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Reverse-Phase High-Performance Liquid Chromatography/Mass Spectrometry (RP-LC-MS/MS, Label-Free Method) for Component Analysis of Gonadotropin Drugs

ABSTRACT: 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—exceptional specificity, absolute quantification capabilities, independence from specialized reagents, powerful multiplexing potential, and ability to provide structural insights—are 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, Ovidrel), three domestic HMG products (Le Baode, Man Fuxin, 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.

ORGANISM(S): Homo Sapiens

SUBMITTER: Xin Dong

PROVIDER: PXD072543 | iProX | Wed Dec 31 00:00:00 GMT 2025

REPOSITORIES: iProX

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Project description:This study was designed to provide a genome-wide analysis of the effects of luteinizing hormone (LH) ablation/replacement versus steroid ablation/replacement on gene expression in the developed corpus luteum (CL) in primates during the menstrual cycle. Naturally cycling, female rhesus monkeys were left untreated (Control; n = 4) or received one of the following treatments for three days beginning on Day 9 of the luteal phase: daily injection of the gonadotropin-releasing hormone (GnRH) antagonist (Antide; n = 5), Antide + recombinant human LH (A+LH; n = 4), Antide + LH + the 3b-HSD antagonist Trilostane (A+LH+TRL; n = 4), and Antide + LH + TRL + progesterone replacement with a synthetic progestin R5020 (A+LH+TRL+ R5020; n = 5). On Day 12 of the luteal phase, CL were removed and samples of RNA from individual CL were fluorescently labeled and hybridized to Affymetrix™ rhesus macaque total genome microarrays. The greatest number of altered transcripts was associated with the ablation/replacement of LH, while ablation/replacement of progestin affected fewer transcripts. Replacement of LH during Antide treatment restored expression of most transcripts to control levels. Real-time PCR validation of a subset of transcripts revealed that most expression patterns were similar between microarray and real-time PCR. Analysis of protein levels were subsequently determined for 2 of the transcripts differentially expressed by real-time PCR. This is the first genome-wide analysis of LH and steroid regulation of gene transcription in the developed primate CL. Further analysis of novel transcripts identified in this data set can clarify the relative role for LH and steroids in CL maintenance and luteolysis. Keywords: LH/steroid ablation/replacement in primate mid-late luteal phase corpora lutea 22 samples from Rhesus Macaque corpus luteum hybridized to individual Rhesus Affymentrix Gene Chip Arrays. 5 treatment groups, with at least 4 replicates per treatment.

Project description:Goal of the experiment: To examine differential gene expression in granulosa cells of women undergoing ovarian stimulation with either recombinant human follicle stimulating hormone or purified human menopausal gonadotropin for in vitro fertilization. Brief description of the experiment: The study was designed to test the hypothesis that human granulosa cell (GC) gene expression response differs between pure recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens. Women < 35 years-old undergoing in vitro fertilization for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, Gonadotropin releasing hormone (GnRH) agonist long protocol plus individualized dosages of (1) recombinant follicle stimulating hormone (rFSH) (Gonal-F®) (n=4) or (2) purified human menopausal gonadotropin (hMG; Menopur®; 75 IU FSH/75 IU LH per vial) (n=3). Follicle development was monitored by ultrasound and serum estradiol levels. When two follicles were ≥ 17mm, human chorionic gonadotropin (hCG) (10,000 IU; Novarel®) was administered. Oocytes were retrieved 35 h post-hCG. Following oocyte recovery, GC were immediately collected in RNALater®. Pooled GC from individual patients were washed, centrifuged over 40% Percoll®, resuspended in RNALater, and snap-frozen in LN. Total RNA was extracted and stored at -70C. Biotinylated cRNA was synthesized from total RNA and each sample was run individually on CodeLink Whole Human Genome Bioarrays (Applied Microarrays). Unnamed genes and genes with <2-fold difference in expression were excluded from further analysis. After exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, basigin, and hypoxia-inducible factor (HIF)-1 alpha. The results suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

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Reverse-Phase High-Performance Liquid Chromatography/Mass Spectrometry (RP-LC-MS/MS, Label-Free Method) for Component Analysis of Gonadotropin Drugs

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