Small RNA profile alterations as a potential mechanism for sperm quality improvement following varicocelectomy.

A varicocele is a pathological enlargement of the pampiniform plexus (testicular veins) in the scrotum. It is a frequent cause of male infertility and affects approximately 15%–20% of adult males. 1 Among male patients with primary infertility, up to 40% are diagnosed with varicoceles, and this proportion is as high as 80% in patients with secondary infertility. 2 Varicoceles can lead to spermatogenetic damage, resulting in impairment of the affected testicle and changes in sperm quality. 3 Surgical treatment by varicocelectomy is often recommended to reduce the damage to sperm and improve male fertility. 4 5 Following varicocelectomy, sperm quality is improved, with restoration of sperm concentration, motility, and morphology. 6 7 Spontaneous pregnancy is also reported in 20%–40% of cases involving varicoceles. 8 Numerous studies have explored the genetic factors underlying male infertility. However, the identified causes explain only 15% of cases. 7 9 10 Therefore, further research is necessary to fully understand the underlying mechanisms of the pathophysiology of male infertility in patients with varicoceles. Researches have indicated that epigenetic changes may play a significant role in male reproductive health. Small noncoding RNAs (sncRNAs) are abundantly expressed in sperm and have been shown to exhibit critical functions in male fertility and varicoceles. 10 11 12 MicroRNAs (miRNAs) are the most extensively studied sncRNAs; they are typically 19–23 nucleotides (nt) in length and modulate gene expression by suppressing translation and destabilizing target mRNAs. 13 miRNAs influence a range of physiological and developmental processes, including cell differentiation, proliferation, and fertility. 14 15 16 Studies have shown that several miRNAs, such as miR-210-3p, miR-21, miR-34a, miR-122a, miR-181a, miR-34c, and miR-192a, show differential expression levels in the semen of patients with varicoceles compared with healthy individuals. Hekim et al . 17 found that the expression of hsa-miR-145 in sperm was decreased in infertile males with varicoceles. The levels of hsa-miR-192a in seminal plasma and testicular tissues were elevated in patients without spermatozoa after varicocelectomy compared with those with spermatozoa. 18 miRNAs not only play important roles in male fertility but also in zygote development. 19 Our prior studies identified several differentially expressed miRNAs (hsa-miR-101-3p, hsa-miR-132-3p, hsa-miR-191-5p, hsa-miR-29a-3p, and hsa-miR-520a-5p) in human sperm that correlate with embryo quality following in vitro fertilization. 12 Together, these findings indicate that the expression profiles of sncRNAs may be altered in males with infertility following surgical intervention for varicoceles and may also contribute to an improved fertility outcome. In this study, we assessed the relationship of sperm quality and sperm miRNAs in infertile males who underwent surgical varicocele treatment. We screened 63 pairs of semen samples from varicocele patients before and after varicocelectomy and found that semen parameters, including sperm concentration, progressive motility, and normal morphology, improved significantly after surgery. Using high-throughput deep sequencing of sperm sncRNAs, we identified 10 miRNAs whose expression levels significantly changed after varicocelectomy (3 were upregulated: hsa-miR-486-3p, hsa-miR-486-5p, and hsa-miR-874-3p; and 7 were downregulated: hsa-miR-132-3p, hsa-miR-202-3p, hsa-miR-34c-5p, hsa-miR-499a-5p, hsa-miR-499b-3p, hsa-miR-520a-3p, and hsa-miR-92b-3p). Further in vitro embryo analysis showed that the abnormal elevation of these miRNAs in zygotes is detrimental to early embryonic development. These results indicate that abnormal miRNA expression may be a potential mechanism underlying varicocele-associated sperm quality impairment.

JHL carried out the experiments. JYL followed up patients and collected sample information. GYZ, HGH, and XW carried out sperm quality indices test. GWC, GQL, and JLH performed varicocelectomy on patients. JNT and XMW performed the oocytes collection and cultured embryos in vitro . MMH drafted the manuscript. TCZ and XZ revised the manuscript. HJS designed the experiments. JZ conceived and supervised the project. All authors read and approved the final manuscript.

