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Altered micro-ribonucleic acid expression profiles of extracellular microvesicles in the seminal plasma of patients with oligoasthenozoospermia

      Objective

      To determine whether microRNA (miRNA) expression profile is different in extracellular microvesicles collected from seminal plasma of men with oligoasthenozoospermia, to gain further insight into molecular mechanisms underlying male infertility.

      Design

      Microarray with quantitative real-time polymerase chain reaction validation and Western blot analysis confirmation.

      Setting

      University research and clinical institutes.

      Patient(s)

      A total of 24 men, including 12 oligoasthenozoospermic subfertile men and 12 normozoospermic men.

      Intervention(s)

      None.

      Main Outcome Measure(s)

      Statistically significant altered miRNA expression profiles in oligoasthenozoospermic subfertile men compared with normozoospermic fertile men.

      Result(s)

      Extracellular microvesicles including exosomes were isolated from seminal plasma by ultracentrifugation. Presence of exosome-specific proteins was confirmed by Western blotting. In the extracellular microvesicles, we analyzed 1,205 miRNAs by microarray and identified 36 miRNAs with altered expression levels in oligoasthenozoospermic compared with normozoospermic fertile men. Seven miRNAs were overexpressed and 29 miRNAs were underexpressed in oligoasthenozoospermic men. Using quantitative real-time polymerase chain reaction as an independent method, we confirmed the significantly higher expression levels of miR-765 and miR-1275 and the significantly lower expression level of miR-15a in oligoasthenozoospermic subfertile men as compared with the normozoospermic men.

      Conclusion(s)

      We identified altered expression levels of miRNAs in extracellular microvesicles from seminal plasma as part of the molecular events in the male genital tract. These miRNAs may help to understand the molecular mechanisms underlying male infertility.

      Key Words

      Infertility and problems in conception are longstanding clinical problems that affect approximately 15% of couples worldwide, with male infertility contributing to approximately 50% of a couple's inability to conceive. Idiopathic male infertility occurs in approximately 60%–75% of all cases in patients without previous fertility problems and with normal findings by physical examination (
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      Many studies identified candidate genes indicating or even possibly causing spermatogenetic impairments. Among these genes, some are associated with genetic causes of male infertility and are necessary for normal spermatogenesis, like CFTR (cystic fibrosis transmembrane conductance regulator) (
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      Semen is a complex viscous fluid made up of a combination of spermatozoa and seminal plasma that contains high levels of subcellular lipid-bound microparticles. In seminal plasma these microparticles are thought to be mainly produced by the luminal prostatic epithelial cells under both physiologic and pathologic conditions. They are expelled with prostatic secretions at ejaculation and likely play a role in the exocrine regulation of cellular functions (
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      ).
      Studies have shown that microvesicles including exosomes contain RNAs, including microRNAs. MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression on a posttranscriptional level by inhibiting translation of their respective target genes (
      • Bartel D.P.
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      ). Therefore, they are important regulators during the different stages of normal spermatogenesis (
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      Dramatic changes in 67 miRNAs during initiation of first wave of spermatogenesis in Mus musculus testis: global regulatory insights generated by miRNA-mRNA network analysis.
      ) and are also involved in many pathologic aspects of spermatogenesis (
      • Stegmayr B.
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      Promotive effect on human sperm progressive motility by prostasomes.
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      • Poliakov A.
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      • Mobley J.A.
      Structural heterogeneity and protein composition of exosome-like vesicles (prostasomes) in human semen.
      ). In testicular tissue, Lian et al. (
      • Lian J.
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      ) identified 173 differentially expressed miRNAs in testis of men with different forms of nonobstructive azoospermia (NOA) compared with controls, including 19 up- and 154 down-regulated miRNAs. Muñoz et al. (
      • Munoz X.
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      Altered miRNA signature of developing germ-cells in infertile patients relates to the severity of spermatogenic failure and persists in spermatozoa.
      ) reported a decreased cellular miRNA content depending on the efficacy of the spermatogenic process. They showed a widely altered miRNA expression profile in developing germ cells of men with spermatogenic failure at different stages of germ cell development. Furthermore, they provide evidence that spermatozoa of men with mild spermatogenic failure retain the deregulated miRNA patterns found in the developing germ cells (
      • Munoz X.
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      Altered miRNA signature of developing germ-cells in infertile patients relates to the severity of spermatogenic failure and persists in spermatozoa.
      ). Additionally, three studies report alterations of miRNA expression patterns in seminal plasma of men with different forms of NOA using either miRNA microarrays (
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      Genome–wide microRNA expression profiling in idiopathic non-obstructive azoospermia: significant up-regulation of miR-141, miR-429 and miR-7-1-3p.
      ) or Solexa sequencing analysis along with quantitative real-time polymerase chain reaction (qRT-PCR) validation (
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      Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility.
      ). Liu et al. (
      • Liu T.
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      • Liu Z.
      Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities.
      ) identified a group of differentially expressed miRNAs in semen of men with subfertility compared with fertile controls. More recently, other miRNAs, such as the miR-34 family (miR-34b, miR-34b*, and miR-34c-5p), miR-15b, miR-122, and miR-429 were found to be differentially expressed in purified spermatozoa of patients with spermatogenic impairments and in testicular tissue of men with different forms of NOA compared with controls (
      • Munoz X.
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      Altered miRNA signature of developing germ-cells in infertile patients relates to the severity of spermatogenic failure and persists in spermatozoa.
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      ). Taken together, the aforementioned studies suggest that deregulation in miRNA expression patterns at the testicular level may contribute to several types of reproduction abnormalities.
      The aim of this study was to determine whether miRNA expression profile is different in extracellular microvesicles collected from seminal plasma of subfertile men with oligoasthenozoospermia compared with normozoospermic men, to gain further insight into molecular mechanisms underlying male infertility.

