Proteomic analysis of seminal plasma from normal volunteers and post-vasectomy patients identifies over 2000 proteins and candidate biomarkers of the urogenital system

Seminal plasma is a fluid that originates from the testis, epididymis,prostate, and seminal vesicles, and hence, proteomic studies may identify potential markers of infertility and other diseases of the genito-urinary tract. We profiled the proteomes of pooled seminal plasma from fertile Control and post-vasectomy (PV) men. PV seminal plasma samples are void of proteins originating from the testis and the epididymis due to ligation of the vas deferens, and hence, comparative analysis of Control and PV data sets allows for identification of proteins originating from these tissues. Utilizing offline MudPIT and high-resolution mass spectrometry, we were able to identify over 2000 proteins in Control and PV pools each and over 2300 proteins all together. With semiquantitative analysis using spectral counting, we catalogued 32 proteins unique to Control, 49 at lower abundance in PV, 3 unique to PV, and 25 at higher abundance in PV. We believe that proteins unique to Control or at lower abundance in PV have their origin in the testis and the epididymis. Public databases have confirmed that many of these proteins originate from the testis and epididymis and are linked to the reproductive tract. These proteins may serve as candidate biomarkers for future studies of infertility and urogenital diseases.     

非梗阻性无精子症的遗传学基础及研究进展

非梗阻性无精子症(non-obstructive azoospermia,NOA)是导致男性不育的重要原因,影响着约0.6%的男性或10%的不育男性.NOA是一种由多因素引起的具有高度遗传异质性和表型异质性的复杂疾病,其中遗传学病因包括染色体异常、Y染色体微缺失、基因突变以及表观遗传修饰等.目前临床上针对NOA患者的遗传学检测,还仅限于结合附睾和睾丸穿刺活检的核型分析及Y染色体微缺失检测,而且一直缺乏理想的治疗方案.因此,深入解析NOA的具体分子机理,对阐明NOA的病因、提高男性不育的临床诊断和治疗具有重要意义.本综述将从NOA的遗传学基础、NOA的病理特征、临床诊断及治疗等方面进行系统的探讨.     

Efficacité et innocuité du prélèvement percutané de sperme testiculaire dans la prise en charge de l’infertilité masculine

Objective

To assess the efficacy and safety of percutaneous testicular biopsy to provide sperm cells for ICSI in male patients with azoospermia not amenable to surgical treatment.

Materials and methods

From October 1995 to December 2001, 175 biopsies were performed in men with azoospermia to provide material for intracytoplasmic sperm injection. Azoospermia was obstructive (OA) in 41 cases and non-obstructive (NOA) in 134 cases. Open biopsy was performed in the first 15 patients in the series and percutaneous biopsy was performed on an outpatient basis, under local anesthesia, with a Biopty Gun® (14G needle), in the subsequent patients as the first step in management. Open surgical biopsies were performed in another 15 patients following a sperm cell-negative percutaneous biopsy.

Results

All biopsies performed for OA were positive, but only 51/134 biopsies (38%) were positive in the NOA group. The material provided by percutaneous biopsy, when positive for sperm cells, was always sufficient to perform ICSI. When percutaneous biopsy was negative, open surgical biopsy failed to give better results. Five men developed minor complications (acute hematocele) following percutaneous biopsies requiring reoperation for hemostasis (3.12%). No major complications were observed. Results were comparable in terms of fertilization and pregnancy rates whether fresh or frozen-thawed sperm was used.

Conclusion

Percutaneous testicular sperm extraction is a safe, well-tolerated and cost-effective procedure in the management of male-factor infertility related to azoospermia.     

