Glycoprotein hormones, including follicle-stimulating hormone (FSH), luteinizing hormone (LH), and thyroid-stimulating hormone (TSH), play essential roles in the reproductive endocrinology of teleosts. However, research on these hormones in non-model species such as the Japanese eel (Anguilla japonica) has been constrained by the lack of specific immunological detection tools. Recombinant A. japonica FSH, LH, and TSH were expressed using a Bombyx mori-based silkworm-baculovirus system. Polyclonal antibodies were then generated using two distinct immunogen strategies: (i) chimeric constructs fusing β-subunits with a murine α-subunit, and (ii) synthetic peptides representing unique, non-conserved β-subunit epitopes. Both approaches induced antibody responses, but only peptide-based immunogens yielded polyclonal antisera with high specificity and sensitivity. These antibodies distinguished homologous glycoprotein hormones without cross-reactivity and retained target selectivity in mixtures simulating physiological serum conditions. Peptide-derived antibodies provide a robust and precise tool for the immunological discrimination of FSH, LH, and TSH in A. japonica. This strategy establishes a scalable framework for species-specific hormone assay development and offers translational potential for endocrine monitoring, reproductive management, and diagnostic applications in aquaculture biotechnology.

In assisted reproductive technology (ART), controlled ovarian hyperstimulation (COS) plays an important role in guiding treatment progress and determining success. Many ovulation-inducing agents are used in this process, including human menopausal gonadotropin (HMG), recombinant follicle-stimulating hormone (r-FSH), recombinant luteinizing hormone (r-hLH), urinary human chorionic gonadotropin (u-hCG), and recombinant human chorionic gonadotropin (r-hCG). HMG, which comes from the urine of postmenopausal women, mainly contains FSH, LH, and hCG. While bioactivity-based potency units and immunoassay-based ELISA methods remain the gold standard for quantifying protein therapeutics, LC-MS/MS is emerging as a game-changing technology. Its unique strengths are exceptional specificity and absolute quantification capabilities, independence from specialized reagents, powerful multiplexing potential, and ability to provide structural insights, establishing it as an essential tool for both orthogonal verification and cutting-edge research in the field. This study used reverse-phase liquid chromatography with mass spectrometry (RP-LC-MS/MS) and a label-free quantitative proteomics method to analyze imported r-hFSH, r-hLH, r-hCG (Gonal-F, Luveris, and Ovidrel), three domestic HMG products (Le Baode, Man Fuxin, and Xin Yunle), and u-hCG (Li Zhu). The results showed that the purity of the main component in imported gonadotropin drugs was higher than 98.55%. On the other hand, the main element of domestic u-HMG is FSH, with small amounts of hCG and very little LH. These results reveal significant differences in the composition of imported and domestic gonadotropin products, underscoring the need to further evaluate their biological activity and clinical effectiveness. This study provides important insights into drug selection for ART, with practical value for improving the quality of HMG drugs and making better use of them in clinical settings.

Glycoprotein hormones alpha 2 (GPA2) and beta 5 (GPB5) are considered to be ancestral members of the glycoprotein hormone (GPH) family. Despite their potential roles in regulating thyroxine production, the immune response, and ovarian function, their roles in the testis remains largely unknown. To further explore this regulation, catshark testicular explants containing late spermatid stages were treated in vitro with recombinant GPA2, GPB5 and single-chain GPB5#GPA2. The comparative proteomic analysis of the treated explants revealed that ScGPB5 and ScGPB5#ScGPA2 were the most effective, modulating abundance of 449 and 525 proteins, respectively. In contrast, ScGPA2 only modulated 212 proteins. Among the differentially abundant proteins (DAPs), 103 showed substantial abundance changes in response to at least one treatment, with mean Log2ratios of 2.5 and - 2.5. Manual functional annotation linked these proteins to spermiogenesis and immunity. Key proteins and candidates involved in spermiogenesis were highlighted, including SPINK2, which is involved in acrosome protection, and LRRC69, which is related to human spermatogenic failure. These results reinforce the hypothesis of a GPA2/GPB5 paracrine regulation of catshark spermiogenesis. They also underscore the necessity of functional and comparative studies to elucidate the physiological functions of GPA2/GPB5 across a broad spectrum of taxa, from non-vertebrates to mammals. SIGNIFICANCE: To our knowledge, this study is the first to report the effects of stimulation with recombinant ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2 on vertebrate testicular tissue in vitro. A large-scale, untargeted proteomic analysis revealed for the first time that all three-ScGPA2, ScGPB5, and single-chain ScGPB5#ScGPA2-can modulate the abundance of 103 of the 7619 identified proteins, including proteins related to spermiogenesis and immunity, as well as others that are less well characterized. This report reinforces the importance of functionally characterizing GPA2 and GPB5 by showing the first results toward the characterization of their spermatogenesis related function in a specie of evolutive interest, the small-spotted catshark. In the future, a complete characterization of GPA2 and GPB5 will further our understanding of the functional evolution of glycoprotein hormones-a keystone group of hormones for vertebrate physiology and critical targets for fertility modulation in both livestock and humans.

