Current Status And Progress Of Male Seminal Plasma Metabolomics Research Ⅱ

2. Research on seminal plasma metabolomics in the field of male reproductive health

Male sperm quality is closely related to the critical window of spermatogenesis (epididymal storage, sperm motility development and spermatogenesis). Innate genetic factors (such as congenital bilateral absence of the vas deferens, cystic fibrosis gene mutation, Y chromosome deletion, Kallmann syndrome and chromosomal abnormalities leading to deterioration of testicular function) and acquired factors (male reproductive diseases such as varicocele, environmental or occupational exposure to toxic chemicals such as heavy metals and endocrine disruptors, and various lifestyle factors such as smoking and drinking) may affect the spermatogenesis process, thereby leading to abnormal sperm quality [2].

HERB CISTANCHE FOR IMPROVE MALE SPERM QUALITY

Due to the key role of seminal plasma in the male reproductive system, the use of seminal plasma metabolomics to explore potential biomarkers and pathogenic mechanisms of male reproductive diseases has great potential. Male reproductive system diseases are highly complex and require the combined use of multiple biomarkers to achieve prediction, diagnosis and etiology analysis. Figure 1 shows the seminal plasma metabolic pathways disturbed by diseases or environmental factors, mainly involving energy metabolism, lipid metabolism, amino acid metabolism, and steroid metabolism pathways. Appendix 3 specifically shows the changes in different metabolites in male infertility. Red indicates up-regulation of metabolites, and blue indicates down-regulation of metabolites. These changes mainly involve changes in amino acid and lipid metabolites and their metabolites. This article will combine the seminal plasma metabolomics research summarized in Table 1 to specifically introduce the application status of seminal plasma metabolomics from the perspectives of different male reproductive diseases and environmental exposure factors.

2.1 Application of seminal plasma metabolomics in the diagnosis and mechanism research of male infertility

2.1.1 Oligospermia, asthenospermia, teratospermia and idiopathic infertility

Oligospermia refers to a sperm concentration lower than 1.5 × 107/mL[37] and is the most common disease in male infertility. Seminal plasma metabolomics based on LC-MS and 1H-NMR have shown that there are significant differences in the levels of amino acids and choline in the seminal plasma of oligospermic men and normal men [12, 15, 18, 32].

A 1H-NMR-based seminal plasma metabolomics study found that the levels of arginine and aspartate in the seminal plasma of oligospermia patients were reduced, which was significantly correlated with the decrease in sperm count [15, 18, 38]. Another LC-MS-based study found that in addition to the decrease in aspartate levels, the levels of glutamate, methionine, tryptophan, proline, and alanine in the seminal plasma of oligospermia patients were also significantly reduced [32]. In addition to amino acids, choline metabolism is also crucial for spermatogenesis. The lack of choline-related enzymes can lead to decreased sperm function, and choline supplementation may be beneficial to male reproductive health. However, there is some controversy about the positive effect of choline supplementation on sperm quality in vivo, which may be due to the lack of a placebo control group or the choline dosage [39]. Further population studies are still needed.

Asthenospermia is also a common cause of male infertility and is characterized by a significant decrease in sperm motility. Seminal plasma metabolomic analysis based on GC-MS showed that the levels of seminal plasma oleic acid, palmitic acid, and valine in patients with asthenozoospermia were significantly increased, suggesting that abnormalities in fatty acid metabolism may affect sperm motility [23]. The sperm membrane contains a variety of fatty acids, and excessive oleic acid levels in the seminal plasma may disrupt the phospholipid metabolism process in the sperm membrane, further leading to a decrease in sperm motility [40]. High levels of fatty acids (especially palmitic acid and arachidonic acid) [14, 41] in sperm membranes are also important factors for low sperm quality. Arachidonic acid (AA) plays an important role in lipid metabolism, but the effect of AA on semen quality remains unclear [42-43]. Seminal plasma targeted metabolism

Group analysis revealed changes in AA metabolic pathways in the seminal plasma of patients with asthenozoospermia [28]. It was found that AA metabolic disorders can be further induced by lipoxygenase (LOX), cytochrome P450 (CYP450) and cyclooxygenase. (cyclooxygenase, COX) metabolic pathway activates P38 mitogen-activated protein kinase, thereby reducing sperm motility. In addition to seminal plasma fatty acids, metabolites involved in various metabolic pathways such as energy, purine, methionine cycle, and branched-chain amino acid metabolism are also significantly altered in the seminal plasma of patients with asthenospermia [14, 30]. The energy required for sperm movement comes from aerobic decomposition pathways such as glycolysis or tricarboxylic acid cycle (TCA). Inhibition of this pathway will lead to reduced ATP production, insufficient energy supply, and reduced sperm motility.

Teratospermia is a disease characterized by the presence of large numbers of abnormally morphological sperm in semen, the pathogenesis of which is unknown [44]. A 1H-NMR-based metabolomic study found that 18 tricarboxylic acid cycle-related metabolites were significantly dysregulated in the seminal plasma of patients with teratozoospermia [17], indicating that energy metabolism may be the main cause of abnormal sperm morphology. The amino acid levels in the seminal plasma of patients with teratozoospermia are also altered, especially taurine, which has an antioxidant effect [45]. Abnormal sperm morphology may be related to the excessive production of reactive oxygen species or the reduction of antioxidants.