基于高通量转录组测序技术的无精子症患者睾丸组织比较转录组分析

本研究探讨不同程度无精子症患者睾丸组织的转录组差异,了解差异表达基因(DEG)在功能、分类和代谢通路的不同,揭示无精子症患者精子发生分子机制,为促进男性不育研究的发展提供理论基础.选取1份非梗阻性无精子症和4份梗阻性无精子症患者睾丸组织样品(从无精子到有精子),进行RNA提取和文库构建,利用Illumina HiSeqTM2500高通量测序,构建无精子症患者睾丸组织转录组文库,并用生物信息学方法进行分析.结果发现,样品比对基因组数据库的平均比对率为94.38%,共检测2 242个属于预测新的蛋白质编码基因的转录本.得到差异表达基因统计结果为:NOA vs. OA1基因上调8 045,下调1 150;OA1 vs. OA2基因上调1 538,下调420;OA2 vs. OA3基因上调1 275,下调1 690;OA3 vs. OA4基因上调1 834,下调1 853.比较5例无精子症睾丸组织的差异基因KEGG,主要富集在RNA降解通路、基底细胞瘤通路、癌通路、黑色素生成通路和调节干细胞多能性等信号通路. PRM1、PRM2、TNP1、UBXN6、CXCL16、NUPR2、CCDC136和CRISP2等基因的表达呈递增趋势,并具有时序特异性.此外,5例无精子症睾丸组织的表达基因有不同程度的基因融合.综上,基因融合可能和无精子症相关,并且不同程度无精子症患者睾丸组织的差异表达基因数量、功能、分类和代谢通路不同.本研究筛选出精子发生、精子运动等差异表达基因,丰富了无精子症患者睾丸组织转录组信息,为开展无精子症患者睾丸组织相关基因及分子调控机制的研究奠定基础.     

Successful microdissection testicular sperm extraction for men with non-obstructive azoospermia

Non-obstructive azoospermia (NOA) is the most severe form of male infertility, defined by lack of spermatozoa in the ejaculate caused by impaired spermatogenesis. The chance of biological fatherhood of these men has been improved since the introduction of microdissection testicular sperm extraction (MD-TESE) combined with intracytoplasmic sperm injection. A thorough patient evaluation preoperatively is essential to recognize any underlying conditions, and to assist in patient counseling on the sperm recovery rate and pregnancy results. This review article summarizes the present data on MD-TESE to reach optimal results is treating men with NOA.     

Comparison of label-free methods for quantifying human proteins by shotgun proteomics

Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic analyses. We describe Serac, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using standard proteins spiked into a complex sample. Linearity and good agreement between observed versus expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio estimates. Peak intensity values useful for protein quantitation ranged from 10(7) to 10(11) counts with no obvious saturation effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (+/-2sigma) when >or=3 peptides/protein were shared between samples. Protein ratios were determined with high confidence from spectral counts when maximum spectral counts were >or=4 spectra/protein, and replicates showed equivalent measurements well within 95% confidence limits. In further tests, complex samples were separated by gel exclusion chromatography, quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios determined by spectral counting agreed well with those determined from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall spectral counting proved to be a more sensitive method for detecting proteins that undergo changes in abundance, whereas peak area intensity measurements yielded more accurate estimates of protein ratios. Finally these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells stimulated under conditions that promote cell differentiation by mitogen-activated protein kinase pathway activation. Protein changes identified with p<0.1 showed good correlations with parallel measurements of changes in mRNA expression.     

Xenogeneic transplantation of human spermatogonia

In the last 3 years, several studies have shown that xenogeneic transplantation of rodent spermatogonia is feasible. The treatment of infertile patients with spermatogenic arrest using the injection of immature germ cells has yielded only poor results. We attempted to establish a complete spermatogenetic line in the testes of mutant aspermatogenic (W/Wv) and severe combined immunodeficient mice (SCID) transplanted with germ cells from azoospermic men. Spermatogenic cells were obtained from testicular biopsy specimens of men (average age of 34.3 +/- 9 years) undergoing infertility treatment because of obstructive and non-obstructive azoospermia. Testicular tissue was digested with collagenase to promote separation of individual spermatogenic cells. The germ cells were injected into mouse testicular seminiferous tubules using a microneedle (40 microm inner diameter) on a 10 ml syringe. To assess the penetration of the cell suspension into the tubules, trypan blue was used as an indicator. Mice were maintained for 50 to 150 days to allow time for germ cell colonisation and development prior to them being killed. Testes were then fixed for histological examination and approximately 100 cross-sectioned tubules were examined for human spermatogenic cells. A total of 26 testicular cell samples, 16 frozen and 10 fresh, were obtained from 24 men. The origin of the azoospermia was obstructive (OA) in 16 patients and non-obstructive (NOA) in 8 patients. The concentration of spermatogenic cells in the OA group was 6.6 x 10(6) cells/ml, and 1.3 x 10(6) cells/ml in the NOA group (p < 0.01). The different spermatogenic cell types were distributed equally in the OA samples, ranging from spermatogenia to fully developed spermatozoa, but in the NOA group the majority of cells were spermatogonia and spermatocytes. A total of 23 testes from 14 W/Wv mice and 24 testes from 12 SCID mice were injected successfully, as judged by the presence of spermatogenic cells in histological sections of testes removed immediately after the injection. However, sections from the remaining testes examined up to 150 days after injection showed tubules lined with Sertoli cells and xenogeneic germ cells were not found. The reason why the two strains of mouse used as recipients did not allow the implantation of human germ cells is probably due to interspecies specificity involving non-compatible cell adhesion molecules and/or immunological rejection.     

