In this study, we first performed UHPLC-QE-MS untargeted metabolomics to detect metabolites in the seminal plasma from controls, varicocele and varicocelectomy groups. Our results clearly show that varicocele significantly affects sperm metabolism, and microsurgical varicocelectomy can reverse this metabolic abnormality. We then identified potential biomarkers to discriminate varicocele from normal controls and to predict the microsurgical outcomes, thus providing tools that can be used to diagnose varicocele earlier. We also identified that certain dipeptides existed in the metabolites of seminal plasma in patient with varicocele; a reduction in the levels of these dipeptides may contribute to the improvement of semen quality after microsurgery.
As showned in Fig. 1A,B,C, the sample quality was good, that the experimental methodology was excellent, and the system stability control was outstanding. Although metabonomics has been applied to the study of male infertility[14–18], so far, it has not been applied to the study of varicocelectomy, especially the change of semen quality after microsurgery. As shown in Fig. 1DEF and Supplementary Table 2, there were marked differences in terms of metabolites in the seminal plasma when compared between the controls, varicocele, and varicocelectomy groups. The reason for this great difference may be that the blood flow is affected by varicocele, resulting in local ischemia and anoxia of testis, and the metabolism of sperm is decreased. Whatever, these results hint that the determination of sperm metabolic changes may be used as a tool for the diagnosis and treatment of varicocele. Our results showed that differences in the levels of metabolites before and after microsurgery were significantly reduced compared with those in patients with varicocele and controls, although there was still strong correlation and clustering properties (Fig. 1). The main reason for these findings may be related to individual genetic background. We compared the same patients before and after surgery; in contrast, patients with varicocele and controls were entirely different individuals.
Our results also showed that the metabolism of glycerophospholipid and sphingolipid were the main changes in sperm metabolism following surgery, and that the main metabolic molecules in these two pathways were significantly up-regulated after microsurgery (Fig. 3). Diphosphatidylglycerol, one of the main phospholipids synthesized by the mitochondrial inner membrane, is related to the assembly and activity of many protein complexes in the mitochondria . These results suggested that the increased mitochondrial activity of sperm may be related to an increase of sperm energy supply and motility. Sphingolipid and its metabolites are not only important structural molecules for the cell membrane, they are also involved in many important signal transduction processes, including cell growth, differentiation, senescence, and programmed cell death . This may explain the improvement of sperm morphology (membrane structure), the reduction in sperm apoptosis, and the increase in sperm concentration, following surgery. Although the differences in metabolic molecules before and after microsurgery was relatively small, these differentially expressed molecules were associated with complex molecular network regulation, thus indicating the importance and influence of these differentially expressed molecules (Fig. 3D,E,F).
8 metabolites and 3 dipeptides were identified to be potential biomarkers for varicocele (Figs. 4 and 5). These 3 dipeptides have not been identified in any other biological fluid before. We found that these dipeptides even existed in the subclinical types of varicocele, and can therefore be used as biomarkers for the early diagnosis of varicocele. In patients with varicocele, the concentration of these dipeptides decreased after operation, but still existed. However, the dipeptides were not detectable in the normal group of males. It was also difficult to detect these dipeptides in the semen of vasectomized patients, indicating that these dipeptides are not derived from the prostate, seminal vesicles, or other accessory gonads (data not shown).
We also found that DPEP3 was expressed in sperm and that levels of this dipeptide were reduced in varicocele patients. This will lead to a reduction in the concentrations of leucine, glutamic acid, tyrosine, phenylalanine, and isoleucine, in the seminal plasma. A reduction in the levels of leucine will result in a reduction in antioxidant and anti-inflammatory ability . A reduction of glutamic acid will lead to a reduction in acid resistance, thus making sperm vulnerable to damage .
The disadvantage of this study is that it is a single center, small sample study. Only the left grade III varicose veins are selected for the study. The selected biomarkers need to be further confirmed in large samples and different stages of disease development.