The objective of this study was to compare embryonic morphokinetic parameters and clinical outcomes of two controlled ovarian stimulation (COS) protocols in women of advanced maternal age (AMA): Pergoveris (r-hFSH:r-hLH, 2:1 ratio) versus r-hFSH monotherapy (GONAL-f). This retrospective, non-interventional descriptive study was conducted at a private university-affiliated IVF center and included 136 ICSI cycles performed in AMA patients between March 2019 and May 2020. Patients were grouped by COS protocol (GONAL-f, n = 64; Pergoveris, n = 72), and embryo morphokinetics and ICSI outcomes were extracted from the clinic's database; the main outcome was time to complete blastulation (tB, hours). Embryos from the Pergoveris group reached tB earlier than those from the GONAL-f group (mean 109.3 h vs 112.6 h), and all morphokinetic milestones occurred sooner with Pergoveris, alongside lower multinucleation rates at the 2-cell and 4-cell stages. Although blastocyst development was higher in the GONAL-f group, Pergoveris was associated with higher oocyte yield and maturity, higher implantation, clinical and ongoing pregnancy rates, and lower miscarriage and OHSS rates; Pergoveris cycles also required higher gonadotropin doses and longer stimulation. Overall, embryos from the r-hFSH:r-hLH group exhibited faster morphokinetic timings and improved implantation and pregnancy outcomes, and prospective studies are warranted to confirm these observations.

Poor ovarian response (POR) remains a major limitation in assisted reproductive medicine. Direct comparisons between r-hFSH alfa + r-LH and r-hFSH delta + hMG in this population are scarce.

Follitropin delta, a recombinant human follicle-stimulating hormone (r-hFSH), is administered using an individualized dosing algorithm based on serum anti-Müllerian hormone (AMH) levels and body weight and offers a different pharmacokinetic (PK) and pharmacodynamic (PD) profile compared to conventional gonadotropins. The retrospective analysis by Eggersmann et al. aimed to compare the cumulative live birth rate (CLBR) of r-hFSH delta versus r-hFSH alfa/beta using data from the German In-vitro Fertilization (IVF) registry (D.I.R.®; Deutsches IVF-Register), encompassing 113,936 stimulation cycles from women aged 24-45 years between 2017 and 2022. The authors found no statistical differences in oocyte yield, pregnancy rate (PR), and live birth rate (LBR), and an increase in cumulative live birth rate (CLBR) in the group stimulated with r-hFSH delta. The study presents several methodological challenges, such as differences in dose exposure, insufficient adjustment for confounding variables, and the inclusion of diverse patient groups, which may affect the clarity of the comparative effectiveness of the treatments. Additionally, the exclusion of patients who did not undergo frozen embryo transfer may limit the interpretability and generalizability of the findings. While Eggersmann et al. added valuable and meaningful insights into the real-world effectiveness of r-hFSH delta, its conclusions warrant thoughtful consideration. Undertaking a comprehensive methodological assessment strengthens causal inferences drawn from observational data, especially when intended to inform clinical decision-making. This real-world study serves as an important piece of the broader evidence base, contributing to our understanding of assisted reproductive technology (ART) outcomes.