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.     

Altered microRNA expression in patients with non-obstructive azoospermia

Background  

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are indicated to play essential roles in spermatogenesis. However, little is known about the expression patterns or function of miRNAs in human testes involved in infertility.     

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.     

Quantitative proteomic analysis by iTRAQ for identification of candidate biomarkers in plasma from acute respiratory distress syndrome patients

Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critical patients. Proteomic analysis of plasma from individuals with ARDS could elucidate new biomarkers for diagnosis and pathophysiology and identify potential ARDS treatment targets. In this study, we recruited 26 patients (15 controls, 11 ARDS). The ARDS group was subdivided into two groups depending on the type of injury: (1) direct lung injury (AD) and (2) indirect lung injury (AI). Using iTRAQ (isobaric tags for relative and absolute quantitation) analysis, we identified 2429 peptides representing 132 plasma proteins. Among these, 16 were differentially expressed in ARDS patients, including 11 overlapping proteins between the AI and AD group and 5 AI-specific proteins. Protein annotation revealed that lipid transport and complement activation were significantly enriched in the biological process category, and lipid transporter, transporter, and serine-type peptidase activities were significantly enriched in the molecular function category. IPA (Ingenuity Pathway Analysis) signaling pathways revealed that the overlapping proteins were involved in a variety of signaling pathways, including those underlying acute phase response; liver X receptor/retinoid X receptor (LXR/RXR) and farnesoid X (FXR)/RXR activation; clathrin-mediated endocytosis; atherosclerosis; interleukin (IL)-12; complement system; and cytokine, nitric oxide, and reactive oxygen species production in macrophages. We present the first proteomic analysis of ARDS plasma using the iTRAQ approach. Our data provide new biomarker candidates and shed light on potential pathological mechanisms underlying ARDS.     

Hormone levels in serum and seminal plasma of men with different types of azoospermia

Hormone concentrations in the serum and seminal plasma of 15 normozoospermic, 17 excretory azoospermic and 14 secretory azoospermic men were measured. The results indicate that: (a) serum FSH and LH levels are markedly elevated in secretory azoospermia, as compared with excretory azoospermia and normozoospermia; (b) serum 17 alpha-hydroxyprogesterone levels are somewhat raised in secretory azoospermia as compared with excretory azoospermia and normozoospermia; (c) serum testosterone levels are lower in both types of azoospermia with respect to normozoospermia; (d) in secretory azoospermia the oestradiol serum levels are relatively high and dihydrotestosterone serum levels relatively low, whereas the serum levels of these hormones in excretory azoospermia are similar to those in normozoospermic men; (e) in the seminal plasma of azoospermic patients the levels of prolactin, progesterone, testosterone, dihydrotestosterone and oestradiol were depressed, but only dihydrotestosterone levels could be of value in differentiating types of azoospermia because they are lower in secretory azoospermia. We suggest that the measurement of FSH, LH, 17 alpha-hydroxyprogesterone, dihydrotestosterone and oestradiol in serum and dihydrotestosterone in seminal plasma may be used in the differential diagnosis between secretory and excretory azoospermia when invasive tests are unavailable.     

Follicle-stimulating hormone may predict sperm retrieval rate and guide surgical approach in patients with non-obstructive azoospermia

Testicular sperm aspiration- (TESA) or micro-dissection testicular sperm extraction- (MD-TESE) combined intracytoplasmic sperm injection (ICSI) was the only option for non-obstructive azoospermia (NOA) patients to have a biological offspring and they had different success rates in sperm retrieval. Our study aimed to find predictor(s) for predicting the sperm retrieval rate (SRR) in NOAs and guide clinicians in choosing different surgical approaches, TESA or MD-TESE for NOAs. 294 NOAs who had undergone TESA or MD-TESE were divided into TESA group and MD-TESE group. Depending on sperm retrieval, each group was divided into two subgroups: successful subgroups and failure subgroups. They respectively were 24 cases and 131 cases, 53 cases and 86 cases. Clinical data, including body mass index (BMI), testicular volume, and serum hormone levels, were analyzed in a retrospective manner. The results showed that follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels and SRR were lower in TESA group as compared to these in MD-TESE group, while testicular volume was higher (P < 0.05). The surgical approach of sperm retrieval significantly affected the SRR (P < 0.05). In TESA subgroups, testicular volume, FSH and LH differed significantly (P < 0.05). In MD-TESE subgroups, the level of FSH and LH differed significantly between both groups (P < 0.05). Using logistics regression, we found a negative correlation (β=-0.083) between FSH and the SRR in TESA group but a positive correlation (β = 0.064) in MD-TESE group (P < 0.05). In conclusion, serum FSH level can predict the SRR of NOAs and guide the clinicians while selecting the suitable surgery approach for NOAs.     

