Male infertility is a complicated multifactorial pathological condition affecting around 7% male population . Particularly, sperm motility is a crucial factor for fertilization, and over 80% of male infertility is induced by sperm motility impairment . Sperm functional or structural defects, deleterious effect of seminal plasma or a combination of these may impair sperm motility . Asthenozoospermia, also known as asthenospermia, is a common condition characterized by low sperm motility . Interestingly, recent studies have reported that sperm RNAs, including a wide variety of mRNA, miRNA and lncRNA, were emerging as critical mechanisms regulating asthenozoospermia . Evidence has revealed the association between asthenozoospermia and specific variations of sperm transcriptome profile and also identified some RNA molecules were differentially expressed in asthenozoospermia patients. Our study aimed to investigate significant dysregulated miRNAs and genes in asthenozoospermia and potential regulatory network in asthenozoospermia pathological condition.
First, we identified 3 overlapped DEMs including miR-374b, miR-193a and miR-34b by screening out previous studies. miR-374b showed a particular behavior and a stable expression in human fertile individuals that could serve as a fertility biomarker. It also seemed to be one of the best normalizing miRNA candidates associated with spermatogenesis and embryogenesis . miR-193a was upregulated in sterile triploid fish which might take part in sperm activity and testicular development by targeting functional genes . miR-34b expression in asthenozoospermia group was also significantly lower compared to control group . In addition, the frequency of methylation of the promoter region of miR-34b in infertile men was higher compared to fertile men, and the highest frequency of methylation was observed in asthenoteratospermia patients . These 3 DEMs were proven to have the potential to become novel molecular biomarkers that could enhance the diagnosis of male infertility. We could further explore the related molecular mechanisms of miR-374b, miR-193a and miR-34b in asthenozoospermia.
We next identified 423 DEGs between GSE22331 dataset and Xiaoning Zhang’s study. Among these DGEs, 250 genes were upregulated, and 173 genes were downregulated in asthenozoospermia. We observed that DGEs enriched in development-related pathways, such as signaling pathways regulating pluripotency of stem cells, wnt signaling pathway and notch signaling pathway. Studies have reported the germ cell lineage originated in the early stage of development and underwent a series of complicate developmental processes that culminate in the generation of the fully matured gametes, the oocytes and the spermatozoa. Human gametogenesis might be reconstituted from pluripotent stem cells, which would facilitate our understanding of germ cell development and fertilization . Another study has indicated that post-transcriptional wnt signaling could affect spermatozoa through GSK3 through inhibiting protein phosphatase 1 to initiate sperm motility . The identification of DEGs and DEMs in asthenozoospermia could provide an insight into the molecular mechanisms driving spermatogenesis and provide new therapeutic targets to repair impaired sperm physiological function and quality.
Further investigation of PPI network, BDNF, NTRK2, HNRNPD, EFTUD2, GSK3B, NGFR, ELAVL2, RPS8, KIT, TRAF6, DVL1, FGF7, SNW1, SYF2, NCL, CRK, NEDD4, TBL3, RRP12, GNL2, NRXN1, KLHL2, LRP5, FBXW8, and CCR7 were identified as hub genes. The most significant gene was BDNF and findings that were similar to previous reports. BDNF was detected in the head, neck, and tail of human spermatozoa , and exogenous BDNF at 0.133 nM could significantly influence viability and motility of human sperm . The expression of miR-4485 was significantly downregulated in the asthenozoospermia patients compared to controls, and KIT, which acts as its target gene, was related to male infertility by bioinformatic analysis . These hub genes enriched in neurotrophin signaling pathway, indicating that they were critical for differentiation of nerve cells as well as playing critical roles in the development of reproductive system and the maintenance of spermatozoa normal function. Interestingly, another neurotrophic factor, NGF (Nerve Growth Factor), had been reported to rescue Sertoli cell viability . Many aspects of fertility depended on intact neurologic function. Therefore, these similar results provided a comprehensive overview that nerve-related genes could also impact male fertilization, and their relationship needs to be investigated further. We also constructed the miRNA-mRNA network, and all of these represented central interactions suggested us new clues to explore underlying mechanisms between miRNA and target mRNA in asthenozoospermia.
Testicular cancer is one of the most common cancers in men with major semen parameter disturbances [40, 41]. We further tested whether these putative hub genes in asthenozoospermia could extensively affect testicular cancer. We compared the mRNA expression of 25 hub genes between testicular cancer patients and normal controls by GEPIA dataset. As expected, EFTUD2, ELAVL2, HNRNPD, KIT, NCL, NRXN1 and RPS8 were significantly varied in testicular cancer patients.
There are still certain limitations in our current study. There was no available data by searching the GEO database for published microarray results of miRNAs in asthenozoospermia. Therefore, we reused microarray assays data of miRNA in asthenozoospermia from 4 published studies by applying a systematic search of PubMed and Web of Science. However, these search results might be incomprehensive, and the inclusion criteria of GEMs were different. Additionally, DGEs in GSE22331 only based on FC ≥ 2 without FDR, since there was only one pooled sample in each group. Hence, the repeatability of the results might not be reliable. The findings of the miRNA-mRNA network did not reveal a high degree of several DEMs, because of our strict including criteria of miRNA and mRNA and limited miRNA target prediction database. In further study, the expressions and associations of DEMs and DEGs require to be validated by dual luciferase reporter assay or other experiments.