Fsh and Lh between Japanese medaka and Nile tilapia. It also showed a higher structural homology and similarity of transmembrane regions of Lhr between both species, in contrast to Fshr, possibly related to the substitution of the conserved cysteine residue in the transmembrane domain 6 in medaka Fshr with glycine. Taken together, this is the first characterization of medaka Fshr and Lhr using homologous ligands, enabling to better understand teleost hormone-receptor interactions and specificities. The data suggest partial ligand promiscuity and cross-species reactivity between gonadotropins and their receptors in medaka and tilapia.

Hexagonally Packed Macroporous Molecularly Imprinted Polymers for Chemosensing of Follicle-Stimulating Hormone Protein.

Homogenous nanostructuration of molecularly imprinted polymer (MIP) films for follicle-stimulating hormone (FSH)-sensing was achieved by using optimized colloidal crystals as a hard mold. Introduction of a heating step after assembling colloidal crystals of silica beads promoted their adhesion. Thus, precise assembling of beads was not disturbed during further multisteps of surface imprinting, and crack-free hexagonal packing was maintained. Scanning electron microscopy imaging confirmed hexagonal packing of silica colloidal crystals as well as homogenous nanostructuration in MIP films. FSH immobilization over silica beads and later its derivatization with electroactive functional monomers was confirmed by X-ray photoelectron spectroscopy analysis. The nanostructured molecular recognition films prepared in this way were combined with an electrochemical transducer in order to design a capacitive impedimetry-based chemosensing system. It was tested for the determination of FSH in the range from 0.1 fM to 100 pM in 10 mM 2-(N-morpholino) ethane sulfonic acid buffer (pH = 4.2). The detection limit of the chemosensor was 0.1 fM, showing a high selectivity with respect to common protein interferences as well as other protein hormones of the gonadotropin family.

Androgen potentiates the expression of FSH receptor and supports preantral follicle development in mice.

Hyperandrogenism is one of the cardinal symptoms in polycystic ovary syndrome and plays a key role in the pathogenesis of polycystic ovary syndrome. However, the precise effects and mechanisms of excess androgen during follicular development are still unclear. Here we investigated the effects of androgen on mouse follicle development in vitro. Androgen did not affect the growth of follicles smaller than 160-180 µm in the presence of follicle-stimulating hormone (FSH). However, in the presence of low FSH, androgen supported the growth of follicles larger than 160-180 µm, a size at which growing follicles acquire FSH-dependency. Androgen did not change the mRNA expression of various growth-promoting factors but did increase mRNA expression of the FSH receptor. We suggest that androgen has a positive impact on follicle development by augmentation of the actions of FSH. Therefore, FSH-responsive but FSH-independent follicles grow in the presence of a certain level of FSH or androgen, and androgen compensates for FSH deficiency in FSH-dependent follicles.

Molecular dynamics simulation of the follicle-stimulating hormone receptor. Understanding the conformational dynamics of receptor variants at positions N680 and D408 from in silico analysis.

Follicle-stimulating hormone receptor (FSHR) is a G-protein coupled receptor (GPCR) and a prototype of the glycoprotein hormone receptors subfamily of GPCRs. Structural data of the FSHR ectodomain in complex with follicle-stimulating hormone suggests a "pull and lift" activation mechanism that triggers a conformational change on the seven α-helix transmembrane domain (TMD). To analyze the conformational changes of the FSHR TMD resulting from sequence variants associated with reproductive impairment in humans, we set up a computational approach combining helix modeling and molecular simulation methods to generate conformational ensembles of the receptor at room (300 K) and physiological (310 K) temperatures. We examined the receptor dynamics in an explicit membrane environment of polyunsaturated phospholipids and solvent water molecules. The analysis of the conformational dynamics of the functional (N680 and S680) and dysfunctional (mutations at D408) variants of the FSHR allowed us to validate the FSHR-TMD model. Functional variants display a concerted motion of flexible intracellular regions at TMD helices 5 and 6. Disruption of side chain interactions and conformational dynamics were detected upon mutation at D408 when replaced with alanine, arginine, or tyrosine. Dynamical network analysis confirmed that TMD helices 2 and 5 may share communication pathways in the functional FSHR variants, whereas no connectivity was detected in the dysfunctional mutants, indicating that the global dynamics of the FSHR was sensitive to mutations at amino acid residue 408, a key position apparently linked to misfolding and variable cell surface plasma membrane expression of FSHRs with distinct mutations at this position.

