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A pilot proteomic study reveals different protein profiles related to testosterone and gonadotropin changes in a short-term controlled healthy human cohort

ABSTRACT: Testosterone deficiency afflicts approximately 30% of men aged from 40-89 years. It is associated with type 2 diabetes, metabolic syndrome and cardiovascular disease. However, the causality of the relationship between low testosterone and metabolic diseases is unclear. We have analysed plasma samples from thirty healthy male volunteers (19-32 years of age) with pharmacologically-induced testosterone deficiency. Blood samples were collected under three conditions: normal testosterone (baseline), ‘zero’ testosterone and ‘restored’ testosterone. As a proof-of-concept, a sample pooling approach was used to obtain one pool per condition, with each pool consisting of 30 individual samples. The proteomic profiles identified significant functional differences, related to the innate immune system, the regulation of insuline-like growth factors, collagen degradation, glycolysis/gluconeogenesis process, among others.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Aniel Sanchez

LAB HEAD: Johan Malm

PROVIDER: PXD016096 | Pride | 2020-08-03

REPOSITORIES: Pride

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A pilot proteomic study reveals different protein profiles related to testosterone and gonadotropin changes in a short-term controlled healthy human cohort.

Pla Indira I Sahlin K Barbara KB Pawłowski Krzysztof K Appelqvist Roger R Marko-Varga György G Sanchez Aniel A Malm Johan J

Journal of proteomics 20200330

PMID: 32240812

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Project description:Background Testosterone (T) is the main androgen playing a crucial role in regulating the function of both reproductive and non-reproductive organs. Current methods for assessing T levels in humans do not reflect the biological androgenic activity (BAA), which may be important for example in the diagnosis of hypogonadism and clarifying pathogenetic mechanisms linking androgen deficiency to various comorbidities. In this study, we aimed to identify new protein markers of BAA in humans and test their predictive value in relation to some androgen deficiency linked pathologies. Methods and Findings Plasma samples (n=90) from 30 healthy chemically castrated young volunteer males (19-32 years) were collected at three different time points: baseline – prior to treatment with GnRH antagonist), three weeks after GnRH antagonist treatment, and at the end of the study, two weeks thereafter. Following the second blood sampling, all men were supplemented with 1000 mg T undecanoate. The samples were analysed by mass spectrometry-based proteomics. A total of 676 proteins were identified among which expression of 46 was statistically significantly associated with T concentration (p<0.05, ANOVA paired). Based on ROC-analysis, 3 candidate proteins were selected, 4-hydroxyphenylpyruvate dioxygenase (4HPPD), insulin-like growth factor-binding protein 6 (IGFBP6) and fructose-bisphosphate aldolase (ALDOB). Together with a mathematically calculated variable (MMA: Multi-Marker Algorithm), based on the combination of 4HPPD and IGFBP6 values, their predictive value in relation to low T levels and cardio-metabolic risk factors was tested in 75 males suffering from infertility, 32-43 years of age. Of these males, four were diagnosed with type 2-diabetes and 14 with the metabolic syndrome. Clinical chemistry data regarding cardio-metabolic parameters, bone mineral density and androgen receptor CAG repeat lengths were assessed. The three proteins significantly distinguished between low (< 8 nmol/L) and normal (> 12 nmol/L) morning fasting T (AUC: 0.70-0.77). The MMA had an even higher accuracy (AUC=0.80) in identifying subjects with low T. 4HPPD, ALDOB and MMA showed higher accuracy than T (AUC=0.85-0.92 vs. AUC=0.55; p<.001) in identifying infertile patients with type 2 diabetes mellitus and metabolic syndrome (AUC=0.74-0.78 vs. AUC=0.56; p<0.05). These markers also showed higher AUC than T in predicting adverse cardiovascular risk lipid profile and insulin resistance patients but the differences were not statistically significant. 4HPPD and MMA distinguished patients with low bone density (z-score in DEXA scan < -1), but no difference was observed when comparing with AUC for T,. The androgenic dependence of 4HPPD and ALDOB were confirmed by finding statistically significant lower levels of these markers in men with androgen CAG repeat lengths < 21 (p=0.002) and >22 (p=0.038) as compared to those with CAG of 21-22. No statistical significances were found for IGFBP6. Conclusions 4HPPD and ALDOB were identified as potential biomarkers for diagnosis of male hypogonadism and in identifying subjects at high risk of some of its clinical sequelae. More extensive testing should be carried out in order to elucidate their usefulness in clinical assessment of androgenic activity, not only in adult males but also in women and in pre-pubertal boys.

Project description:Testosterone is essential to maintain qualitative spermatogenesis. Nonetheless, no studies have been yet performed in humans to analyze the testosterone-mediated expression of the sperm proteins and their importance in reproduction. Thus, this study aimed to identify sperm protein alterations in male hypogonadism using proteomic profiling. We have performed a comparative proteomic analysis comparing sperm from fertile controls (a pool of 5 normogonadic normozoospermic fertile men) versus sperm from patients with secondary hypogonadism (a pool of 5 oligozoospermic hypogonadic patients due to isolated LH deficiency). Sperm protein composition was analyzed, after peptide labelling with Isobaric Tags, via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) on an LTQ Velos-Orbitrap mass spectrometer. LC-MS/MS data were analyzed using Proteome Discoverer. Criteria used to accept protein identification included a false discovery rate (FDR) of 1% and at least 1 peptide match per protein. Up to 986 proteins were identified and, of those, 43 proteins were differentially expressed: 32 proteins were under-expressed and 11 were over-expressed in the pool of hypogonadic patients. Bioinformatic analyses were performed using UniProt Knowledgebase, and the Gene Ontology Consortium database based on PANTHER. Notably, 13 of these 43 differentially expressed proteins have been previously reported to be related to sperm function and spermatogenesis. Western blot analyses for A-Kinase Anchoring Protein 3 (AKAP3) and the Prolactin Inducible Protein (PIP) were used to confirm the proteomics data. In summary, a high-resolution mass spectrometry-based proteomic approach was used for the first time to describe alterations of the sperm proteome in secondary male hypogonadism. A panel of deregulated sperm proteins was identified, including Prosaposin, Ropporin-1B, SERPINA5, SMOC-1, SPANXB1, GSG1, ELSPBP1, fibronectin, 5-oxoprolinase, AKAP3, AKAP4, HYDIN, ROPN1B, ß-Microseminoprotein and Protein S100-A8, which might represent putative physiological in vivo targets for androgen deficiency.

Dataset Information

A pilot proteomic study reveals different protein profiles related to testosterone and gonadotropin changes in a short-term controlled healthy human cohort

Publications

A pilot proteomic study reveals different protein profiles related to testosterone and gonadotropin changes in a short-term controlled healthy human cohort.

Similar Datasets

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