Chromosome abnormalities in spermatozoa of patients with azoospermia and normal somatic karyotype

Since epididymal and testicular spermatozoa of azoospermic patients are frequently used for intracytoplasmic sperm injection (ICSI), many studies have been carried out to evaluate their karyotype. This article will review all published data on this topic. In most of the studies, spermatozoa have been retrieved from the testis or the epididymis of patients with nonobstructive (NOA) or obstructive (OA) azoospermia, respectively. Sperm aneuploidy has been evaluated by fluorescence in situ hybridization using probes for sex chromosomes and an array of autosomes. A significantly higher sperm aneuploidy rate has been reported in patients with NOA and OA compared to ejaculated spermatozoa, mainly for sex chromosomes. The magnitude of the increase varies between studies, probably because of the heterogeneity of case selection as well as of the methodology employed. The majority of the studies reported that patients with NOA have a greater sperm aneuploidy rate compared to OA. The greater frequency of sperm aneuploidy in azoospermic patients increases the risk of transmitting a karyotype abnormality to the offspring generated by ICSI.     

Differential proteomic analysis of renal cell carcinoma tissue interstitial fluid

Renal cell carcinoma (RCC), the most common type of kidney cancer, currently has no biomarker of clinical utility. The present study utilized a mass spectrometry-based proteomics workflow for identifying differentially abundant proteins in RCC by harvesting shed and secreted proteins from the tumor microenvironment through sampling tissue interstitial fluid (TIF) from radical nephrectomies. Matched tumor and adjacent normal kidney (ANK) tissues were collected from 10 patients diagnosed with clear cell RCC. One-hundred thirty-eight proteins were identified with statistically significant differential abundances derived by spectral counting in tumor TIF when compared to ANK TIF. Among those proteins with elevated abundance in tumor TIF, nicotinamide n-methyltransferase (NNMT) and enolase 2 (ENO2) were verified by Western blot and selected reaction monitoring (SRM). The presence of ENO2 and thrombospondin-1 (TSP1) were verified as present and at elevated abundance in RCC patient serum samples as compared to a pooled standard control by enzyme-linked immunosorbent assay (ELISA), recapitulating the relative abundance increase in RCC as compared with ANK TIF.     

Differential protein profiling of synovial fluid from rheumatoid arthritis and osteoarthritis patients using LC-MALDI TOF/TOF

The purpose of this study was to identify those proteins relatively more abundant in the synovial fluid (SF) of patients suffering from rheumatoid arthritis (RA) and osteoarthritis (OA) using high performance liquid chromatography coupled to mass spectrometry. 20 individual SF samples from each disease were pooled into two groups (RA and OA) to reduce the contribution of extreme individual values. Prior to the proteomic analysis, samples were immunodepleted from the top 20 most abundant plasma proteins, to enrich the lower-abundance protein fractions. Then, they were subjected to protein size fractioning and in-gel digestion, followed by reversed-phase peptide separation in a nano-LC system and subsequent peptide identification by MALDI-TOF/TOF. This strategy led to the identification of 136 different proteins in SF, which is the largest number of SF proteins described up to date by proteomics. A relative quantification of the proteins between RA and OA was carried out by spectral counting analysis. In RA, our results show a greater relative abundance of proteins related to complement activation, inflammation and the immune response, such as the major matrix metalloproteinases and several neutrophil-related proteins. In OA, we detected an increase in proteins involved in the formation and remodeling of the extracellular matrix (ECM), such as fibronectin, kininogen-1, cartilage acidic protein 1 and cartilage oligomeric matrix protein. The results obtained for MMP-1, BGH3, fibronectin and gelsolin were verified by immunoblotting analyses. Some of the novel proteins identified in this work might be relevant not only for increasing knowledge on the etiopathogenesis of RA and OA processes, but also as putative disease biomarkers, as their presence in SF is a prior step to their dilution in serum. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

A genome-wide association study reveals that variants within the HLA region are associated with risk for nonobstructive azoospermia

A genome-wide association study of Han Chinese subjects was conducted to identify genetic susceptibility loci for nonobstructive azoospermia (NOA). In the discovery stage, 802 azoospermia cases and 1,863 controls were screened for genetic variants in the genome. Promising SNPs were subsequently confirmed in two independent sets of subjects: 818 azoospermia cases and 1,755 controls from northern China, and 606 azoospermia cases and 958 controls from central and southern China. We detected variants at human leukocyte antigen (HLA) regions that were independently associated with NOA (HLA-DRA, rs3129878, p_combine = 3.70 × 10⁻¹⁶, odds ratio [OR] = 1.37; C6orf10 and BTNL2, rs498422, p_combine = 2.43 × 10⁻¹², OR = 1.42). These findings provide additional insight into the pathogenesis of NOA.     