Institutional Review Board approval (No. 2022001) was obtained from Shanghai Jishengsuo Hospital (Shanghai, China) before initiation of this study, and informed consent was obtained from all participants. Permission for the public release of these data has been granted. Male patients with varicoceles who required surgery at Shanghai Jishengsuo Hospital between August 2022 and August 2024 were enrolled in this study. Exclusion criteria included any type of genetic disease or a cancer diagnosis. The follow-up period for all patients was at least 1 month. A total of 63 patients were enrolled in the study. Semen samples were collected before surgery. All patients were followed up at least 1 month after varicocelectomy, and semen samples were collected again. Samples were obtained from participants by masturbation after 3 days of sexual abstinence. The samples were allowed to liquefy at 37°C for 30 min and were processed immediately. Samples were analyzed for the primary semen parameters defined by the World Health Organization guidelines (5 th edition), 20 including sperm concentration, progressive motility, and normal sperm morphology. Isolation of total RNA was conducted as previously described, 12 with slight modifications. Briefly, total RNA was purified from supernatant of spermatozoa. The precipitated sperm was suspended in somatic cell elimination buffer and placed on ice for 20 min and then washed twice with phosphate-buffered saline (PBS). Next, 700 ml Qiazol Lysis reagent (QIAGEN GmbH, Hilden, Germany) mixed with dithiothreitol (DTT; 80 mmol l −1 ; Merck KGaA, Darmstadt, Germany) was added, and the mixture was incubated for 2 h to ensure complete lysis. The sample was then processed using the Qiagen miRNeasy Mini Kit following the manufacturer’s recommendations. The quantity and quality of the RNA were assessed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Small RNA libraries were constructed as previously described. 21 All libraries were sent for sequencing on either an Illumina HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA). Adapter sequences were removed from the 3’ end of the sequenced reads using fastp (version 0.20.1). 22 After quality filtering, the sequencing reads were mapped to the human genome (hg38) using Bowtie (version 1.2.1.1). 23 The reads were aligned sequentially to known miRNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), and small nuclear RNA (snRNA) sequences using Bowtie, without any mismatch allowed. Reads that aligned to pre-miRNA were identified as miRNAs. The remaining reads of the specified length were extracted and used to identify piwi-interacting RNAs (piRNAs) following the previously described method. 24 The miRWalk database ( http://mirwalk.umm.uni-heidelberg.de/ ; last accessed on 15 November 2024) was used to predict downstream target genes of differentially expressed miRNAs. Analysis was performed on predicted target genes that were identified in all three of the following databases: TargetScan, miRDB, and miRTarBase. Only gene targets with binding probability scores between miRNAs and target genes of at least 0.95 were considered. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to explore the biological function of all predicted target genes using the DAVID bioinformatics resources ( https://david-d.ncifcrf.gov/ ; last accessed on 02 December 2024). Three types of GO term analysis were applied, namely biological process (BP), cellular component (CC), and molecular function (MF). For both GO and KEGG pathway analyses, the thresholds were set as follows: at least two genes must be included in each term or pathway, and the adjusted P value (using the Benjamini–Hochberg correction) was below 0.1. Male and female B6D2F1 mice (6–8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co. (Beijing, China). Mouse zygotes were collected as previously described. 25 Briefly, female mice (8–10 weeks old) were intraperitoneally injected with 10 IU of Pregnant Mare Serum Gonadotropin (PMSG; mimics follicle-stimulating hormone and stimulates follicular development and oocyte maturation). After 48 h, the mice were injected with 10 IU of human chorionic gonadotropin (hCG) and mated with male mice. After 13 h, mated female mice with vaginal plugs were selected. Fertilized zygotes/cumulus masses were then surgically isolated from the oviducts. The zygotes were cultured in drops of K-modified simplex optimized medium (KSOM; Nanjing Aibei Biotechnology Co., Nanjing, China) under oil at 37°C in an atmosphere of 5% CO 2 , and embryo development was monitored at regular intervals. Fertilization was assessed by checking for two pronuclei and polar bodies. The 2-cell stage was checked at 24 h after microinjection (day 2), the 4- to 8-cell stage was assessed at 48 h (day 3), the morulae stage was observed at 72 h (day 3), and the blastocyst stage was checked at day 4. All mouse experiments were reviewed and approved by the Shanghai Institute for Biomedical and Pharmaceutical Technologies (Shanghai, China; Approval No. 2023-03). Mouse zygotes were microinjected with 3–5 pl of miRNA (miR-132-3p, miR-34c-5p, and miR-520a-3p; the sequences are shown in Supplementary Table 1 ) using an inverted microscope (Nikon Ti2; Nikon Corporation, Tokyo, Japan) with