| According to the World Health Organization(WHO),male infertility is defined as the inability to conceive after unprotected intercourse for at least one year owing to the male factors.Epidemiological surveys show that nearly 30 million men worldwide suffer from infertility.In recent years,due to increased long-term mental stress and exposure to environmental toxicants,the incidence of male infertility is still rising.Asthenozoospermia,the most prevalent male infertility disorder,which is caused by the compromised sperm motility,has become an important health issue.Therefore,it is particularly essential to uncover the etiology and potential pathogenesis of asthenozoospermia,and to seek effective treatment targets.Spermatogenesis is a complex physiological process that occurs in a coordinated and orchestrated manner which is regulated at various levels.The coordinated regulation of phase-specifically expressed genes is critical for the normal development of male gametes.In recent decades,mounting studies have demonstrated that the gene regulations occurring at the transcriptional level and post-transcriptional level exist throughout the whole process of spermatogenesis.The role and the regulation mechanisms of small non-coding RNAs(nc RNAs)in the field of spermatogenesis,including mi RNA(micro RNA),pi RNA(PIWI-interacting RNA)and si RNA(Small Interfering RNA),have been widely explored.Additionally,researchers have pointed out that paternal contributions to fertilization not only lie in transporting the haploid paternal genome into the oocyte,but also in delivering paternal RNAs into the oocyte upon fertilization,indicating that spermatozoal RNAs play a role in the early development of embryos.Up to now,the circular RNA,as a novel transcript form,its role in the biogenesis of human male gametes and the early development of embryos remain an underexplored area.Circular RNA constitutes a novel non-coding RNA family with unique structures and largely unanswered biological functions in recent eras.It is a single-stranded and covalently closed circular molecule,formed in a back-splicing manner from a precursor m RNA.Unlike canonical linear m RNA,circular RNA is not equipped with the cap structure in the 5' end and poly A tailing in the 3' end,which empowers it the ability to tolerate the digestion of 5'-3' exonuclease,so that it can stably exist in body fluids such as semen and saliva without being degraded.Also,circular RNA owns a longer half-life than its linear m RNA isoform.This unparalleled stability puts a unique advantage of circular RNAs as a biomarker in disease diagnosis.Additionally,circular RNAs can compete for binding with mi RNA owing to its MRE sites(mi RNA Response Element),therefore,they are capable of regulating targeted gene expression and the abundance of gene products.Based on the above clarified characteristics,circular RNAs are currently reported to be involved in a variety of human diseases,encompassing cancers and non-neoplastic diseases.As for the reproductive system,expression profiles of circular RNAs are identified in human seminal plasma and bovine testis tissues,and certain specific circular RNAs display differential expression under different circumstances(such as health and disease,adult cattle and fetal cattle).These findings reveal that circular RNAs may play important biological functions in the reproductive system,especially in the male reproductive system.Therefore,it is of great significance to map the circular RNA expression profiles in human spermatozoa and to identify differentially expressed circular RNAs in pathological conditions.Relevant studies have not yet been reported.By constructing the expression profile of circular RNAs in human spermatozoa,screening and verifying specific circular RNAs that are related to asthenozoospermia,this study aims at establishing a more sensitive and specific diagnostic indicator than routine semen analysis assay,providing a scientific basis for elucidating the etiology of asthenozoospermia,and shedding new light on the pathogenesis of male infertility.Objectives 1.To identify and characterize the expression of circular RNAs in human spermatozoa,profiling and revealing its distribution features.2.To screen and validate differentially expressed circular RNAs between asthenozoospermia patients and healthy male controls,preliminarily performing functional analysis.3.To explore the underlying mechanism of circular RNA in the occurrence and development of asthenozoospermia.Methods After collecting the spermatozoa from the asthenozoospermia group and the control group,total RNA was extracted by TRIzol reagent,and the linear transcripts were removed by RNase R treatment.The enriched circular RNA was used for transcriptome library preparation,paired-end NGS sequencing and subsequent bioinformatics analysis to profile the expression pattern and distribution characteristics of circular RNA in sperms from asthenozoospermia patients.The online DAVID software