      Materials and methods

       Study Population and Sample Collection

      The study was approved by the institutional review board (no. 195/11) of the University Hospital of Saarland. Informed consent was obtained from each of the participants. A total of 24 men, aged 24–39 years, were included in this study, including 12 oligoasthenozoospermic subfertile men and 12 normozoospermic men (i.e., 6 men with normal semen parameters from couples undergoing infertility treatment and 6 donor men with proven fertility). The inclusion criteria for men were as follows: all men were attending the male infertility clinic for infertility treatment, and all men were evaluated for semen parameters (abnormal spermiogram in case of subfertility and normal in case of fertility). The exclusion criteria were as follows: smoking, drug use, exposure to environmental or occupational toxicants, sexually transmitted diseases, cryptorchidism, genitourinary anomalies, and surgery related to infertility treatment. In addition, we excluded subjects with azoospermia and/or incomplete semen analysis parameters. Samples were obtained from each participant by masturbation after 3 days of sexual abstinence, allowed to liquefy at 37°C for 30 minutes, and then processed immediately according to the 2010 guidelines of the World Health Organization (
      World Health Organization
      WHO laboratory manual for the examination and processing of human semen.
      ). The semen samples were then loaded on to 45%–90% discontinuous Puresperm gradients (Nidacon International) and centrifuged at 500 × g at room temperature for 20 minutes. The upper layer seminal plasma (supernatant) was aspirated and transferred to a new tube for purification of extracellular microvesicles, including exosomes.

       Purification of Extracellular Microvesicles from Seminal Plasma

      Extracellular microvesicles including exosomes were isolated from seminal plasma as described elsewhere (
      • Jansen F.H.
      • Krijgsveld J.
      • van Rijswijk A.
      • van den Bemd G.J.
      • van den Berg M.S.
      • van Weerden W.M.
      • et al.
      Exosomal secretion of cytoplasmic prostate cancer xenograft-derived proteins.
      ) with slight modifications. Briefly, seminal plasma samples were subjected to sequential centrifugation steps at 300 × g for 10 minutes, 2,000 × g for 20 minutes, and 10,000 × g for 30 minutes with each step at 4°C. The supernatant containing the extracellular microvesicles was spun at 68,000 × g at 4°C for 90 minutes using an Optima MAX-E ultracentrifuge (Beckman Coulter). Extracellular vesicle pellets were resuspended in 0.32 M sucrose (Sigma Aldrich) and centrifuged again at 100,000 × g at 4°C for 90 minutes. The extracellular vesicle pellet was then resuspended in FBS and used for total RNA, including miRNA isolation.

       Total RNA, Including miRNAs Isolation

      The miRNeasy Mini kit was used with the QIAcube robot to isolate the total RNA including miRNAs according to the manufacturer's instructions (Qiagen). Total RNA was eluted with 30 μL of RNase-free water and quantified using a Nanodrop ND-2000 spectrophotometer (Thermo Scientific). The integrity was assessed with the Agilent 2100 Bioanalyzer using the RNA Nano 6000 Kit (Agilent Technologies). In agreement with other studies (
      • Mathivanan S.
      • Fahner C.J.
      • Reid G.E.
      • Simpson R.J.
      ExoCarta 2012: database of exosomal proteins, RNA and lipids.
      ,
      • Skog J.
      • Wurdinger T.
      • van Rijn S.
      • Meijer D.H.
      • Gainche L.
      • Sena-Esteves M.
      • et al.
      Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers.
      ,
      • Valadi H.
      • Ekstrom K.
      • Bossios A.
      • Sjostrand M.
      • Lee J.J.
      • Lotvall J.O.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      ), the majority of the RNA content was smaller than 200 nucleotides, indicating that extracellular microvesicles primarily contain short RNA including miRNA and degraded mRNA. The RNA integrity number for samples was <2.8 (Supplemental Fig. 1, available online). To remove DNA contamination, DNase I (Ambion) treatment was carried out according to the manufacturer's instructions. Conventional PCR with exon spanning primers for GAPDH (forward: 5′-CGACCACTTTGTCAAGCTCA-3′; reverse: 5′-AGGGGTCTACATGGCAACTG-3′) was performed to indicate residual DNA in the samples (
      • Salas-Huetos A.
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      New insights into the expression profile and function of micro-ribonucleic acid in human spermatozoa.
      ).

       Western Blot for Extracellular Vesicles

      For Western blot analysis, the precipitated vesicles were resuspended in 1× radioimmunoprecipitation assay buffer at room temperature for 5 minutes (Cell Biolabs), followed by the addition of Laemmli buffer mixed with β-mercaptoethanol (Sigma Aldrich). The solution was heated at 95°C for 5 minutes. Proteins were separated via 10% standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane. The polyvinylidene difluoride membrane was blocked with 5% dry milk in Tris-buffered saline plus 0.05% Tween (TBS-T) for 1 hour and incubated at 4°C overnight with antibodies against exosomal marker proteins, namely CD63, CD81, CD9, and HSP70, each at 1:500 dilution in 5% dry milk in TBS-T (System Biosciences). These exosomal marker proteins are commonly used as confirmation of exosome collection and purity (
      • Vojtech L.
      • Woo S.
      • Hughes S.
      • Levy C.
      • Ballweber L.
      • Sauteraud R.P.
      • et al.
      Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions.
      ,
      • Caby M.P.
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      Exosomal-like vesicles are present in human blood plasma.
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      • Zhao K.
      • et al.
      ExoCarta: a web-based compendium of exosomal cargo.
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      • Thery C.
      • Boussac M.
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      • Garin J.
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      Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.
      ,
      • Wubbolts R.
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      ,
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      ,
      • Zeringer E.
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      Methods for the extraction and RNA profiling of exosomes.
      ). After three times washing with TBS-T (30 minutes each time), membranes were incubated at 4°C for 1 hour with goat anti-rabbit HRP antibody at a 1:10,000 dilution in 5% dry milk in TBS-T (System Biosciences). After incubation, the membranes were again washed three times with TBS-T (30 minutes each time), and proteins were detected using enhanced chemiluminescence substrate (Cell Signaling Technology).