micromanipulators (NARISHIGE ONE-4; Narishige Co., LTD., Tokyo, Japan). The working concentration of miRNA was 1 ng μl −1 . The injection dose was controlled via the operation handle. The injected zygotes exhibited a distension of 20%–30%. Microinjected zygotes were then transferred into KSOM and cultured in vitro as described above. Sequences of synthetic microRNAs miRNA: microRNAs Single zygotic lysates were obtained using the RNA Single Cell Extraction and Purification Kit (Nanjing Your True-life BioTech Development Co., Ltd., Nanjing, China). Briefly, each zygote was washed three times in wash buffer. The zygote was then transferred to RNA extraction buffer and subjected to freezing and thawing in liquid nitrogen three times. miRNA (5 pg) was added, and the mixture was incubated at 37°C for 0 min, 10 min, 30 min, 60 min, or 24 h followed by RNA exaction. At the same time, Cel-miR-39 was added to the lysate as an internal loading control during RNA extraction. The sequence of Cel-miR-39 is listed in Supplementary Table 1 . 26 Reverse transcription for miRNAs (from human sperm and mouse zygotes) was conducted using the miRNA 1 st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s instructions. Briefly, 200 ng of RNA from each human sperm sample or total RNA from a single zygote was used for complementary DNA (cDNA) synthesis. The cDNA was then amplified using specific miRNA primers to examine the expression of particular miRNAs. The relative expression levels of miRNAs in human sperm or in single zygote from mice were analyzed using the miRNA Unimodal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) on the QuantStudio 3 system (QuantStudio 3; Thermo Fisher Scientific Inc.). Primers are listed in Supplementary Table 2 . Sequences of microRNAs for reverse transcription and quantitative real-time polymerase chain reaction miRNA: microRNAs At least three replicates were carried out for each experiment. GraphPad Prism (version 9.5.1; GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Data are presented as mean ± standard deviation (s.d.). P < 0.05 indicated statistical significance. R software (version 4.4.1) was used to analyze the differences in miRNA expression among the tested groups. Differential sncRNA expression analysis was performed using DESeq2 (version 1.46.0) with read counts data. Pathway analysis graphs were generated using the ggplot2 package (version 3.5.1).

This study included 63 male patients who had varicoceles and underwent varicocelectomy for infertility treatment. The patient age (mean ± s.d.) was 31.8 ± 3.9 years, and the body mass index (mean ± s.d.) was 23.98 ± 3.29 kg m −2 . Baseline sperm quality indices of semen parameters, including sperm concentration, progressive motility, and normal sperm morphology, are shown in Table 1 . All sperm parameters were within the normal limits indicated in the World Health Organization guidelines (5 th edition), 20 except for normal sperm morphology (mean ± s.d.: 2.5% ± 2.0%), which was below the standard (4%). Clinical characteristics of patients (n=63) BMI: body mass index; s.d.: standard deviation To investigate the effect of varicocelectomy on improving sperm quality, we assessed differences in all sperm quality indices of the 63 patients before and after surgery. Most patients showed improvement in three sperm quality indices, sperm concentration, progressive motility, and normal sperm morphology with log 2 (fold change) values greater than zero, with marked improvements in sperm concentration and normal sperm morphology ( Figure 1a ). We then analyzed the number of patients with improvements in sperm quality indices (defined as a fold change ≥1.5). We found that a large proportion of patients showed improvements in these indices, including 22 out of 63 patients for sperm concentration, 19 out of 63 for progressive motility, and 24 out of 63 for normal sperm morphology ( Figure 1b ). While 22 patients showed improvement in only one index (6 for sperm concentration, 7 for progressive motility, and 9 for normal sperm morphology), 17 patients showed improvement in two indices and 3 patients showed improvement in all three indices. Overall, among the 63 patients, 43 patients showed improvement in sperm quality after varicocelectomy, with improvement in at least one of the three indices after surgical treatment. Among the 43 patients with improvement, significant differences were observed in sperm concentration ( P < 0.05) and progressive motility ( P < 0.05) after surgery, with the most substantial improvement being in normal sperm morphology ( P < 0.0001; Figure 1c ). Improvement in sperm quality in patients with varicoceles after varicocelectomy. ( a ) Log 2 (fold change) of sperm quality indexes in 63 varicocele patients. Three main indexes were evaluated before and after surgery (before group, n = 63; after group, n = 63): sperm concentration, progressive motility, and normal sperm morphology. ( b ) Venn diagram analysis of the number of patients with improvements in sperm quality indexes (defined as a fold change ≥1.5). ( c ) The differences in sperm quality indexes among the 43 patients with improvements in sperm quality after surgery compared with before surgery. Improvement was defined as improvement in any one