Retro-inverso follicle-stimulating hormone peptide-mediated polyethylenimine complexes for targeted ovarian cancer gene therapy.

BACKGROUND: The development of nanoparticle drug delivery systems with targeted ligands has the potential to increase treatment efficiency in ovarian cancer. METHODS: We developed a 21-amino acid peptide, YTRDLVYGDPARPGIQGTGTF (L-FP21) conjugated to polyethylenimine (PEI) and methoxy polyethylene glycol (mPEG) to prepare a nanoparticle drug vehicle to target follicle-stimulating hormone receptor (FSHR) in ovarian cancer. At the same time, we optimized the ligand of the nanoparticle vehicle using D-peptides, which consist of D-amino acids (D-FP21). Nanoparticle vehicles carrying the therapeutic gene plasmid growth-regulated oncogene alpha (pGRO-α) short hairpin RNA (shRNA) (FP21-PEG-PEI/pGRO-α) were prepared for further investigation. RESULTS: Compared with L-FP21, D-FP21 exhibited improved biological stability and higher uptake rate for FSHR-expressing ovarian cancer cells. The cytotoxicity of the L, D-FP21-PEG-PEI/pGRO-α complexes were significantly lower than that of the PEI/pGRO-α complex. The nanoparticle drug with the targeted ligand showed higher transfection efficiencies and improved anti-proliferation effects for ovarian cancer cells than that without the targeted ligand (mPEG-PEI/pGRO-α). Furthermore, an in vivo evaluation of an antitumor assay indicated that D-FP21-PEG-PEI/pGRO-α inhibited the growth of tumor spheroids considerably more than L-FP21-PEG-PEI/pGRO-α; their tumor inhibition rates were 58.5% and 33.3%, respectively. CONCLUSIONS: D-FP21-PEG-PEI/plasmid DNA is a safe and efficient gene delivery vehicle for ovarian cancer targeted therapy.

Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.

Follicle-stimulating hormone encapsulation in the cholesterol-modified chitosan nanoparticles via molecular dynamics simulations and binding free energy calculations.

Follicle-stimulating hormone (FSH) is widely applied in the modern ovarian stimulation techniques. However, it must be administered daily because of its short half-life. Recently, the cholesterol (CS) modified chitosan (CTS) nanogels have attracted significant interest as promising controlled release protein delivery because of their ability to minimize the aggregation and irreversible denaturation of proteins. Herein, we report a molecular dynamics (MD) simulation investigation on the molecular mechanisms of FSH encapsulation in the CS-CTS nanogels. The MD simulations have been performed using the GROMACS software for up to 200ns simulation time. Furthermore, the binding free energy has been calculated by the molecular mechanics [MM] with Poisson-Boltzmann [PB] and surface area solvation (MM/PBSA) method by using the g_mmpbsa tool. Our findings suggest that the main driving force of the formation of the CS-CTS nanogels is the hydrophobic interactions between the CS-CS moieties in water. The results have also indicated that the CS-CTS nanogel formation can occur through the hydrogen bonding in addition to the hydrophobic interactions. The obtained data demonstrate that the FSH encapsulation into the CS-CTS nanogels is a gradual process driven by the hydrophobic interactions between the hydrophobic patch of FSH and the hydrophobic nanodomains of the nanogel. Our results also reveal that except in the hydrophobic patch region, the flexibility of FSH was reduced in the presence of the nanogel. This study provides the elucidation of the nanogel-FSH interactions at the molecular level and presents new perspective for the ideal design and applications of the CS-CTS nanogel in protein delivery.

Biosimilars to recombinant human FSH medicines: comparable efficacy and safety to the original biologic.

