Expression profiles of circular RNAs in spermatozoa from aging men

Advanced paternal age (APA) is associated with decreased fertility, but the mechanism underlying APA remains unknown. CircRNAs have been reported to be ideal candidate biomarkers for diagnostic and therapeutic applications in many diseases and are also involved in spermatogenesis. Hence, we aimed to assess the circRNA expression profile of spermatozoa from aging men. We recruited 6 subjects, including 3 in the younger group (men age < 40) and 3 in the APA group (men age ≥ 40). RNA sequencing was exploited to identify the expression profiles of circRNAs between the two groups. The expression levels of circRNAs were validated using real-time quantitative polymerase chain reaction (RT–qPCR). Kyoto Encyclopedia of Genes and Genomes biological pathway analysis and Gene Ontology analysis were performed to evaluate the functions of differentially expressed circRNAs (DE-circRNAs) between the two groups. In total, 18,787 circRNAs were sequenced in the spermatozoa of two groups. Our analysis revealed that there were 1056 downregulated circRNAs and 1228 upregulated circRNAs between the two groups, and KEGG analysis showed they were mainly involved in pathways including the DNA repair signaling pathway, meiotic recombination signaling pathway, and PI3K/AKT signaling pathway. In conclusion, our study suggested that circRNAs play a vital role in spermatozoa from aging men and provided a fresh perspective on the specific regulatory mechanism of spermatozoa from aging men.


Introduction
With modern lifestyles, increasing work pressure and the opening of China's three-child policy, the phenomenon of late marriage and late childbearing has become widespread, and advanced childbearing is receiving more attention in the field of reproductive medicine.A number of studies have found that pregnancy rate tends to decrease with increasing female age, in association with decreases in ovarian reserve and egg quality in older women, especially those offspring, such as achondroplasia (ACH) [15], autism spectrum disorder (ASD) [16], schizophrenia and bipolar disorder (BP) [17].However, the molecular mechanisms through which advanced paternal age causes these diseases remain to be elucidated.
Based on this, we sought ideal biomarkers and therapeutic targets in aging men.CircRNAs attracted our attention due to their important properties of high conservation and stability compared with other linear RNAs.CircRNAs are newly discovered closed ring RNA molecules produced by backsplicing that are mainly located in vesicles in the cytoplasm or stored in exosomes [18].Recent research on circRNAs has shown that they are involved in many regulatory processes, including transcriptional modulation, splice interference, miRNA segregation and translation [19], and are related with spermatogenesis, such as flagellum morphogenesis, spermatozoa development, sperm motility, et al [20][21][22].It has also been reported that circRNAs function as miRNA sponges, RNA binding proteins or translation regulators [23].However, there are no reports concerning circRNAs in APA.
Our study first explored the differential expression profiles of circRNAs in spermatozoa between younger (aged < 40 years) and APA (aged ≥ 40 years) men by nextgeneration sequencing (NGS).Furthermore, bioinformatics analysis was employed to investigate the potential functions of differentially expressed circRNAs from the two groups.

Human spermatozoa
Sperm samples were collected from volunteers (n = 6) who met the following criteria: (1) sperm concentration > 15*10 6 /ml, progressive motility ≥ 32%, and normal sperm morphology > 4%; and (2) no other diseases, including acute or chronic inflammation of reproductive glands and gonads, immune factors, etc.After 3-7 days of sexual abstinence, semen samples were produced by masturbation and collected in sterile sample containers.The samples were delivered to the laboratory within 1 h after ejaculation.The sperm samples were allowed to liquefy for 30 min at 37 °C and then analyzed using computer-assisted sperm analysis (CASA) technology.Then, the sperm samples were purified on a Percoll density gradient and stored in liquid nitrogen until RNA purification was performed.All subjects gave their informed consent for inclusion in the study.The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Nanjing Maternity and Child Healthcare Hospital, affiliated to Nanjing Medical University.

Total RNA extraction and RT-qPCR
Total RNA was collected from all samples, and the cDNA library was constructed following a previous report [24].Furthermore, new sperm samples were collected to performed RT-qPCR according to a previous report, and the primers are shown in Table 1.

CircRNA NGS
CircRNA NGS analysis of 6 sperm samples, including 3 sperm samples from the younger group and 3 sperm samples from the APA group, was implemented by Biotechnology (Shanghai, China) and the protocol was conducted according to previous report [25].

Gene Ontology and Kyoto Encyclopedia of Genes and Genomes biological pathway analysis
To further understand the biological functions and functional characteristics of the parental genes of abnormally expressed circRNAs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using DAVID.GO terms and KEGG pathways that met the criterion of adjusted p < 0.05 were considered significantly enriched.

Statistical analysis
All data were analyzed by GraphPad Prism 6.0 and the statistical significance of the data was analyzed by Student's t test.When the P value was < 0.05, the difference was considered to be statistically significant.