of three sperm quality indexes. * P < 0.05; **** P < 0.0001. We next explored the effects of varicocelectomy on sncRNA composition in sperm. We sequenced sperm small RNA libraries from the 63 patients before and after surgery. Approximately 14 418 149 and 8 641 890 raw reads were generated from samples before and after surgery, respectively, and 12 720 369 and 7 480 375 useful reads (15–30 nt) were obtained. A total of 8 920 511 and 5 522 580 unique reads were mapped to the reference genome ( Table 2 ), with a mapped ratio exceeding 70%. The length distribution of small RNA reads ranged from 17 nt to 40 nt in both groups, indicating a high proportion of sncRNAs in sperm ( Supplementary Figure 1 ). These results demonstrated that the reliability and quality of the reads were adequate for further analysis. Overview of small RNA sequencing in sperm from 63 patients before and after varicocelectomy The composition of the identified sncRNAs is shown in Figure 2 . Deep sequencing of sperm RNAs of the two groups identified several populations of sncRNAs mapping to the human genome, with varying proportions of expression across different samples ( Supplementary Table 3 ). The proportion of sperm sncRNAs varied slightly after varicocelectomy ( Figure 2a ). There were four major sncRNAs (mean ± s.d.) in sperm of before-surgery group: tRNA-derived small RNA (tsRNA; 74.0% ± 21.7%), rRNA-derived small RNA (rsRNA; 10.9% ± 11.5%), piRNA (5.5% ± 5.9%), and miRNA (4.11% ± 3.05%; Supplementary Table 4 ). The content of sncRNAs was consistent with that in sperm from individuals with normal fertility. 12 Expression profile of sncRNAs in sperm from the 63 patients. ( a ) The average composition of sncRNA categories in sperm in 63 patients before and after varicocelectomy. ( b ) The percentages of different sncRNAs were altered after varicocelectomy. miRNAs showed significant changes. * P < 0.05. ( c ) Venn diagram of the numbers of miRNAs expressed before and after surgery. sncRNA: small noncoding RNA; tsRNA: tRNA-derived small RNA; rsRNA: rRNA-derived small RNA; piRNA: piwi-interacting RNAs; miRNA: microRNAs; snoRNA: small nucleolar RNA; others: those cannot be assigned to any known small-RNA class. The proportions of mapped small noncoding RNAs in 63 patients Before: before-surgery group; After: after-surgery group; tsRNA: tRNA-derived small RNA; rsRNA: rRNA-derived small RNA; piRNA: piwi-interacting RNAs; miRNA: microRNAs; snoRNA: small nucleolar RNA; Others: those cannot be assigned to any known small-RNA class The average proportion of small noncoding RNAs before and after varicocelectomy sncRNAs: small noncoding RNAs; NS: not significant, tsRNA: tRNA-derived small RNA, piRNA: piwi-interacting RNAs, miRNA: microRNAs, snoRNA: small nucleolar RNA, rsRNA: rRNA-derived small RNA The mean proportion of most sncRNAs was unchanged before and after surgery. But miRNA significantly dropped from 4.1% to 3.2% ( P = 0.03470; Figure 2b ), with a decrease of 23.4%. The mean of piRNA showed a similar yet non-significant decline, from 5.5% to 4.3% (a decrease of 21.6%, P = 0.0660), possibly owing to wide inter-patient variability. Annotated analysis showed that 189 of the 232 identified miRNAs were present in samples before and after surgery; 43 miRNAs were uniquely expressed in samples before surgery and no new miRNAs emerged after varicocelectomy ( Figure 2c ). To analyze the mechanism by which varicocelectomy improves sperm quality, we first examined the overall miRNA expression patterns. We further divided both the before- and after-surgery groups into two subgroups by improvements in sperm quality: the not-improved group (NIM; fold change <1.5, n = 20) and the improved group (IM; fold change ≥1.5, n = 43). Patients were then stratified into four groups: Before-NIM ( n = 20), After-NIM ( n = 20), Before-IM ( n = 43), and After-IM ( n = 43). The expression levels of the top 50 miRNAs are shown in Figure 3a . miRNAs were expressed at the lowest level in the After-IM group, indicating that miRNA expression levels decreased after surgery and these miRNAs may be associated with sperm quality. The other three types of sncRNAs did not show similar differences ( Supplementary Figure 2 for tsRNA, Supplementary Figure 3 for rsRNA, and Supplementary Figure 4 for piRNA). The differentially expressed miRNAs in patients with improved sperm quality. ( a ) Heatmap of the top 50 expressed miRNAs in 63 patients. The 63 patients from before- and after-surgery groups were divided by improvement in sperm quality: the not-improved (NIM; fold change <1.5; n = 20) and the improved groups (IM; fold change ≥1.5; n = 43). ( b ) The differentially expressed miRNAs in 43 patients whose sperm quality was improved after varicocelectomy. The threshold was absolute fold change ≥2. * P < 0.05; ** P < 0.01; **** P < 0.0001. Up: the miRNA expression was significantly upregulated; down: the miRNA expression was significantly downregulated. ( c ) GO term enrichment analysis of the differentially expressed miRNA target genes. ( d ) KEGG pathway enrichment analysis of target genes of the differentially expressed miRNA. The top 10 pathways are shown, according to the ratio of involved target genes. Count: number of genes enriched in this pathway; RPM: reads