Idiopathic male infertility refers to unexplained male infertility in which routine semen analysis results are within the normal range and physical and endocrine abnormalities are excluded. Metabolomic analysis based on Raman spectroscopy found differences in the expression of oxidative stress-related metabolites in seminal plasma samples from idiopathic infertile men and fertile men [21].

Metabolome research based on GC-MS found 44 differentially expressed metabolites in the seminal plasma of men with idiopathic infertility [25], which are mainly involved in amino acid metabolism and oxidative stress processes. Amino acid catabolism related to sperm quality is increased. Antioxidant-related metabolites are reduced. The above studies indicate the important role of oxidative stress in idiopathic infertility, suggesting that metabolic status can be improved through corresponding supplements.

2.1.2 Varicocele

The incidence of varicocele in infertile men is very high, with approximately 40% of primary infertile men and 80% of secondary infertile men suffering from the disease [46]. Patients with varicocele have increased production of reactive oxygen species and higher levels of oxidative stress [46], which affect lipids, proteins and nucleic acids in semen, leading to sperm abnormalities [48]. 1H-NMR-based seminal plasma metabolomics analysis found 19 important differential metabolites involving changes in amino acid, lipid and energy metabolism, mainly involved in oxidative stress processes [16]. Another non-targeted seminal plasma metabolomics study based on LC-MS also showed that the levels of 8 metabolites related to amino acid, lipid and energy metabolism in the seminal plasma of patients with varicocele were changed. For example, decreased leucine levels may lead to decreased antioxidant or anti-inflammatory capacity, leading to sperm abnormalities. Surgical resection can reverse the abnormal metabolic status of varicocele, mainly manifested in the restoration of glycerophospholipid and sphingolipid levels [33]. Glycerol phospholipids are closely related to mitochondrial activity, while sphingolipids are important components of cell membranes and participate in multiple signal transduction pathways. The restorative upregulation of these two types of lipid molecules may be the reason for the improvement of sperm morphology after surgical intervention, and also confirms the feasibility of seminal plasma metabolites as markers of varicocele [33].

2.2 Seminal plasma metabolomics is used to reveal the molecular mechanism of environmental exposure affecting sperm quality

Exposure to various harmful or beneficial substances in the environment can significantly affect sperm quality by interfering with the metabolic process of seminal plasma. The MIMA (meet-in-metabolite analysis) analysis method we proposed previously is suitable for studying the biochemical pathways that lead to adverse outcomes of environmental exposure and revealing the complex relationship between exposure and health [49]. We used this method to conduct the first study on the association between environmental arsenic exposure and male reproductive diseases, and found that arsenic exposure at normal environmental concentrations not only showed a dose-effect relationship with the incidence of male infertility, but also was significantly correlated with a series of disease metabolite markers (acylcarnitine, aspartic acid, estradiol metabolites and uridine, etc.) [50]. This laid the foundation for using seminal plasma metabolomics to explore the molecular mechanism of environmental exposure leading to decreased sperm quality. We analyzed for the first time the complex association between the internal exposure levels of endocrine disruptors phthalates, perfluorinated compounds and various metal and non-metal elements, the seminal plasma metabolome and sperm quality parameters [7, 26, 29]. The results show that seminal plasma metabolites are ideal research objects for studying the relationship between environmental exposure and sperm quality [7]; endocrine disruptors phthalates can affect sperm quality by affecting the content of polyunsaturated fatty acids, acylcarnitines and amino acids [26, 29], while environmental zinc and selenium increase sperm concentration by affecting the content of acylcarnitines [7], and inorganic arsenic reduces sperm concentration by affecting fatty acid and carnitine metabolism [29].

3 Conclusion and Prospect

Recent studies on the seminal plasma metabolome have shown that abnormal metabolism of seminal plasma carbohydrates, amino acids, lipids and carnitine may be the main pathways of male reproductive system diseases and environmental exposure reproductive toxicology. However, research in this field still faces huge challenges.

The biological interpretation of metabolomics data is highly dependent on the background knowledge of the discovered molecular pathways, but the physiological effects of seminal plasma molecules have not yet been fully elucidated. Therefore, it is still impossible to comprehensively and deeply analyze the specific molecular pathways by which seminal plasma metabolites play physiological roles through metabolomics technology. In addition, the critical window of spermatogenesis is sensitive and complex, with huge differences between individuals. Individual metabolomes also have temporal and spatial differences, making it difficult to obtain a single, specific metabolic marker. Therefore, the use of a combination of markers is a feasible method to significantly enhance disease prediction capabilities [51]. Finally, there is an urgent need to effectively integrate multi-level seminal plasma omics data (including transcriptomics, proteomics, and lipidomics) to conduct in-depth mechanistic studies of reproductive-related diseases and provide a basis for clinical intervention measures to improve male semen quality.

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