Verification of male infertility biomarkers in seminal plasma by multiplex selected reaction monitoring assay

Seminal plasma is a promising biological fluid to use for noninvasive clinical diagnostics of male reproductive system disorders. To verify a list of prospective male infertility biomarkers, we developed a multiplex selected reaction monitoring assay and measured the relative abundance of 31 proteins in 30 seminal plasma samples from normal, nonobstructive azoospermia and post-vasectomy individuals. Median levels of some proteins were decreased by more than 100-fold in nonobstructive azoospermia or post-vasectomy samples, in comparison with normal samples. To follow up the most promising candidates and measure their concentrations in seminal plasma, heavy isotope-labeled internal standards were synthesized and used to reanalyze 20 proteins in the same set of samples. Concentrations of candidate proteins in normal seminal plasma were found in the range 0.1-1000 μg/ml but were significantly decreased in nonobstructive azoospermia and post-vasectomy. These data allowed us to select, for the first time, biomarkers to discriminate between normal, nonobstructive azoospermia, and post-vasectomy (simulated obstructive azoospermia) seminal plasma samples. Some testis-specific proteins (LDHC, TEX101, and SPAG11B) performed with absolute or nearly absolute specificities and sensitivities. Cell-specific classification of protein expression indicated that Sertoli or germ cell dysfunction, but not Leydig cell dysfunction, was observed in nonobstructive azoospermia seminal plasma. The proposed panel of biomarkers, pending further validation, could lead to a clinical assay that can eliminate the need for testicular biopsy to diagnose the category of male infertility, thus providing significant benefits to patients as well as decreased costs associated with the differential diagnosis of azoospermia.     

Recueil chirurgical de spermatozoïdes dans un groupe de 50 patients avec un antécédent de cryptorchidie bilatérale: facteurs pronostics d’extraction

Bilateral cryptorchidism is a severe factor of male infertility. We evaluated the results of testicular sperm extraction in 50 men with nonobstructive azoospermia associated with cryptorchidism between 1995 to 2001. We evaluated clinical parameters predictive of successful sperm extraction. Serum follicle stimulating hormone (FSH) was elevated in 67% of cases and testicular volume was decreased in 70% of cases, confirming the secretory origin of the azoospermia. Serum follicle stimulating hormone (FSH), testicular volume and histological parameters were examined as predictive factors for sperm recovery. The positive sperm recovery rate was 68%. As in the population of men with nonobstructive azoospermia, the sperm recovery rate for patients with a history of orchidopexy is approximately 68% and there are currently no clinical parameters predicting successful sperm retrieval in this subpopulation of patients.     

A label-free quantification method by MS/MS TIC compared to SILAC and spectral counting in a proteomics screen

In order to assess the biological function of proteins and their modifications for understanding signaling mechanisms within cells as well as specific biomarkers to disease, it is important that quantitative information be obtained under different experimental conditions. Stable isotope labeling is a powerful method for accurately determining changes in the levels of proteins and PTMs; however, isotope labeling experiments suffer from limited dynamic range resulting in signal change ratios of less than approximately 20:1 using most commercial mass spectrometers. Label-free approaches to relative quantification in proteomics such as spectral counting have gained popularity since no additional chemistries are needed. Here, we show a label-free method for relative quantification based on the TIC from peptide MS/MS spectra collected from data-dependent runs can be used effectively as a quantitative measure and expands the dynamic range over isotope labeling experiments allowing for abundance differences up to approximately 60:1 in a screen for proteins that bind to phosphotyrosine residues.     

Noninvasive evaluation of testicular function by measuring oxygen saturation of scrotal-testicular tissue

We conducted a single-center cross-sectional study to determine whether scrotal-testicular tissue oxygen saturation (S-T StO₂) measured by finger-mounted near-infrared spectroscopy is useful for the evaluation of testicular function. S-T StO₂ was significantly higher in patients with nonobstructive azoospermia (NOA, p< 0.05), and showed a positive correlation with luteinizing hormone levels (LH) even in participants without NOA (r = 0.34, p< 0.05), suggesting that elevated S-T StO₂ is associated with a reduction in testicular function.     