per million; GO: Gene Ontology; BP: biological process; CC: cellular component; MF: molecular function; KEGG: Kyoto Encyclopedia of Genes and Genomes; MAP: mitogen-activated protein; I-SMAD: inhibitory SMA- and MAD-related protein; ATP: adenosine triphosphate; DNA: deoxyribonucleic acid; RNA: ribonucleic acid; MAPK: mitogen-activated protein kinase; SMAD2 : SMA- and MAD-related protein 2; BCL2L11 : BCL-2-like 11; CDK6 : cyclin-dependent kinase 6; ITGAV : integrin subunit alpha V; E2F3 : E2F transcription factor 3; ELOC : elongin C; GNB5 : guanine nucleotide-binding protein subunit beta-5; PIK3R1 : phosphoinositide-3-kinase regulatory subunit 1; ESR1 : estrogen receptor 1; PHLPP2 : PH domain and leucine-rich repeat protein phosphatase 2; SGK3 : serum/glucocorticoid-regulated kinase 3; ELK4 : ETS-like transcription factor 4; MAP3K3 : mitogen-activated protein kinase kinase kinase 3; NFYB : nuclear transcription factor Y subunit beta; WEE1 : WEE1 G2 checkpoint kinase. Next, we analyzed the differential expression of miRNAs between the Before-IM and After-IM groups, using an absolute fold change ≥2 and P < 0.05 as the screening criteria. Analysis of the differentially expressed miRNAs showed that 10 miRNAs were significantly changed in the After-IM group ( Figure 3b , Supplementary Figure 5 , and Supplementary Table 5 ). Three miRNAs were upregulated (hsa-miR-486-3p, hsa-miR-486-5p, and hsa-miR-874-3p), while 7 were downregulated (hsa-miR-132-3p, hsa-miR-202-3p, hsa-miR-34c-5p, hsa-miR-499a-5p, hsa-miR-499b-3p, hsa-miR-520a-3p, and hsa-miR-92b-3p). The differentially expressed microRNAs logFC: the log of fold change of miRNA expression; logCPM: the log of miRNA counts per million; Sig: the significant change of miRNA expression Analysis of the 10 differentially expressed miRNAs using miRWalk online databases predicted 97 target genes ( Supplementary Table 6 ). GO term enrichment analysis of the predicted target genes was performed across the three main GO categories: BP, CC, and MF ( Figure 3c ). In the BP category, regulation of transcription by RNA polymerase II was the most significantly and frequently identified term, followed by positive regulation of transcription by RNA polymerase II, apoptotic process, and other cellular basic processes. In the CC category, most of the transcripts were associated with the cytosol, followed by nucleus, cytoplasm, and nucleoplasm. In the MF category, DNA-binding transcription factor activity was the most significant enrichment term identified, followed by protein binding, cis -regulatory region sequence-specific DNA binding, and sequence-specific DNA binding. The predicted target genes of 10 differentially expressed microRNAs To further analyze the potential pathways affected by these target genes, KEGG pathway enrichment analysis was performed. The top 10 pathways based on the ratio of involved target genes are shown in Figure 3d . The results indicate that the genes are enriched in several important pathways related to the phosphatidylinositol 3-kinase-protein kinase B signaling pathway, cell cycle, and cellular senescence. Additionally, genes such as phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and E2F transcription factor 3 (E2F3) play roles in multiple pathways. To validate the sequencing results, the expression of the 10 miRNAs were evaluated by qRT-PCR in sequenced samples. After reviewing RNA content for each sample, 15 pairs of samples were selected from 43 pairs of sequenced samples. The expression levels of five miRNAs were consistent with the deep sequencing results; hsa-miR-132-3p, hsa-miR-34c-5p, and hsa-miR-520a-3p were decreased and hsa-miR-486-3p and hsa-miR-874-3p were increased after surgery treatment ( Figure 4a ). U6 was used as an internal control. Aberrantly expressed miRNAs affected early embryonic development. ( a ) Quantitative real-time polymerase chain reaction (qRT-PCR) validation of differentially expressed miRNAs in 15 pairs of sequenced samples. U6 was used as an internal control. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. ( b ) Outcomes of early embryonic development after microinjection of synthesized miRNAs in zygotes. Control: no injection; random: injection of a random synthesized control; miR-132-3p: injection of synthesized miR-132-3p; miR-34-5p: injection of synthesized miR-34c-5p; miR-520a-3p: injection of synthesized miR-520a-3p. ( c ) The development of the early embryo from zygote to blastocyst after microinjection. Day 1 was the zygote stage, day 2 was the 2-cell stage, day 3 was the 4- to 8-cell stage, day 4 was the morulae stage, and day 5 was the blastocyst stage. Scale bar=100 μm. NS: not significant. The three downregulated miRNAs were selected for further analysis, including miR-132-3p, miR-34c-5p, and miR-520a-3p. We then evaluated the effects of these miRNAs on embryonic development. We synthesized mimics of the three miRNAs ( Supplementary Table 1 ) and microinjected them (100 nmol l −1 ) into normal zygotes. A random sequence was used as a negative control (Random), and we also included a control of zygotes without microinjection (Control). After microinjection, the zygotes were cultured in vitro under standard conditions. To ensure that the microinjected synthetic miRNAs were