HLA-DPB1*04:01 allele is associated with non-obstructive azoospermia in Japanese patients

Azoospermia is defined by absence of sperm in the semen and can either be caused by obstruction of the seminal tract (obstructive azoospermia) or by defects in spermatogenesis (non-obstructive azoospermia, NOA). Previous studies reported that specific alleles and single nucleotide polymorphisms (SNPs) in the human leukocyte antigen (HLA) region were associated with NOA in East Asians. We attempt to expand upon previous findings by genotyping more HLA genes and to replicate SNP associations by focusing on Japanese NOA patients. HLA typing of six genes (HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1) was done on 355 NOA patients using SSO-Luminex assay while genotyping of two previously reported SNPs (rs498422 and rs3129878) was done on 443 patients and 544 fertile males using TaqMan assay. Association between the HLA alleles and SNP with NOA was assessed with Chi squared and logistic regression tests. We found that HLA-DPB1*04:01 [corrected p value, P _c 7.13 × 10^?6; odds ratio (OR) 2.52], DRB1*13:02 (P _c 4.93 × 10^?4, OR 1.97), DQB1*06:04 (P _c 8.94 × 10^?4, OR 1.91) and rs3129878 (p value 3.98 × 10^?4; OR 1.32) showed significant association with NOA, however, these loci are in linkage disequilibrium with each other. The conditional logistic regression tests showed that DPB1*04:01 is independently associated with NOA, confirming the involvement of the HLA region in the etiology of NOA in Japanese patients.     

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Screening for microdeletions in human Y chromosome--AZF candidate genes and male infertility

About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.     

High-resolution mass spectrometry proteomics for the identification of candidate plasma protein biomarkers for chronic obstructive pulmonary disease

Although cigarette smoking is recognized as the most important cause of chronic obstructive pulmonary disease (COPD), the pathophysiological mechanisms underlying the lung function decline are not well understood. Using off-line strong cation exchange fractionation with RP-LC-ESI-MS/MS and robust database searching, 1758 tryptic peptides were identified in plasma samples from cigarette smokers. Using two statistical approaches, 30 peptides were identified to be associated with the annualized rate of lung function decline over 5 years among smokers with COPD characterized as having rapid (n?=?18) or slow (n?=?18) decline and 18 smokers without COPD. The identified peptides belong to proteins that are involved in the complement or coagulation systems or have antiprotease or metabolic functions. This research demonstrates the utility of proteomic profiling to improve the understanding of molecular mechanisms involved in cigarette smoking-related COPD by identifying plasma proteins that correlate with decline in lung function.     

非梗阻性无精子症患者睾丸体积、生殖激素水平与睾丸穿刺取精结果的相关性研究

目的:探讨非梗阻性无精子症患者睾丸体积、生殖激素水平与睾丸穿刺取精术(TESA)结果的相关性,以及可用于预测TESA结果的睾丸体积、生殖激素水平的切点值,从而为非梗阻性无精子症患者进一步诊疗提供重要资料.方法:121例研究对象均为非梗阻性无精子症患者(NOA),测定其睾丸体积和生殖激素水平,并根据TESA结果分为无精子组和有精子组.结果:无精子组和有精子组的左侧睾丸体积(ml)、右侧睾丸体积(ml)、泌乳素(PRL,ng/ml)、卵泡刺激素(FSH,mIU/ml)、黄体生成素(LH,mIU/ml)、雌二醇(E2,pmol/L)、血清总睾酮(TT,nmol/L)水平分别为7.07±1.06和11.75±1.38、7.37±1.37和11.70±1.98、12.43±11.69和9.60±4.55、15.77±10.84和8.01±7.43、6.12±2.92和8.11±20.11、119.36±43.52和141.12±48.33、11.43±4.05和12.46 ±4.60.无精子组血清FSH和PRL水平平均值高于有精子组,并且有显著的统计学差异.虽然无精子组的睾丸体积平均数小于有精子组,但两组之间没有统计学差异.对于年龄、血清E2和Tr水平,两组之间也没有统计学差异.利用ROC曲线优选的睾丸体积切点值为9ml,此点其敏感性为93.8％/89.6％(左/右),特异性为100％/94.3％(左/右),睾丸体积ROC曲线的AUC为0.984/0.961(左/右),表明其诊断准确性较高;优选的血清FSH水平切点值为8.18 mIU/ml,此点其敏感性为71.2％,特异性为75.0％,FSH水平ROC曲线的AUC为0.743,表明其诊断准确性中等.结论:睾丸体积和FSH水平对于预测NOA患者TESA结果具有重要意义,并且睾丸体积诊断准确性明显优于FSH.     