       MiRNA Microarray Analysis

      In the screening phase, miRNA expression profiles of oligoasthenozoospermic subfertile men (n = 6) and normozoospermic fertile men (n = 6) were determined using Sureprint G3 Human v16 miRNA, 8×60K microarray platform (Agilent Technologies). All procedures were carried out according to the manufacturer's recommendations. Briefly, 100 ng input of total RNA from each sample was dephosphorylated by incubation with calf intestinal phosphatase at 37°C for 30 minutes and denatured using 100% dimethyl sulfoxide at 100°C for 5 minutes. Samples were labeled with pCp-Cy3 using T4 ligase at 16°C incubation for 2 hours. Each labeled RNA sample was hybridized onto an individual 8×60K format Agilent miRNA array slide, with each array containing probes for 1,205 human miRNAs. Hybridizations were performed in SureHyb chambers (Agilent Technologies) at 55°C for 20 hours with rotation. Arrays were washed and dried according to the manufacturer's recommendations and scanned at a resolution of 3 μm in double-pass mode using an Agilent G2565BA scanner. Data were generated by Agilent AGW Feature Extraction software version 10.10.11 (Agilent Technologies).

       Reverse Transcription and qRT-PCR of miRNA

      The expression level of miRNAs in extracellular microvesicles was determined for all 24 samples using the miScript SYBR Green Kit (Qiagen) by qRT-PCR. Ribonucleic acid (200 ng) was reverse transcribed using the miScript Reverse Transcription kit (Qiagen) according to the manufacturer's recommendations. The complementary DNA was diluted 1:10, and 2 μL of complementary DNA was mixed with 10 μL 2× QuantiTect SYBR Green PCR Master Mix, 2 μL 10× miScript Universal Primer, 2 μL 10× miScript Primer Assay for eight selected miRNAs (miR-30b, miR-20a, miR-148a, miR-765, miR-1299, miR-26b, miR-1275, and miR-15a), and RNU6B as an endogenous control (Qiagen) in a total volume of 20 μL. All qRT-PCR reactions were set up using a QIAgility automated PCR setup robot (Qiagen) before performing qRT-PCR using a StepOnePlus system (Life Technologies). Melting curve analysis was used to control for the specificity of qRT-PCR products. Data were analyzed with SDS Relative Quantification Software version 2.3 (Life Technologies). The miRNA reverse transcription control was used to assess the performance of a reverse transcription reaction by detecting template synthesized from the kit's built-in control RNA (Qiagen).

       MiRNA Target Prediction and Pathway Analysis

      Enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses was performed using DIANA-miRPath version 3.0 software based on predicted targets by DIANA-microT-CDS (
      • Vlachos I.S.
      • Zagganas K.
      • Paraskevopoulou M.D.
      • Georgakilas G.
      • Karagkouni D.
      • Vergoulis T.
      • et al.
      DIANA-miRPath v3.0: deciphering microRNA function with experimental support.
      ). Targets of miRNAs with a score of more than 0.8 were selected. Only KEGG pathways with a P value <.05 and a false discovery rate (FDR) <0.05 were retained.

       Statistical Analysis

      We used the freely available R statistical environment version 2.14.2 (
      ) to analyze the differences of miRNA expression in extracellular microvesicles of oligoasthenozoospermic subfertile men compared with normozoospermic fertile men. Raw data generated by Agilent Feature Extraction image analysis software were quantile normalized. The raw data indicate that the majority of miRNAs are normally distributed. The Shapiro-Wilk test was carried out for each miRNA separately. We analyzed significance levels of miRNAs by applying an unpaired two-tailed t test and computed area under the curve values for each miRNA. The parametric t test is a valid hypothesis test for our scenario because the t test is not sensitive to deviations from the normality, especially when the deviations are moderate (
      • Altman D.G.
      • Bland J.M.
      Statistics notes: the normal distribution.
      ). For two-tailed t tests with a desired α error likelihood of 0.01 and a statistical power of 0.95, we determined a required cohort size of 12 samples. The relative quantitative method of 2−ΔΔCq was used to measure the dynamic change of specific selected miRNAs (
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
      ). Real-time PCR and clinical data were tested for significance using the Student t test and nonparametric Mann-Whitney U test, respectively. A P value of <.05 was considered significant, and data are presented as the mean ± SD.

      Results

       Characteristics of Study Subjects

      We analyzed a total of 24 seminal plasma samples from oligoasthenozoospermic subfertile men and normozoospermic men. The selected demographics and the characteristics of the men are summarized in Table 1. As determined by nonparametric analysis, there were no overall significant differences of the mean age, the semen volume, or the semen pH between normozoospermic men and oligoasthenozoospermic subfertile men. However, there were significant differences between normozoospermic men and oligoasthenozoospermic men concerning the count, motility, and morphology of the spermatozoa.
      Table 1Patient and semen characteristics.
      CharacteristicNormozoospermic men (n = 12)Oligoasthenozoospermic men (n = 12)P value
      Age (y)28.17 ± 4.0428.17 ± 3.97.9070
      Volume (mL)2.50 ± 0.762.05 ± 0.46.0701
      pH8.08 ± 0.468.34 ± 0.31.2411
      Count (106/mL)65.37 ± 17.935.94 ± 2.50<.0001
      Motility (% motile)59.92 ± 11.8421.83 ± 5.83<.0001
      Morphology (%)42.58 ± 22.0223.75 ± 14.53.0109
      Note: Data presented as mean ± SD; P<.05 was considered to be significant; P<.0001 was considered to be highly significant (nonparametric Mann-Whitney U test).