was applied for GO analysis.Meanwhile,the Circ Base and Circ Net databases were utilized to predict the circ RNA-mi RNA-m RNA co-interaction regulatory network.Finally,five circular RNAs highly expressed in the human reproductive system were selected from the top 200 differentially expressed circular RNAs among two groups.PCR assay was performed with divergent primers and convergent primers,and PCR products were recovered for Sanger sequencing to seek the back-splicing junction.Also,expression levels of five selected circular RNAs among groups were compared by RT-q PCR,as well as expression levels of circular RNA and their linear isoforms encoded by the same gene in patients.Results 1.Expression characteristics and distribution of human spermatozoa derived circular RNAs-Almost 83% of spermatozoa circular RNAs were originated from exons and the majority of these exonic circular RNAs consisted of 2-4 exons.Host genes from all 24 chromosomes were capable of producing circular RNAs,but chromosome 1 encoded the most circular RNAs.2.Gene Ontology enrichment analysis of differentially expressed spermatozoal circular RNAs(1)Cell component(CC): significantly enriched in sperm flagella,primary cilium,flagellar axoneme.(2)Biological process(BP): significantly enriched in ciliary morphogenesis,regulation of ciliary beating frequency,determination of left/right asymmetry of the body axis,epithelial ciliary movement,assembly of axoneme dynamic protein arm complex.(3)Molecular function(MF): significantly enriched in methylation histone binding,histone acetyltransferase activity and histone methyltransferase activity.3.Preliminary validation of five differentially expressed candidate circular RNAs The coding genes corresponding to the selected five circular RNAs were all highly expressed in the human reproductive system.hsa_circ_MYO9B,hsa_circ_WHSC1(8),hsa_circ_WHSC1(6)and hsa_circ_CAMSAP1,hsa_circ_FAM213A were successfully amplified by PCR with divergent primers.Sanger sequencing confirmed their back-splicing junction,but hsa_circ_FAM213A failed to Sanger sequencing.4.Differential expression analysis of five candidate circular RNAs(1)Differential expression among asthenozoospermia patients and their matched healthy counterparts Compared with their healthy control,the expression levels of hsa_circ_WHSC1(8),hsa_circ_WHSC1(6)and hsa_circ_CAMSAP1 were significantly up-regulated in asthenozoospermia patients(p <0.05).Meanwhile,the expression level of hsa_circ_FAM213A in asthenozoospermia patients showed a tendency of up-regulation,but the difference was not statistically significant(p >0.05).Conversely,the expression level of hsa_circ_MYO9B was significantly down-regulated in asthenozoospermia patients(p <0.01).(2)Differential expression among circular RNAs and their linear isoforms in patients The abundance of five candidate circular RNAs were significantly lower than their linear isoforms(p<0.01).However,considering the fact that under physiological conditions,the yield of circular transcripts was usually less than one twentieth of their linear counterparts,four(hsa_circ_FAM213A,hsa_circ_WHSC1(8 hsa_circ_WHSC1(6)and hsa_circ_CAMSAP1)of five candidate circular RNAs dramatically up-regulated when compared with physiological conditions.On the contrary,the expression abundance of hsa_circ_MYO9B was significantly down-regulated.5.Prediction analysis of interaction network between the five candidate circular RNAs and targeted mi RNAs as well as m RNAs(1)mi RNAs interacting with hsa_circ_MYO9B There were 13 mi RNAs capable of targeting MYO9 B m RNA,which eventually formed two m RNA-mi RNA-circ RNA axes,with mi RNA let-7e-5p and mi RNA-484 being the connecting nodes.(2)mi RNAs interacting with hsa_circ_CAMSAP1 There were 40 mi RNAs capable of targeting CAMSAP1 m RNA,and a total of three m RNA-mi RNA-circ RNA axes are formed.The node mi RNAs were: mi RNA-33a-3p,mi RNA-4303 and mi RNA-4468,respectively.(3)mi RNAs interacting with the remaining three candidate circular RNAs There were 40 mi RNAs capable of targeting WHSC1 m RNA,but no negative feedback loop was found.Meanwhile,hsa_circ_WHSC1(6)and hsa_circ_FAM213A failed to identify potential targeted mi RNAs in the present database.Conclusions 1.This study confirmed the presence of circular RNAs in human spermatozoa and constructed its expression profile.circular RNAs were differentially expressed among asthenozoospermia patients and their matched controls.Besides,circular RNAs and linear transcripts encoded by the same gene showed different expression levels.2.Differentially expressed circular RNAs were related to sperm motility.3.Sanger sequencing solidly validated the presence of back-splicing sites of five candidate circular RNAs,which was widely accepted as the most direct evidence for the presence of circular RNA.4.Circular RNAs acted in the m RNA-mi RNA-circ RNA interaction network,regulating expression abundance of the targeted genes. 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