Two recombinant follicle-stimulating hormone (rFSH)-bearing similar biological medicines (biosimilars) have been authorized by the European Commission. Biosimilar is a regulatory concept alluding to the evidence-based high-standard comparability studies needed to demonstrate its equivalence to a reference original biologic. Because biosimilar development represents a shift from the long-lasting existing paradigms, a thorough understanding of the science behind it will contribute to helping prescribers make informed treatment choices. Contrary to chemically-synthesized medicines, biologics are subject to an inherent molecular variability. From the experience with original biologics, regulatory authorities have accumulated a wealth of knowledge as to what minor batch-to-batch physicochemical variations may be therapeutically acceptable in a given product. Furthermore, in spite of analytically detectable differences, the two original rFSH-bearing medicines currently on the market share fundamentally the same therapeutic profile. Unlike those original medicines, a biosimilar of an rFSH product and the corresponding reference biologic share essentially the same active pharmaceutical ingredient. They are also administered via the same route, at the same dose, and for the same indications. This article revises the background evidence over which the biosimilarity principle has been built, and highlights the therapeutic potential for follitropin biosimilars in order to reassure physicians on their benefit.

Manufacturing of Recombinant Human Follicle-Stimulating Hormone Ovaleap® (XM17), Comparability with Gonal-f®, and Performance/Consistency.

Ovaleap® (XM17) is a recombinant human follicle-stimulating hormone to treat infertility by inducing ovulation or controlled ovarian stimulation for assisted reproductive technology (ART) procedures. Ovaleap® (follitropin-α) was approved by the European Medicines Agency in 2013 as a biosimilar medicinal product to the reference medicine, Gonal-f®. Information is often not easily accessible and/or publicly available regarding the rigorous manufacturing procedures for biosimilars. Objectives of the current analysis were to report on validation procedures for the Ovaleap® manufacturing process, to compare the characteristics of Ovaleap® versus Gonal-f®, and to describe the performance and consistency of Ovaleap®. Formal validation of the Ovaleap® manufacturing process was performed at full commercial scale, consisting of several consecutive fermentation and purification runs. Comparison with Gonal-f® involved numerous techniques to determine molecular structure, isoform distribution, biological activity, and product-related impurities. The stability of the multidose application system, targeted for long-term stability at ambient temperature, was assessed and demonstrated. All analyses showed the manufacturing process of Ovaleap® to be robust and consistent. Ovaleap® was found to have similar characteristics when compared with Gonal-f®. This analysis supports the role of Ovaleap® as a biosimilar to Gonal-f®, thus providing patients and clinicians with another therapeutic option during ART procedures.

Two complementary methods to control gonadotropin-releasing hormone vaccination (Improvac®) misuse in horseracing: Enzyme-linked immunosorbent assay test in plasma and steroidomics in urine.

Since the availability on the European market of the vaccine Improvac® dedicated to male pig immunological castration, the risk of misuse of this product in horses is now considered as a threat for the horseracing industry. Immunological castration is not allowed by the racing codes (immune system, Article 6). Indeed, this vaccination against the hypothalamic hormone luteinizing hormone-releasing hormone or gonadotropin-releasing hormone (GnRH) will prevent the release from the anterior pituitary of luteinizing hormone and follicle stimulating hormone, which are required for the development and activity of gonads in males (testes) and female (ovaries) and therefore all their subsequent physiological functions. This treatment will induce a strong hormonal variation resulting in a behaviour modification of the animals. In this work, four male standardbreds treated with Improvac® vaccine (two intramuscular injections within 4 weeks) were studied. Monitoring of the total scrotal width showed a decrease of the scrotum size (37%) and production of anti-GnRH antibodies was detected up to 200 days after the first injection. Anti-GnRH antibodies were detected in plasma after caprylic acid precipitation followed by an enzyme-linked immunosorbent assay (ELISA) as a rapid and efficient screening method applicable to routine analysis. These results were correlated to a switch of the sexual status from male group to gelding/female group obtained by a steroidomic approach with urine based on ten endogenous compounds. Copyright © 2017 John Wiley & Sons, Ltd.