       Detection of Seminal Plasma Extracellular Microvesicles Including Exosomes

      We isolated extracellular microvesicles from seminal plasma collected from the oligoasthenozoospermic and the normozoospermic men. To confirm the presence of exosomes, proteins were isolated, separated, and probed with antibodies against exosomal marker proteins. For this analysis, we included samples from six oligoasthenozoospermic men and eight normozoospermic men, with each of the samples analyzed by Western blotting. In all cases we detected CD63, CD81, CD9, and HSP70, indicating the presence of exosomes in the ultracentrifuged pellet (Supplemental Fig. 2).

       Differentially Expressed miRNAs in Seminal Plasma Extracellular Microvesicles

      To evaluate the expression level of miRNAs in extracellular vesicles, we determined the miRNA expression profiles of six normozoospermic fertile men and six oligoasthenozoospermic subfertile men by microarrays. Out of 1,205 mature human miRNAs that were represented on each microarray, 388 miRNAs (32.20%) were identified in at least one seminal plasma sample. By applying an unpaired two-tailed t test, we found 36 miRNAs that showed a significant difference of the expression level in oligoasthenozoospermic subfertile men as compared with normozoospermic fertile men. In detail, we found seven miRNAs with a higher and 29 miRNAs with a lower expression level in oligoasthenozoospermic compared with normozoospermic fertile men. MiR-1275 showed the highest fold change (3.23) among the overexpressed miRNAs, and miR-26b showed the highest fold change (14.08) among the underexpressed miRNA (Table 2). The distinct expression signatures of oligoasthenozoospermic subfertile men and normozoospermic fertile men are visualized in the heatmap in Supplemental Figure 3.
      Table 2Significantly altered miRNAs in oligoasthenozoospermic subfertile men as compared with normozoospermic fertile men as determined by microarray.
      ExpressionmiRNAsFold changeP valueArea under the curve
      Overexpressed miRNAsmiR-12753.23.0260.889
      miR-42983.13.0270.861
      miR-3675-3p2.70.0260.931
      miR-7652.33.0180.875
      miR-483-5p2.17.0360.847
      miR-12991.72.0280.944
      miR-7661.54.0230.833
      Underexpressed miRNAsmiR-43061.71.0210.111
      miR-28-5p2.42.0440.167
      miR-42862.74.0350.167
      miR-962.83.0290.111
      miR-1852.92.0430.139
      miR-4252.96.0360.167
      miR-1003.09.0430.139
      miR-30e3.14.0430.167
      miR-331-3p3.82.0450.139
      miR-374a4.16.0390.222
      miR-15b4.19.0320.139
      miR-193b4.64.0420.139
      miR-30c4.76.0380.139
      miR-254.81.0300.139
      miR-27a5.23.0370.194
      miR-23a5.24.0480.167
      miR-27b5.34.0320.194
      miR-15a5.58.0260.139
      miR-935.76.0400.167
      miR-374b6.07.0300.167
      miR-200b6.87.0430.167
      miR-23b8.14.0380.222
      miR-20a8.33.0340.167
      miR-218.44.0470.167
      miR-148a8.99.0270.167
      miR-179.22.0490.139
      miR-30b10.88.0310.167
      miR-36311.44.0440.125
      miR-26b14.08.0280.222
      Note: P<.05 was considered to be significant (Student t test).

       Validation of Differentially Expressed miRNAs using qRT-PCR

      For validation purposes we re-examined the expression level of eight miRNAs, namely miR-30b, miR-20a, miR-148a, miR-765, miR-1299, miR-26b, miR-1275, and miR-15a, using the samples of 12 oligoasthenozoospermic subfertile men and 12 normozoospermic men, including the samples that were used for the microarray analysis. The miRNAs were selected according to their expression level in oligoasthenozoospermic subfertile men as compared with normozoospermic fertile men and according to their known associations with spermatogenesis (
      • Lian J.
      • Zhang X.
      • Tian H.
      • Liang N.
      • Wang Y.
      • Liang C.
      • et al.
      Altered microRNA expression in patients with non-obstructive azoospermia.
      ,
      • Liu T.
      • Cheng W.
      • Gao Y.
      • Wang H.
      • Liu Z.
      Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities.
      ,
      • Abu-Halima M.
      • Backes C.
      • Leidinger P.
      • Keller A.
      • Lubbad A.M.
      • Hammadeh M.
      • et al.
      MicroRNA expression profiles in human testicular tissues of infertile men with different histopathologic patterns.
      ,
      • Abu-Halima M.
      • Hammadeh M.
      • Schmitt J.
      • Leidinger P.
      • Keller A.
      • Meese E.
      • et al.
      Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments.
      ,
      • Curry E.
      • Safranski T.J.
      • Pratt S.L.
      Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility.
      ,
      • Ji Z.
      • Lu R.
      • Mou L.
      • Duan Y.G.
      • Zhang Q.
      • Wang Y.
      • et al.
      Expressions of miR-15a and its target gene HSPA1B in the spermatozoa of patients with varicocele.
      ,
      • Kaczmarek K.
      • Studencka M.
      • Meinhardt A.
      • Wieczerzak K.
      • Thoms S.
      • Engel W.
      • et al.
      Overexpression of peroxisomal testis-specific 1 protein induces germ cell apoptosis and leads to infertility in male mice.
      ,
      • Liu Y.
      • Liu W.B.
      • Liu K.J.
      • Ao L.
      • Cao J.
      • Zhong J.L.
      • et al.
      Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields.
      ,
      • Niu Z.
      • Goodyear S.M.
      • Rao S.
      • Wu X.
      • Tobias J.W.
      • Avarbock M.R.
      • et al.
      MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells.
      ,
      • Novotny G.W.
      • Belling K.C.
      • Bramsen J.B.
      • Nielsen J.E.
      • Bork-Jensen J.
      • Almstrup K.
      • et al.
      MicroRNA expression profiling of carcinoma in situ cells of the testis.
      ,
      • Kotaja N.
      MicroRNAs and spermatogenesis.
      ). In detail, we selected the most overexpressed and underexpressed miRNAs (i.e., miR-1275 and miR-26b, respectively). In addition, we included moderately underexpressed miRNAs (i.e., miR-30b, miR-20a, miR-148a, and miR-15a) and moderately overexpressed miRNAs (i.e., miR-765 and miR-1299). For all of the eight miRNAs the qRT-PCR showed the same direction of expression changes as the microarray analysis. In detail, miR-30b, miR-20a, miR-148a, miR-26b, and miR-15a showed a lower expression level and miR-765, miR-1299, and miR-1275 showed an increase expression level in oligoasthenozoospermic men as compared with normozoospermic men. The significance of the altered expression was confirmed for three miRNAs (i.e., miR-765, miR-1275, and miR-15a; P<.05, FDR corrected). No significant differences were found for miR-30b, miR-20a, miR-148a, miR-1299, and miR-26b. In summary, the qRT-PCR confirmed the results of the microarray analysis for three miRNAs with regard both to the direction (over- and underexpression) and to the significance of the altered expression (P<.05) between oligoasthenozoospermic and normozoospermic men (Table 3).
      Table 3Confirmation of the differential expression of miRNAs in the extracellular microvesicles from oligoasthenozoospermic subfertile men as compared with normozoospermic men, using qRT-PCR.
      miRNAFold changeP valueAdjusted P valueExpression
      miR-30b1.40.0064.0501Under
      miR-20a1.87.0868.5164Under
      miR-148a1.23.2503.9002Under
      miR-7652.23.0011.0088Over
      miR-12991.57.0141.1074Over
      miR-26b1.29.1880.8110Under
      miR-12751.63.0034.0269Over
      miR-15a2.80.0059.0462Under
      Note: P<.05 was considered to be significant (Student t test).