functional, we first detected their stability and conservation after incubation with lysate of a single zygote. The synthetic miRNAs showed a trend of degradation over time in the lysate, as detected by qRT-PCR ( Supplementary Figure 6 ). After incubation for only 10 min, approximately 82% of the miRNAs was still detected. We then monitored early embryonic development of the injected zygotes. We found that the miRNA-injected zygotes showed changes in embryonic development compared with the Control and Random groups ( Supplementary Table 7 ). miR-34c-5p showed the most significant effects ( P < 0.0001), impacting embryonic development as early as the 2-cell stage. Only 34.4% of these zygotes reached the 2-cell stage (compared with 100.0% in the Control and Random groups), and only 9.3% developed to the blastocyst stage (compared with 94.7% in the Control group and 92.2% in the Random group; Figure 4b ). For the other two miRNAs (miR-132-3p and miR-520a-3p), the most pronounced effects were observed at the blastocyst stage, with development rates of 89.8% for miR-132-3p and 73.1% for miR-520a-3p. Notably, the blastocyst development rate of miR-520a-3p-injected zygotes was significantly decreased compared with that of the Control group ( P < 0.01). Moreover, abnormally increased expression of miRNA affected the development of the early embryo and severely reduced the embryo quality in the miR-34c-5p group ( Figure 4b ). The early embryo quality was also decreased in the miR-132-3p and miR-520a-3p groups, whereas the Random and Control groups were unaffected. These results indicated that aberrant expression of these miRNAs is detrimental to early embryonic development. Outcomes of early embryonic development after the microinjection of synthesized microRNAs into zygotes ** P <0.01; **** P <0.0001

This study indicates that sperm quality is significantly improved following varicocelectomy. Furthermore, the expression profiles of sncRNAs in sperm are altered, with 10 miRNAs (3 upregulated and 7 downregulated) showing significant changes post-surgery. These changes correlate with the improvements in sperm quality. Microinjection experiments demonstrated that abnormally elevated expression of specific miRNAs (miR-132-3p, miR-34c-5p, and miR-520a-3p) negatively affected zygote development, suggesting that these miRNAs may play significant roles in the early phases of embryonic development. These findings provide evidence that the aforementioned miRNAs may serve as noninvasive markers for sperm quality and as potential biomarkers to identify different types of defective spermatozoa. This, in turn, may enable proper treatment through varicocelectomy and lead to better fertility outcomes.

Varicoceles have been shown to be associated with reduced male fertility potential. 27 28 Surgical treatment is the most common and effective clinical option for affected patients. 29 30 Multiple studies have reported improvement in semen parameters following varicocelectomy. 31 32 33 While most previous studies compared healthy individuals in the varicocele group, in this study, we compared the changes in sperm quality in patients with varicoceles before and after varicocelectomy. We found that varicoceles affect semen parameters, and these semen parameters improved after at least 1 month following varicocelectomy. We further found that the expression of sncRNAs changed, especially miRNAs, in the sperm of patients who had undergone varicocelectomy. Additionally, the abnormal expression of certain miRNAs (hsa-miR-132-3p, hsa-miR-34c-5p, and hsa-miR-520a-3p) identified as differentially expressed after varicocelectomy were shown to affect zygotic development. Overall, this study reveals that the expression of miRNAs changes with sperm quality and these miRNAs may be relevant to early embryo development. Mammalian sperm serve as carriers of both the paternal genome and epigenome, which includes DNA methylation, retained histones, and sncRNAs. Abundant sncRNA has been detected in spermatozoa, including tsRNAs, rsRNAs, piRNAs, and miRNAs. 12 34 miRNAs are the most extensively studied, followed by piRNAs and tsRNAs. Numerous miRNAs have been shown to correlate with infertility. The fertilization of sperm is heavily dependent on post-transcriptional regulatory processes, and miRNAs have emerged as critical regulators of these processes. Thus, changes in miRNA expression may contribute to male infertility. 35 36 The purpose of this study was to investigate whether the expression of sncRNA in sperm is affected in patients with varicoceles and whether sncRNA composition changes after surgery. By examining the profiles of the four major types of sperm sncRNAs, we found that miRNAs exhibited the greatest changes in both expression proportion and expression levels ( Figure 2b and 2c , Figure 3a , and Supplementary Figure 2 – 4 ). Differential expression analysis revealed that 10 miRNAs (3 upregulated and 7 downregulated) were differentially expressed in the After-IM group compared with the Before-IM group. These miRNAs were associated with the improvement of sperm quality indices. Some have been previously reported to be involved in spermatogenesis and male infertility, such as miR-34c, miR-449, and miR-202. 