Characterization of circRNAs
We employed next-generation sequencing to profile cir-cRNAs in sperm samples from the younger group (men < 40 age) and APA group (men ≥ 40 age).We detected a total of 18,787 circRNAs, 61% of which are already known according to circBase (http://circrna.org/),and 39% of which are novel (Fig. 1a).The circRNAs of sperm from the younger group and the APA group were described (Fig. 1b), and these circRNAs were distributed on all chromosomes, excluding mitochondrial chromosome (Fig. 1c).As shown in Fig. 1d, the majority of circRNAs were exonic in origin.

Overview of differentially expressed circRNAs (DE-circRNAs)
A hierarchical clustering plot showed DE-circRNAs (Fig. 2a) among the sperm samples, and a volcano plot was generated to distinguish DE-circRNAs according to the following criteria: fold change cutoff ≥ 1.5 and p value cutoff ≤ 0.05 (Fig. 2b).In addition, as shown in Fig. 2c, the largest numbers of DE-circRNAs were distributed on chromosomes 1, 2, and 3. Most of the upregulated and downregulated circRNAs were derived from exons (Fig. 2d).

Construction of circRNA-miRNA interaction network
CircRNA-miRNA interactions were predicted by popular target prediction software, and an interaction network was constructed by Cytoscape software.Specific predictions were made based on miRanda (Fig. 6).

Discussion
Recently, much attention has been given to advanced paternal age, which not only reduces conception rate and increases the likelihood of spontaneous miscarriage [26] but also increases the risk of psychiatric and developmental disorders in the offspring [27].In our study, we first identified circRNAs connected with APA and analyzed the differences

Functional analysis of DE-circRNAs
We performed GO analysis, and with respect to biological process terms, we identified that DE-circRNAs were mainly involved in organelle organization, cell projection assembly, chromosome organization, microtubule-based processes, etc.With respect to cellular component terms, DE-circRNAs were associated with the microtubule organizing center, cilia, axonemes, intraciliary transport particle B, etc.With respect to molecular function terms, DE-cir-cRNAs were related to zinc ion binding, ATP binding, tubulin binding, motor activity, cyclin binding, etc. (Fig. 4).Further, KEGG analysis was performed and demonstrated  4 Gene Ontology (GO) classification of the parental genes of the differentially expressed circRNAs.Fig. 6 The network of circRNAs and the predicted binding partner miRNAs.Fig. 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the pathways of the parental genes of the differentially expressed circRNAs. 1 3 in circRNA expression profiles between younger and APA men.
We mapped the changes in circRNA expression patterns in human spermatozoa and found significant differences between young and older men.We found 1228 upregulated and 1056 downregulated circRNAs between the younger group and APA group.To more fully understand the mechanism of APA, we performed GO and KEGG enrichment analyses.Gene Ontology analysis showed that DE-cir-cRNAs between the younger group and APA group were mainly associated with ATP binding, tubulin binding, motor activity, and cyclin binding, and KEGG pathway analysis demonstrated that DE-circRNAs are involved in several pathways, including the DNA repair signaling pathway, meiotic recombination signaling pathway, and PI3K/AKT signaling pathway.
PI3 kinase (PI3K) and its target AKT (protein kinase B) have been reported to protect spermatozoa from oxidative stress-induced apoptosis [28].The balance of reactive oxygen species (ROS) cluster products and biological antioxidants in spermatozoa is necessary to maintain functions including sperm motility, energy acquisition, the acrosome reaction and sperm-egg fusion [29].However, Mazur et al. demonstrated reduced antioxidant capacity and excessive ROS in sperm from aging males [30].The overproduction of ROS can trigger damage to spermatozoa, such as lipid peroxidation, single-stranded or double-stranded DNA breaks, reduced mitochondrial membrane potential, and ultimately impact sperm-egg fusion [31].Furthermore, Qiao et al. reported that APA could cause adverse pregnancy outcomes and birth defects [32].Therefore, we need to consider paternal age in addition to maternal age.Our study highlights several possible mechanisms underlying the reduced function of spermatozoa in aging men.However, there are some limitations in our research, such as the small volume of sperm samples and a lack of in-depth functional studies.Therefore, we will explore the detailed mechanisms involved in APA in future studies.
In conclusion, our study showed the DE-circRNAs between younger (< 40 years) and APA (≥ 40 years) men based on the analysis of expression profiles, elucidating potential signaling pathways involved in APA and further predicting the potential interactions of the DE-circRNAs with miRNAs.Further exploratory experiments and mechanistic studies need to be performed to understand the mechanisms by which circRNAs regulate the expression of related genes.

Fig. 1 Fig. 3
Fig. 1 Overview of circular circRNAs isolated in sperm from the younger group and APA group.a The percentage of known circRNAs and new circRNAs identified in the two groups.b All evaluated cir-cRNAs from the two groups.The height of the column represents the

Fig. 2
Fig. 2 Changes in the circRNA expression profile in aging men. a Hierarchical clustering analysis of significantly upregulated or downregulated circRNAs.b Heatmap of differentially expressed circRNAs.The volcano plot was generated to highlight circRNAs with a fold change > 2.0 and P < 0.05.The red dots in the figure represent the sta-

Table 1
Primer sequences and annealing temperatures for circRNAs.

Table 2
Essential characteristics of the 6 randomly selected circRNAs.