       Comparative Pathway Analysis

      We used the DIANA-mirPath algorithm to gain insights to the biological pathways of the miRNAs that were altered in the extracellular microvesicles of oligoasthenozoospermic men as compared with normozoospermic men. On the basis of the microarray analysis we identified 75 and 12 KEGG pathways that were significantly enriched (P<.05, FDR corrected) for targets of over- and underexpressed miRNAs, respectively (Supplemental Table 1). As shown in Table 4, the target genes of the altered miRNAs are mostly involved in “Signaling pathways,” “Cellular community,” “Cell growth and degradation,” and “Immune system.” Enrichment analysis applying the KEGG database displayed the highest correlation for signaling pathways, providing further evidence that extracellular microvesicles target proteins that are frequently involved in the intracellular signaling pathways.
      Table 4The KEGG pathways significantly enriched for target genes of over- and underexpressed miRNAs in extracellular microvesicles in oligoasthenozoospermic subfertile men as compared with normozoospermic fertile men.
      Mode of interactionKEGG pathwayP value, FDR correctedNo. genesNo. miRNAs
      Signaling pathwaysRas signaling pathway (hsa04014)8.79E+0611729
      MAPK signaling pathway (hsa04010).0009313129
      ErbB signaling pathway (hsa04012)5.69E+065828
      Hippo signaling pathway (hsa04390)2.36E-078428
      Rap1 signaling pathway (hsa04015).0012311028
      cAMP signaling pathway (hsa04024).0038310328
      PI3K-Akt signaling pathway (hsa04151).0003217228
      Wnt signaling pathway (hsa04310)7.83E+067827
      FoxO signaling pathway (hsa04068)3.43E-078427
      mTOR signaling pathway (hsa04150).000254027
      cGMP-PKG signaling pathway (hsa04022).011598427
      GnRH signaling pathway (hsa04912).046554726
      Sphingolipid signaling pathway (hsa04071).001666426
      HIF-1 signaling pathway (hsa04066).032365426
      TGF-beta signaling pathway (hsa04350)2.10E+055024
      Cellular communitySignaling pathways regulating pluripotency of stem cells (hsa04550)2.03E+058329
      Focal adhesion (hsa04510).0015894526
      Adherens junction (hsa04520).0015894526
      Gap junction (hsa04540).0023164925
      Tight junction (hsa04530).038820356
      Cell growth and degradationProtein processing in endoplasmic reticulum (hsa04141).0309628228
      Ubiquitin mediated proteolysis (hsa04120).0016787726
      p53 signaling pathway (hsa04115).0029384125
      Immune systemT cell receptor signaling pathway (hsa04660).0049615827
      Fc epsilon RI signaling pathway (hsa04664).0437473826
      CancersProstate cancer (hsa05215)7.83E+065527
      Pancreatic cancer (hsa05212).003833926
      Non-small cell lung cancer (hsa05223).005823126
      Small cell lung cancer(hsa05222).032894526
      Glioma2.06E+064026
      Colorectal cancer (hsa05210).007783622