37 38 39 miR-34 family members play a crucial role in spermatogenesis. miR-34c has been identified as highly expressed in spermatocytes and spermatids. Numerous studies have demonstrated that the expression levels of miR-34 are significantly lower in sperm of infertile men than those fertile men, particularly in conditions such as oligoasthenoteratozoospermia and non-obstructive azoospermia. 40 41 42 Another study revealed that the expression of several miRNAs, including miR-34c-5p, is markedly lower in azoospermia but higher in asthenozoospermia. 43 Here, we observed that miR-34c-5p levels were lower after surgical treatment with the improvement of sperm quality indices. Researches have shown that miR-34c promotes apoptosis in murine male germ cell by targeting activating transcription factor 1 ( ATF1 ). 44 45 One hypothesis is that a high level of sperm apoptosis may be associated with varicoceles, leading to a relatively higher expression level of miR-34c in sperm. After varicocelectomy, apoptosis decreases and sperm quality improves, accompanied by reduced miR-34c levels. This also suggests that miR-34c-5p may serve as a potential noninvasive biomarker for diagnosing different pathological types of infertility. The miR-34/449 family is crucial for the function of spermatozoa. Abnormal expression of miR-34/449 family members has been implicated in the development of infertility by targeting the E2F transcription factor-phosphorylated retinoblastoma protein (E2F-pRb) pathway, and these miRNAs exhibit similar expression patterns; they are all specifically expressed in the mouse testis and show a pronounced increase upon meiotic initiation during spermatogenesis. 46 We also observed significant reduction in the expression levels of miR-34c-5p, miR-449a-5p, and miR-449b-3p in sperm of patients, with improvement in sperm quality after surgery. miR-202 is also an important miRNA for spermatogenesis. The expression levels of several miRNAs (miR-202-5p, miR-34c-5p, miR-10b, miR-191, and miR-126) were found to be significantly lower in patients with Sertoli cell-only syndrome compared with healthy individuals. 47 In our study, miR-202-3p was downregulated in the After-IM group, which exhibited improved sperm quality. This aligns with the findings of Yang et al. , 48 which demonstrated that the expression of miR-202-3p is elevated in Sertoli cells derived from Sertoli cell-only syndrome patients, contrasting with those from patients with obstructive azoospermia who have normal spermatogenesis. miRNA-202-3p modulates spermatogenesis through its effects on Sertoli cells by targeting low-density lipoprotein receptor-related protein 6 ( LRP6 ) and cyclin D1 in the Wingless-type MMTV integration site family (Wnt)/β-catenin signaling pathway. Notably, the expression profiles of many miRNAs in our study were not consistent with previous studies. This discrepancy may be due to the different types of samples used in our study. We used a self-control approach for each patient, comparing the before- and after-surgery status, with the before-surgery sample as control. Other studies typically compared fertile and infertile individuals. Our design may maximally remove the differences caused by the individual’s factors and give greater consideration to the effects of varicoceles and surgical treatment on sncRNA expression profiles. This suggests that changes in sncRNAs may reflect the impact of varicocelectomy on men with varicoceles, as this is also a type of paternal stress, rather than changes observed in men with normal fertility. The pathways related to the miRNA target genes identified in this study are frequently related to infertility. In our study, PI3K-Akt was the signaling pathway with a large number of enriched genes. One study showed that regulation of the PI3K pathway in semen is responsible for sperm motility. 49 50 This indicates that sperm motility may be improved through the PI3K-Akt pathway. The proportion of tumor- and infection-related pathways in our results is relatively high. This may be related to the high correlation between varicoceles and inflammation. 51 52 53 PIK3R1 and E2F3 genes were present in most pathways. E2F transcription factors bind to retinoblastoma proteins and are responsible for testicular development and spermatogenesis. 54 Yue et al . 55 found that the circular RNA Circ_0001495 influences the development of endometriosis through the miR-34c-5p/E2F3 axis, which suggests that this gene may also be involved in spermatogenesis in a similar way. Previous studies on paternal epigenetic inheritance have highlighted an intricate connection between paternal health and offspring development. Argaw-Denboba et al . 56 revealed that disruptions in the paternal microbiome can negatively influence offspring fitness by impairing the function of the gut–germline axis. Moreover, numerous studies have shown that paternal stress, such as exercise, diet and infections, can have a significant impact on the health of offspring through mechanisms involving sperm sncRNAs and epigenetic inheritance. For instance, Tomar et al . 