Chromosomal evaluation in a group of Tunisian patients with non-obstructive azoospermia and severe oligozoospermia attending a Tunisian cytogenetic department

Male infertility is the cause in half of all childless partnerships. Numerous factors contribute to male infertility, including chromosomal aberrations and gene defects. Few data exist regarding the association of these chromosomal aberrations with male infertility in Arab and North African populations. We therefore aimed to evaluate the frequency of chromosomal aberrations in a sample of 476 infertile men with non-obstructive azoospermia (n = 328) or severe oligozoospermia (n = 148) referred for routine cytogenetic analysis to the department of cytogenetics of the Pasteur Institute of Tunis. The overall incidence of chromosomal abnormalities was about 10.9%. Out of the 52 patients with abnormal cytogenetic findings, sex chromosome abnormalities were observed in 42 (80.7%) including Klinefelter syndrome in 37 (71%). Structural chromosome abnormalities involving autosomes (19.2%) and sex chromosomes were detected in 11 infertile men. Abnormal findings were more prevalent in the azoospermia group (14.02%) than in the severe oligozoospermia group (4.05%). The high frequency of chromosomal alterations in our series highlights the need for efficient genetic testing in infertile men, as results may help to determine the prognosis, as well as the choice of an assisted reproduction technique. Moreover, a genetic investigation could minimize the risk of transmitting genetic abnormalities to future generations.     

Separation, characterization and identification of boar seminal plasma proteins

Methods used for the isolation, separation and characterization of boar seminal plasma proteins are discussed, as well as techniques applied to study their binding properties. Attention is paid to interactions of these proteins with different types of saccharides and glycoconjugates, with membrane phospholipids, and to interactions between proteins. Boar seminal plasma contains different types of proteins: spermadhesins of the AQN and AWN families; DQH and PSP proteins belong to the most abundant. Some of these proteins are bound to the sperm surface during ejaculation and thus protein-coating layers of sperm are formed. Sperms coated with proteins participate in different types of interactions occurring in the course of the reproduction process, e.g. formation of the oviductal sperm reservoir, sperm capacitation, oocyte recognition and sperm binding to the oocyte.     

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n?=?5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p?=?0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.     

Immunochemical identification and physico-chemical characteristics of specific proteins of seminal plasma

Two specific antigens of human spermatic plasma have been identified: thermostable alpha 1-globulin and alpha 2-microglobulin of fertility. The thermostable alpha 1-globulin presents a glycoprotein containing sialic acids with a molecular weight of 60000 +/- 7000. In addition to the sperm, it has been also identified in the saliva of men and women. Alpha 2-microglobulin of fertility is identical to placental alpha 2-microglobulin. This protein is specific for the reproductive system of men and women. The physicochemical properties of the test antigens are described.     

Cell-free and intracellular nucleic acids: new non-invasive biomarkers to explore male infertility

Male infertility is a devastating problem that affects many couples worldwide. However, the molecular mechanisms and causes of idiopathic male infertility remain unclear. Circulating cell-free nucleic acids have an important role in human physiology and emerging evidence suggests that they play a role in male infertility. This review summarizes recent results on cell-free and intracellular nucleic acids in male infertility and discusses their potential use as biomarkers of male infertility in the clinical practice.     

Proteomic identification of plasma biomarkers in uterine leiomyoma

Recent progresses in quantitative proteomics have offered opportunities to discover plasma proteins as biomarkers for tracking the progression and for understanding the molecular mechanisms of uterine leiomyomas. In the present study, plasma samples were analyzed by fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 20 proteins have been firmly identified representing 13 unique gene products. These proteins mainly functioned in transportation (such as apolipoprotein A-I) and coagulation (such as fibrinogen gamma chain). Additionally, our quantitative proteomic approach has identified numerous previous reported plasma markers of uterine leiomyomas such as alpha-1-antitrypsin. On the contrary, we have presented several putative uterine leiomyomas biomarkers including afamin, apolipoprotein A-I, carbonic anhydrase 1, fibrinogen beta chain, fibrinogen gamma chain, gelsolin, hemopexin, leucine-rich alpha-2-glycoprotein, serotransferrin and vitamin D-binding protein which have not been reported and may be associated with the progression and development of the disease. In summary, we report a comprehensive patient-based proteomic approach for the identification of potential plasma biomarkers for uterine leiomyomas. The potential of utilizing these markers for screening and treating uterine leiomyomas warrants further investigations.