      Discussion

      We identified an altered miRNA expression profile in oligoasthenozoospermic men when compared with the normozoospermic men by both microarray and qRT-PCR analyses. Although there are no previous studies on miRNAs from extracellular microvesicles isolated from seminal plasma of men with spermatogenic impairments, few studies analyzed the miRNA content directly from semen and whole cell-free seminal fluid (
      • Wu W.
      • Hu Z.
      • Qin Y.
      • Dong J.
      • Dai J.
      • Lu C.
      • et al.
      Seminal plasma microRNAs: potential biomarkers for spermatogenesis status.
      ,
      • Wu W.
      • Qin Y.
      • Li Z.
      • Dong J.
      • Dai J.
      • Lu C.
      • et al.
      Genome–wide microRNA expression profiling in idiopathic non-obstructive azoospermia: significant up-regulation of miR-141, miR-429 and miR-7-1-3p.
      ,
      • Wang C.
      • Yang C.
      • Chen X.
      • Yao B.
      • Yang C.
      • Zhu C.
      • et al.
      Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility.
      ,
      • Li H.
      • Huang S.
      • Guo C.
      • Guan H.
      • Xiong C.
      Cell-free seminal mRNA and microRNA exist in different forms.
      ). These studies report distinctive miRNA signatures for semen collected from infertile men and for seminal plasma from men with azoospermia compared with fertile control men. Specifically, miR-374b was markedly underexpressed in the seminal plasma of men with azoospermia (
      • Wang C.
      • Yang C.
      • Chen X.
      • Yao B.
      • Yang C.
      • Zhu C.
      • et al.
      Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility.
      ), and miR-20a was overexpressed in seminal plasma of men with NOA (
      • Wu W.
      • Qin Y.
      • Li Z.
      • Dong J.
      • Dai J.
      • Lu C.
      • et al.
      Genome–wide microRNA expression profiling in idiopathic non-obstructive azoospermia: significant up-regulation of miR-141, miR-429 and miR-7-1-3p.
      ). Although miR-23a, miR-23b, miR-30b, miR-27a, and miR-100 showed an underexpression in semen samples of men with infertility, miR-1275 and miR-483 showed an overexpression in this group as compared with fertile control men (
      • Liu T.
      • Cheng W.
      • Gao Y.
      • Wang H.
      • Liu Z.
      Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities.
      ). The alteration of the expression direction (i.e., over- and underexpression) of these miRNAs are in agreement with our data: our microarray analysis identified the same changes of expression direction for miR-374b, miR-23a, miR-23b, miR-30b, miR-27a, and miR-100, and our qRT-PCR analysis confirmed the changes of expression direction for miR-1275, each for the comparison between oligoasthenozoospermic and normozoospermic men. We compared the miRNAs identified in extracellular microvesicles collected from seminal plasma with previously reported miRNAs from purified spermatozoa of the same patients (
      • Abu-Halima M.
      • Hammadeh M.
      • Schmitt J.
      • Leidinger P.
      • Keller A.
      • Meese E.
      • et al.
      Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments.
      ). Of the 36 miRNAs that were differentially expressed in extracellular microvesicles from men with oligoasthenozoospermia as compared with men with normozoospermia, we found 30 miRNAs that were also differentially expressed in purified spermatozoa samples from men with oligoasthenozoospermia or asthenozoospermia as compared with men with normozoospermia (
      • Abu-Halima M.
      • Hammadeh M.
      • Schmitt J.
      • Leidinger P.
      • Keller A.
      • Meese E.
      • et al.
      Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments.
      ). The remaining six miRNAs, namely miR-28-5p, miR-93, miR-96, miR-100, miR-483-5p, and miR-4306, were differentially expressed in the extracellular microvesicles but not in purified spermatozoa (Supplemental Table 2). As for the 30 miRNAs that were differentially expressed both in extracellular microvesicles and in purified spermatozoa, we found 13 miRNAs showing the same direction of expression changes in extracellular microvesicles and in purified spermatozoa. In detail, three miRNAs, including miR-1275, miR-765, and miR-766, were overexpressed both in extracellular microvesicles of oligoasthenozoospermic men as compared with normozoospermic men and in purified spermatozoa of men with oligoasthenozoospermia as compared with normozoospermic fertile men. The remaining 10 miRNAs (miR-15a, miR-15b, miR-17, miR-20a, miR-23a, miR-25, miR-30c, miR-374a, miR-374b, and miR-425) were underexpressed again both in extracellular microvesicles of oligoasthenozoospermic men and in spermatozoa of men with oligoasthenozoospermia or asthenozoospermia as compared with normozoospermic fertile men (
      • Abu-Halima M.
      • Hammadeh M.
      • Schmitt J.
      • Leidinger P.
      • Keller A.
      • Meese E.
      • et al.
      Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments.
      ). We found seven miRNAs, including miR-1275 and miR-483-5p that were overexpressed and miR-100, miR-27a, miR-23b, miR-23a, and miR-30b that were underexpressed, both in extracellular microvesicles of oligoasthenozoospermic men and in semen samples from infertile men compared with fertile controls (
      • Liu T.
      • Cheng W.
      • Gao Y.
      • Wang H.
      • Liu Z.
      Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities.
      ). Only one miRNA, miR-25, was underexpressed both in extracellular microvesicles of oligoasthenozoospermic men as compared with normozoospermic men and spermatozoa from oligozoospermic men compared with normozoospermic fertile controls (
      • Munoz X.
      • Mata A.
      • Bassas L.
      • Larriba S.
      Altered miRNA signature of developing germ-cells in infertile patients relates to the severity of spermatogenic failure and persists in spermatozoa.
      ). Furthermore, miR-374b was underexpressed in extracellular microvesicles of oligoasthenozoospermic men and also underexpressed in whole cell-free seminal fluid samples from men with azoospermia compared with fertile controls (Supplemental Table 2) (
      • Wu W.
      • Qin Y.
      • Li Z.
      • Dong J.
      • Dai J.
      • Lu C.
      • et al.