Protein abundance ratios for global studies of prokaryotes

The use of multidimensional capillary HPLC combined with MS/MS has allowed high qualitative and quantitative proteome coverage of prokaryotic organisms. The determination of protein abundance change between two or more conditions has matured to the point that false discovery rates can be very low and for smaller proteomes coverage is sufficiently high to explicitly consider false negative error. Selected aspects of using these methods for global protein abundance assessments are reviewed. These include instrumental issues that influence the reliability of abundance ratios; a comparison of sources of nonlinearity, errors, and data compression in proteomics and spotted cDNA arrays; strengths and weaknesses of spectral counting versus stable isotope metabolic labeling; and a survey of microbiological applications of global abundance analysis at the protein level. Proteomic results for two organisms that have been studied extensively using these methods are reviewed in greater detail. Spectral counting and metabolic labeling data are compared and the utility of proteomics for global gene regulation studies are discussed for the methanogenic Archaeon Methanococcus maripaludis. The oral pathogen Porphyromonas gingivalis is discussed as an example of an organism where a large percentage of the proteome differs in relative abundance between the intracellular and extracellular phenotype.     

Identification and functional analysis of spermatogenesis-associated gene modules in azoospermia by weighted gene coexpression network analysis

Nonobstructive azoospermia (NOA) or testicular failure is the most severe form of male infertility. A variety of conditions, both acquired and congenital, can cause azoospermia. However, in a large number of azoospermia patients who are classified as idiopathic cases, the etiology remains poorly understand mainly due to the lack of knowledge of all the genetic causes and molecular mechanisms responsible for spermatogenesis failure. Identification of the key gene modules and pathways-related spermatogenesis failure might help to reveal the mechanisms of idiopathic azoospermia. Therefore, the expression patterns of spermatogenesis-associated genes in NOA were analyzed by weighted gene coexpression network analysis (WGCNA) based on two public microarray data sets (GSE45885 and GSE45887), which included 51 samples and 32,321 genes. We identified a module (turquoise) that was significantly related to the Johnsen score of the testicular samples. In addition, the results of function and pathway enrichment analyses based on the online bioinformatics database Metascape revealed that genes in the turquoise module were mainly related to the process of spermatogenesis and spermatid development. To further identify spermatogenesis-associated genes, a microarray data set (GSE926) of murine testis at different developmental time points was analyzed by WGCNA. The blue module in GSE926 was significantly related to the time of murine testis development. The overlap study and k-core analysis based on protein–protein interaction network revealed that spermatogenesis- and spermatid development–associated genes, including glyceraldehyde-3-phosphate dehydrogenase, ADAM metallopeptidase domain 2, transition protein 1, testis-specific serine kinase 2, transition protein 2, and germ cell-associated 1 (GSG1), were further identified in the selected modules. The expression profile of GSG1 in human testis was chosen for further study using immunochemistry staining. Taken together, these screened gene modules and pathways provided a more detailed genetic and molecular mechanism underlying spermatogenesis failure occurrence and holds promise as potential diagnosis biomarkers and therapeutic targets.     

Exploration du plasma seminal humain: Nouvelle approche par la resonance magnetique nucleaire du proton (RMN-1H)

Human seminal plasma glycerylphosphorylcholine (GPC), glycerylphosphorylethanolamine (GPE), citric acid (Cit) and lactic acid (Lac) were analzed by measuring the peak area of proton nuclear magnetic resonance (NMR) spectra in four groups of patients: 21 men with secretory azoospermia; 14 men with excretory azoospermia after vasectomy; seven patients presenting with very severe oligoasthenozoospermia; and 18 normozoospermic subjects. When comparing secretory and excretory azoospermia cases with normozoospermic men, seminal plasma GPC, Cit and Lac peak areas were lower in azoospermic cases compared to normozoospermic men (16.79, 8.18 and 2.28 versus 23.38, 10.58 and 4.3. arbitrary units respectively, P <0.01). The peak area ratios Cit/Lac and GPC/lac were significantly different between normozoospermic men and either secretory or excretory azoospermic groups (P<0.01). A significant difference was also found in the GPE/GPC peak intensity ratio between secretory and excretory azoospermia cases (P<0.001). These results demonstrate that quantitative markers such as GPC, GPE, Cit and Lac may be useful in the examination of human seminal plasma by proton NMR in infertility investigations.