Screening, identification and interaction analysis of key MicroRNAs and genes in Asthenozoospermia

Introduction

Male infertility is a severe problem in human reproduction health ^{1, 2}. Abnormal sperm morphology, poor sperm motility and low sperm count are identified as common causes of male infertility. According to the criteria reported by the World Health Organization, asthenozoospermia is characterized by total motility < 40% and progressive motility < 32% in fresh semen samples ³. Several factors have been confirmed to be associated with asthenozoospermia, as shown in Figure 1. Chromosomal abnormality was correlated with sperm deficiency including asthenozoospermia ⁴. The multiple morphological abnormalities of the sperm flagella (MMAF) is one of the most known causes of asthenozoospermia. Mutations in the mitochondrial genome have been reported in MMAF, such as DNAH1, CFAP43, CFAP44, CFAP69, and QRICH2 ^5-8, and study have shown compelling evidence that the loss-of-function mutations of these genes could induce MMAF and lead to male infertility. Hormone could also modulate spermatogenesis and impact fertilization. In a clinical trial, most of 786 subfertility patients were characterized as idiopathic oligo or asthenozoospermia who improved their semen quality and concentration by receiving hormonal treatment ⁹. Sperm motility was under the control of flagellum ¹⁰ and any structural or functional defects in flagellum would lead to reduced motility and induce asthenozoospermia ¹¹. Moreover, the anti-sperm antibodies played key roles in female and male immunological infertility and sperm-immobilizing antibodies that bound to the surface of ejaculated sperm could impact the sperm motility ¹². There were other exogenous sources could result in asthenozoospermia, for example, drinking alcohol, smoking, toxicant and physical activity also have effect on the quality of sperm ^13-17. However, the epigenetic signatures which affect asthenozoospermia are not well understood and require to be further investigated.

Currently, increasing genetic factors contributing to male infertility were reported. Alterations were observed between fertile men and asthenozoospermia patients' transcriptomes, including pathways and genes which were associated with this disease. SEMG1 mRNA and protein expression were upregulated in asthenozoospermia patients compared with that in normozoospermia group ¹⁸. CABYR and ROPN1 mRNA were significantly downregulated in asthenozoospermia patients' samples and a positive correlation was confirmed between the expression of the two genes, indicating that the co-expression of CABYR and ROPN1 was a prerequisite for normal sperm motility and flagellar function ¹⁹. In addition, miRNAs also have dysregulated expressions in testicular biopsy samples and sperms of infertile men. The expression of miR-34b in the control group was significantly higher than that in the asthenozoospermia group, demonstrating that miR-34b might serve as a novel biomarker of male subfertility ²⁰. The downregulated expression of miR-525 and upregulated expression of miR-151a were also associated with asthenozoospermia and male infertility ^{18, 21}.

In this study, we explored the differential expressed miRNAs (DEMs) and differentially expressed genes (DEGs) in asthenozoospermia patients through reanalyzing the data from published studies and a GEO dataset. Then, we conducted pathway and gene ontology analysis of DEGs using DAVID. The protein-protein interaction (PPI) network was constructed by STRING and DEMs' target genes were predicted by TargetScan. Our results proposed a novel insight into the related biological characteristics and molecular pathways of the DEMs and DEGs in asthenozoospermia. A miRNA-mRNA network further exhibited the potential mechanisms linked to asthenozoospermia.

Methods

Results

Discussion

Male infertility is a complicated multifactorial pathological condition affecting around 7% male population ²⁸. Particularly, sperm motility is a crucial factor for fertilization, and over 80% of male infertility is induced by sperm motility impairment ⁸. Sperm functional or structural defects, deleterious effect of seminal plasma or a combination of these may impair sperm motility ²⁹. Asthenozoospermia, also known as asthenospermia, is a common condition characterized by low sperm motility ³⁰. Interestingly, recent studies have reported that sperm RNAs, including a wide variety of mRNA, miRNA and lncRNA were emerging as critical roles regulating asthenozoospermia ³¹. Evidence has revealed certain associations 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 explore the 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 seemed to be one of the best normalizing miRNA candidates associated with spermatogenesis and embryogenesis, and it also exhibited a particular behavior and a stable expression in human fertile individuals ³². In addition, decreased miR-374b expression could act as a first indication of increased sperm DNA fragmentation index, indicating that miR-374b would potentially become a diagnostic biomarker of idiopathic infertile males ³³. miR-193a was upregulated in sterile triploid fish which might take part in sperm activity and testicular development by targeting functional genes ³⁴. The expression of miR-34b in asthenozoospermia group was significantly lower than control group ²⁰. Besides, the frequency of methylation of the promoter region of miR-34b in infertile men was higher than fertile men, and the highest frequency of methylation was observed in asthenoteratospermia patients ³⁵. These 3 DEMs were proven to be associated spermatogenesis and they were promising for severing as novel biomarkers that could enhance the diagnosis of male infertility. We could further explore the epigenetic regulatory 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 were 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 ³⁶. Another study has indicated that post-transcriptional Wnt signaling could affect spermatozoa through GSK3 through inhibiting protein phosphatase 1 to initiate sperm motility ³⁷. The identification of DEGs and DEMs in asthenozoospermia could provide an insight into the molecular mechanisms driving spermatogenesis and open the possibility of 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, a member of the nerve growth factor family. BDNF was detected in the head, neck, and tail of human spermatozoa ³⁸, and exogenous BDNF at 0.133 nM could significantly influence viability and motility of human sperm ³⁹. Interestingly, another neurotrophic factors, NGF, has been reported to rescue Sertoli cell viability ⁴⁰. NGFR, a low affinity receptor which can bind to NGF, exerted a role in the processes of rat Leydig cell postnatal differentiation and in the regulation of functional activities ⁴¹. The expression of miR-4485 was significantly downregulated in the asthenozoospermia patients compared to controls, and KIT, which acted as its target gene, was related to male infertility by bioinformatic analysis ⁴². A recent study has uncovered a decrease in abundancy of CCR7 receptor in the high-fertile boars ⁴³. In particular, these hub genes were enriched in the 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. Many aspects of fertility depended on intact neurologic function. Therefore, these similar results provided a comprehensive overview that nerve-related genes might also impact male fertilization, and their relationships required to be investigated further. We also constructed the miRNA-mRNA network, and all of these central interactions suggested us new clues to explore underlying mechanisms between miRNA and its target mRNA in asthenozoospermia.

Testicular cancer is one of the most common cancers in men with major semen parameter disturbances ^{44, 45}. 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.

In our study, the promising miRNAs and mRNA in asthenozoospermia were filtered. They might be dysregulated and exhibited diagnostic values in asthenozoospermia. Additionally, we constructed the miRNA-mRNA network which revealed the underlying mechanism in asthenozoospermia. The development of therapies through direct targeting this network might contribute to the effective treatment of asthenozoospermia. Overall, our findings provided several clues to future diagnosis and clinical treatment of asthenozoospermia.

There were 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 miRNAs 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.

Conclusion

In summary, we were the first to pool published studies and a GEO dataset to identify potential DEMs and DEGs that were involved in asthenozoospermia. Importantly, we also clearly demonstrated the relationship between DEMs and DEGs and suggested regulatory pathways in asthenozoospermia including signaling pathways regulating pluripotency of stem cells, Wnt signaling pathway and Notch signaling pathway. We provided key information to profoundly understand the pathological process of asthenozoospermia and presented theoretical basis to discover novel therapeutic interventions. Further studies in vivo and in vitro are required to confirm these intriguing mechanistic possibilities.

Supplementary Material