      Genome–wide microRNA expression profiling in idiopathic non-obstructive azoospermia: significant up-regulation of miR-141, miR-429 and miR-7-1-3p.
      ,
      • Wang C.
      • Yang C.
      • Chen X.
      • Yao B.
      • Yang C.
      • Zhu C.
      • et al.
      Altered profile of seminal plasma microRNAs in the molecular diagnosis of male infertility.
      ). Of the six miRNAs that were only expressed in the extracellular microvesicles, miR-28 and miR-4306 were previously reported in seminal plasma and serum-derived exosomes, respectively (
      • Vojtech L.
      • Woo S.
      • Hughes S.
      • Levy C.
      • Ballweber L.
      • Sauteraud R.P.
      • et al.
      Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions.
      ,
      • Madhavan B.
      • Yue S.
      • Galli U.
      • Rana S.
      • Gross W.
      • Muller M.
      • et al.
      Combined evaluation of a panel of protein and miRNA serum-exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity.
      ). Vojtech et al. (
      • Vojtech L.
      • Woo S.
      • Hughes S.
      • Levy C.
      • Ballweber L.
      • Sauteraud R.P.
      • et al.
      Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions.
      ) identified miR-28 as one of the lower abundant miRNAs in seminal plasma-derived exosomes from healthy men. MiR-28-5p has not yet been associated with spermatogenesis or its related processes. Our data show a distinct population of miRNAs that is common to extracellular microvesicles collected from seminal plasma. It is legitimate to hypothesize that this miRNA pattern mirrors to some extend the miRNA pattern in spermatozoa, but it can also be influenced by the number of germ cells produced in the testis and by the expression pattern of microvesicles secreted from other organs of the reproductive tract (i.e., epididymis and prostate). As part of these processes, miRNAs are loaded from the cells of the genital tract into exosomes (
      • Vojtech L.
      • Woo S.
      • Hughes S.
      • Levy C.
      • Ballweber L.
      • Sauteraud R.P.
      • et al.
      Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions.
      ,
      • Renneberg H.
      • Konrad L.
      • Dammshauser I.
      • Seitz J.
      • Aumuller G.
      Immunohistochemistry of prostasomes from human semen.
      ,
      • Ronquist G.
      • Brody I.
      The prostasome: its secretion and function in man.
      ). With regard to the general function of the miRNA in extracellular microvesicles of seminal plasma, several reports indicate the transfer of extracellular messenger RNA and miRNA from one cell to another through extracellular communication (
      • Valadi H.
      • Ekstrom K.
      • Bossios A.
      • Sjostrand M.
      • Lee J.J.
      • Lotvall J.O.
      Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
      ,
      • Kosaka N.
      • Iguchi H.
      • Yoshioka Y.
      • Takeshita F.
      • Matsuki Y.
      • Ochiya T.
      Secretory mechanisms and intercellular transfer of microRNAs in living cells.
      ,
      • Wang K.
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      • Baxter D.
      • Galas D.J.
      Export of microRNAs and microRNA-protective protein by mammalian cells.
      ,
      • Yuan A.
      • Farber E.L.
      • Rapoport A.L.
      • Tejada D.
      • Deniskin R.
      • Akhmedov N.B.
      • et al.
      Transfer of microRNAs by embryonic stem cell microvesicles.
      ). Moreover, the extracellular RNAs in the male reproductive tract not only commute between cells but are also able to regulate gene expression by endocrine-like cell-to-cell communication (
      • Belleannee C.
      Extracellular microRNAs from the epididymis as potential mediators of cell-to-cell communication.
      ,
      • Laffont B.
      • Corduan A.
      • Ple H.
      • Duchez A.C.
      • Cloutier N.
      • Boilard E.
      • et al.
      Activated platelets can deliver mRNA regulatory Ago2*microRNA complexes to endothelial cells via microparticles.
      ,
      • Vickers K.C.
      • Palmisano B.T.
      • Shoucri B.M.
      • Shamburek R.D.
      • Remaley A.T.
      MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.
      ).
      We used qRT-PCR to confirm the expression levels of 8 of the 36 miRNAs, which showed significant differences between oligoasthenozoospermic subfertile men and normozoospermic fertile men in the microarray assay. These miRNAs were selected according to their altered expression between oligoasthenozoospermic subfertile men and normozoospermic fertile men and on the basis of their known associations with spermatogenesis (
      • Lian J.
      • Zhang X.
      • Tian H.
      • Liang N.
      • Wang Y.
      • Liang C.
      • et al.
      Altered microRNA expression in patients with non-obstructive azoospermia.
      ,
      • Liu T.
      • Cheng W.
      • Gao Y.
      • Wang H.
      • Liu Z.
      Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities.
      ,
      • Abu-Halima M.
      • Backes C.
      • Leidinger P.
      • Keller A.
      • Lubbad A.M.
      • Hammadeh M.
      • et al.
      MicroRNA expression profiles in human testicular tissues of infertile men with different histopathologic patterns.
      ,
      • Abu-Halima M.
      • Hammadeh M.
      • Schmitt J.
      • Leidinger P.
      • Keller A.
      • Meese E.
      • et al.
      Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments.
      ,
      • Curry E.
      • Safranski T.J.
      • Pratt S.L.
      Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility.
      ,
      • Ji Z.
      • Lu R.
      • Mou L.
      • Duan Y.G.
      • Zhang Q.
      • Wang Y.
      • et al.
      Expressions of miR-15a and its target gene HSPA1B in the spermatozoa of patients with varicocele.
      ,
      • Kaczmarek K.
      • Studencka M.
      • Meinhardt A.
      • Wieczerzak K.
      • Thoms S.
      • Engel W.
      • et al.
      Overexpression of peroxisomal testis-specific 1 protein induces germ cell apoptosis and leads to infertility in male mice.
      ,
      • Liu Y.
      • Liu W.B.
      • Liu K.J.
      • Ao L.
      • Cao J.
      • Zhong J.L.
      • et al.
      Overexpression of miR-26b-5p regulates the cell cycle by targeting CCND2 in GC-2 cells under exposure to extremely low frequency electromagnetic fields.
      ,