57 demonstrated that mitochondrial tRNAs in epididymal spermatozoa serve as sensors for acute high-fat diet exposure and influence offspring metabolism by epigenetic inheritance. The research group led by Hannan has also made notable contributions to this field. They discovered that alterations in the paternal gut microbiome, immune activation, bacterial infection, glucocorticoid exposure, and even exercise can alter the content of sperm sncRNAs, ultimately affecting offspring behavior and physiological functions in mice. 58 59 60 61 62 These findings reveal the diverse mechanisms through which paternal health can influence the health of offspring via sperm sncRNAs. Other studies have reported an impact of sperm sncRNA on offspring. Huang et al . 63 found that male mice subjected to psychological stress of chronic unpredictable mild stimulation exhibited significant changes in sperm sncRNAs and reproductive function, and the offspring not only displayed depressive-like behavior but also showed a marked decrease in survival rate. These findings suggest that paternal stress can have profound consequences for the development of offspring. Chen et al . 26 and Sharma et al . 64 identified sperm tsRNAs as key epigenetic factors that may influence preimplantation development and contribute to the intergenerational inheritance of diet-induced metabolic disorders. Chen et al . 26 demonstrated that microinjecting sperm tsRNA fractions from high-fat diet male mice into normal zygotes could result in offspring with metabolic abnormalities. These studies collectively emphasize the important role of sperm sncRNAs in affecting the development and health of offspring through epigenetic mechanisms. An increasing number of studies have revealed that paternal miRNAs play crucial roles in early embryonic development. 65 The most studied sperm miRNA in humans is miR-34c, which has been shown to have a complex role in preimplantation embryonic development. 66 Liu et al . 67 demonstrated that sperm-borne miR-34c is essential for the first cleavage division of mouse zygotes. However, another study indicated that miR-34c is not required for mouse fertilization or embryo development, suggesting conflicting roles in different contexts. 68 Our previous research demonstrated that miRNAs in sperms are associated with embryo quality following in vitro fertilization. 12 Furthermore, studies in mice have indicated that sperm miRNA expression is altered by traumatic stress in early life, and behavioral and metabolic changes were observed across generations through microinjecting purified sperm RNAs into wild-type fertilized mouse oocytes. 69 Similarly, Rodgers et al . 70 found that miRNAs levels were elevated in the sperm of stressed mice. Upon microinjecting these miRNAs into zygotes, the resulting offspring exhibited stress dysregulation phenotypes, including reactivity of the hypothalamic–pituitary–adrenal stress axis, similar to their fathers. 70 71 In the current study, we microinjected synthetic miRNAs into normal zygotes and assessed their effects on early embryonic development using early cleavage rates as a metric. We found a correlation between aberrant miRNA expression and early cleavage rates. The lowest percentage of good-quality embryos was observed in the miR-34c group, starting from the 2-cell stage. The miR-132-3p and miR-520a-3p groups also exhibited lower cleavage rates, particularly at the blastocyst stage ( Figure 4 ). These findings suggest that these aberrantly expressed miRNAs may negatively impact early embryonic development. Therefore, the expression of these miRNAs in sperm may serve as a promising predictor for the clinical diagnosis of male infertility. Additional studies are needed to confirm the results of this study. In future research, we plan to validate the miRNA regulation pathways in animal varicocele models. These findings will help elucidate the specific roles of sncRNAs in sperm quality. It will also strengthen the evidence base for clinical applications of varicocele surgery.

All authors declare no competing interests.

The length distribution of small RNAs in sperm. Before: before-surgery group; after: after-surgery group. tsRNA: tRNA-derived small RNA; rsRNA: rRNA-derived small RNA; piRNA: piwi-interacting RNAs; miRNA: microRNAs; snoRNA: small nucleolar RNA; others: those cannot be assigned to any known small-RNA class. Heatmap of top 50 tsRNAs with the highest expression in 63 patients. Both in Before and after groups (before- and after-surgery), 63 patients were subdivided by improvement in sperm quality: the not-improved (NIM; fold change <1.5, n = 20) and the improved groups (IM; fold change ≥1.5, n = 43). Four subgroups: before-NIM ( n = 20), after-NIM ( n = 20), before-IM ( n = 43), and after-IM ( n = 43). tsRNA, tRNA-derived small RNA. Heatmap of top 50 rsRNAs with the highest expression in 63 patients. rsRNA: rRNA-derived small RNA. Heatmap of top 50 piRNAs with the highest expression in 63 patients. piRNA: piwi-interacting RNAs. Volcano plots of the differentially expressed miRNAs in 43 patients whose sperm quality improved after varicocelectomy. The threshold was absolute fold change ≥2, P < 0.05. Up: the expression of miRNA was upregulated significantly; down: the expression of miRNA was downregulated significantly; none: the expression of miRNA was not changed significantly. The relative expression of miRNAs incubated in single zygote lysate. **** P < 0.0001.