      • Niu Z.
      • Goodyear S.M.
      • Rao S.
      • Wu X.
      • Tobias J.W.
      • Avarbock M.R.
      • et al.
      MicroRNA-21 regulates the self-renewal of mouse spermatogonial stem cells.
      ,
      • Novotny G.W.
      • Belling K.C.
      • Bramsen J.B.
      • Nielsen J.E.
      • Bork-Jensen J.
      • Almstrup K.
      • et al.
      MicroRNA expression profiling of carcinoma in situ cells of the testis.
      ,
      • Kotaja N.
      MicroRNAs and spermatogenesis.
      ). Several of them have been identifed in seminal plasma and semen, in purified spermatozoa, or in both seminal plasma/semen and spermatozoa. The qRT-PCR confirmed the results of the microarray analysis for three miRNAs with regard both to the direction (over- and underexpression) and to the significance of the altered expression (P<.05) between oligoasthenozoospermic and normozoospermic men. These three miRNAs were differentially expressed in testis with spermatogenic disorders. Specifically, miR-1275 was significantly overexpressed in men with Sertoli cell-only syndrome as compared with control men, as shown by microarray analysis (
      • Abu-Halima M.
      • Backes C.
      • Leidinger P.
      • Keller A.
      • Lubbad A.M.
      • Hammadeh M.
      • et al.
      MicroRNA expression profiles in human testicular tissues of infertile men with different histopathologic patterns.
      ), in normal spermatozoa as determined by TaqMan arrays (
      • Salas-Huetos A.
      • Blanco J.
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      New insights into the expression profile and function of micro-ribonucleic acid in human spermatozoa.
      ), and in semen of men with infertility as determined by microarray analysis, qRT-PCR, and Northern blotting (
      • Liu T.
      • Cheng W.
      • Gao Y.
      • Wang H.
      • Liu Z.
      Microarray analysis of microRNA expression patterns in the semen of infertile men with semen abnormalities.
      ). MiR-15a was significantly underexpressed in the spermatozoa of men with varicocele (
      • Ji Z.
      • Lu R.
      • Mou L.
      • Duan Y.G.
      • Zhang Q.
      • Wang Y.
      • et al.
      Expressions of miR-15a and its target gene HSPA1B in the spermatozoa of patients with varicocele.
      ). Notably, the miR-15a target Cyclin T2 gene is involved in early spermatogenesis (
      • Teng Y.
      • Wang Y.
      • Fu J.
      • Cheng X.
      • Miao S.
      • Wang L.
      Cyclin T2: a novel miR-15a target gene involved in early spermatogenesis.
      ). In addition, miR-765 and miR-1275 were overexpressed in spermatozoa of subfertile men with oligoasthenozoospermia (
      • Abu-Halima M.
      • Hammadeh M.
      • Schmitt J.
      • Leidinger P.
      • Keller A.
      • Meese E.
      • et al.
      Altered microRNA expression profiles of human spermatozoa in patients with different spermatogenic impairments.
      ). Bioinformatics analysis by DIANA-mirPath predicted several KEGG biological pathways that were significantly enriched (P<.05, FDR correct-ed) for differentially over- or underexpressed miRNAs in extracellular microvesicles. Among the predicted pathways were the Ras, MAPK, ErbB, cAMP, PI3K-Akt, Wnt, and Hedgehog signaling pathways, focal adhesion, and junctional complexes including tight, adherens, and gap junctions. All of these biological pathways have a role in spermatogenesis or its related processes. The aforementioned studies mostly analyzed seminal plasma, semen and/or spermatozoa. Because these sources contain significant amounts of miRNAs, it can be difficult to identify specific miRNAs as biomarkers for male infertility. The extracellular microvesicles with their cargo of miRNAs provide an enriched population of miRNAs largely free of endogenous RNA contaminants like degradation products of larger RNAs. Hence, the extracellular microvesicles are increasingly proposed as a source for disease-specific miRNA signatures (
      • Vojtech L.
      • Woo S.
      • Hughes S.
      • Levy C.
      • Ballweber L.
      • Sauteraud R.P.
      • et al.
      Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions.
      ,
      • Gallo A.
      • Tandon M.
      • Alevizos I.
      • Illei G.G.
      The majority of microRNAs detectable in serum and saliva is concentrated in exosomes.
      ). In summary, we report miRNAs in the extracellular microvesicles with significantly altered expression levels between oligoasthenozoospermic subfertile men and normozoospermic men. There is a considerable overlap between differential miRNAs in extracellular microvesicles collected from seminal plasma, miRNAs in semen, and purified spermatozoa. Although it is important to bear in mind that the miRNA expression profiles of extracellular microvesicles were obtained from a small number of men, the altered expression levels in oligoasthenozoospermic subfertile men compared with normozoospermic men have been confirmed for three miRNAs by qRT-PCR. The alteration of the expression of miRNAs will help to understand the molecular mechanisms underlying male infertility.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1Examples of RNA content by capillary electrophoresis by using an Agilent 2100 Bioanalyzer and an RNA Nano chip. The RNAs isolated from extracellular microvesicles collected from seminal plasma show very low integrity, with RNA integrity number <2.8.
      Figure thumbnail fx2
      Supplemental Figure 2Western blot analysis of the exosomal marker proteins CD63 (approximately 53 kDa), CD81 (approximately 26 kDa), CD9 (approximately 28 kDa), and HSP70 (approximately 70 kDa) for samples of oligoasthenozoospermic and normozoospermic men. Microvesicles isolated with ultracentrifugation protocols were separated on a 10% standard SDS-PAGE. Standard Western blot procedures with anti-CD63, anti-CD81, anti-CD9, and anti-HSP70 antibodies were used to detect exosomal protein markers.
      Figure thumbnail fx3
      Supplemental Figure 3Heatmap based on the differential expression of the 36 altered miRNAs in oligoasthenozoospermic and normozoospermic men. Columns represent seminal plasma microvesicle samples, and rows represent miRNAs. Dark blue indicates high expression, and dark red low expression.

      Supplementary data

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