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Stable-protein Pair Analysis as A Novel Strategy to Identify Proteomic Signatures: Application To Seminal Plasma From Infertile Patients*

  • Author Footnotes
    ** These authors contributed equally.
    Ferran Barrachina
    Footnotes
    ** These authors contributed equally.
    Affiliations
    From the ‡Molecular Biology of Reproduction and Development Research Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain and Biochemistry and Molecular Genetics Service, Hospital Clínic, Barcelona, Spain;
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  • Author Footnotes
    ** These authors contributed equally.
    Meritxell Jodar
    Footnotes
    ** These authors contributed equally.
    Affiliations
    From the ‡Molecular Biology of Reproduction and Development Research Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain and Biochemistry and Molecular Genetics Service, Hospital Clínic, Barcelona, Spain;
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  • David Delgado-Dueñas
    Affiliations
    From the ‡Molecular Biology of Reproduction and Development Research Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain and Biochemistry and Molecular Genetics Service, Hospital Clínic, Barcelona, Spain;
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  • Ada Soler-Ventura
    Affiliations
    From the ‡Molecular Biology of Reproduction and Development Research Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain and Biochemistry and Molecular Genetics Service, Hospital Clínic, Barcelona, Spain;
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  • Josep Maria Estanyol
    Affiliations
    Proteomics Unit, Scientific Technical Services, University of Barcelona, Barcelona, Spain;
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  • Carme Mallofré
    Affiliations
    Department of Pathology, University of Barcelona, Hospital Clínic, Barcelona, Spain;
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  • Josep Lluís Ballescà
    Affiliations
    Clinic Institute of Gynaecology, Obstetrics and Neonatology, Hospital Clínic, Barcelona, Spain
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  • Rafael Oliva
    Correspondence
    To whom correspondence should be addressed:Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Carrer Casanova 134, 08036 Barcelona, Spain. Tel.:+34 934021877;
    Affiliations
    From the ‡Molecular Biology of Reproduction and Development Research Group, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la Recerca Biomèdica, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain and Biochemistry and Molecular Genetics Service, Hospital Clínic, Barcelona, Spain;
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  • Author Footnotes
    * This work was supported by grants to R.O. from the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competividad; fondos FEDER ‘una manera de hacer Europa’ PI13/00699, PI16/00346), from Fundación Salud 2000 (SERONO 13-015), from EUGIN-UB (EUREP 2014), and from EU-FP7-PEOPLE-2011-ITN289880. F.B. is granted by Spanish Ministry of Education, Culture and Sports (Ministerio de Educación, Cultura y Deporte para la Formación de Profesorado Universitario, FPU15/02306). M.J. is granted by Government of Catalonia (Generalitat de Catalunya, pla estratègic de recerca i innovació en salut, PERIS 2016–2020, SLT002/16/00337).
    This article contains supplemental material.
    ** These authors contributed equally.
Open AccessPublished:December 05, 2018DOI:https://doi.org/10.1074/mcp.RA118.001248
      Our aim was to define seminal plasma proteome signatures of infertile patients categorized according to their seminal parameters using TMT-LC-MS/MS. To that extent, quantitative proteomic data was analyzed following two complementary strategies: (1) the conventional approach based on standard statistical analyses of relative protein quantification values; and (2) a novel strategy focused on establishing stable-protein pairs. By conventional analyses, the abundance of some seminal plasma proteins was found to be positively correlated with sperm concentration. However, this correlation was not found for all the peptides within a specific protein, bringing to light the high heterogeneity existing in the seminal plasma proteome because of both the proteolytic fragments and/or the post-translational modifications. This issue was overcome by conducting the novel stable-protein pairs analysis proposed herein. A total of 182 correlations comprising 24 different proteins were identified in the normozoospermic-control population, whereas this proportion was drastically reduced in infertile patients with altered seminal parameters (18 in patients with reduced sperm motility, 0 in patients with low sperm concentration and 3 in patients with no sperm in the ejaculate). These results suggest the existence of multiple etiologies causing the same alteration in seminal parameters. Additionally, the repetition of the stable-protein pair analysis in the control group by adding the data from a single patient at a time enabled to identify alterations in the stable-protein pairs profile of individual patients with altered seminal parameters. These results suggest potential underlying pathogenic mechanisms in individual infertile patients, and might open up a window to its application in the personalized diagnostic of male infertility.

      Graphical Abstract

      Infertility is a worldwide frequent problem that affects ∼15% of reproductive-aged couples. Around the 50% of the fertility problems is because of a male factor and, from those, the 40–60% of the infertile patients present some alterations in at least one of the seminal parameters assessed by a routine semen analysis (sperm concentration, motility and morphology) (
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      ). According to these seminal parameters, infertile patients are categorized in: (i) patients with low or absent sperm concentration (oligozoospermia or azoospermia, respectively), (ii) patients with defective sperm motility (asthenozoospermia) and/or (iii) patients with abnormal sperm morphology (teratozoospermia) (
      • World Health Organization
      ). The semen evaluation, together with a complete medical history and physical examination, will determine whether the initial assessment needs to be complemented with genetic and/or hormonal analyses, urinalysis or testicular biopsies. Unfortunately, the current available tools for the evaluation of male fertility are limited and insufficient, and the development of new methodologies to better discern the male factor etiology is required (
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      • Yamakawa K.
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      Comparative analysis of interindividual variations in the seminal plasma proteome of fertile men with identification of potential markers for azoospermia in infertile patients.
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      ).
      Therefore, the analysis of the seminal plasma proteome mimicking the infertility classification according to seminal parameters is warranted, because it might help to decipher potential pathogenic mechanisms resulting in these sperm alterations. However, it is important to consider a wide range of factors may alter both sperm and seminal plasma compositions, leading to a high heterogeneity within patients sharing the same phenotype. This is challenging for the conventional analysis of the quantitative proteomic data based on the search of differential proteins between groups of patients with similar characteristics. Therefore, the main aim of the present study was to define the seminal plasma proteome signatures of infertile patients categorized according to their seminal parameters (normozoospermia, NZ
      The abbreviations used are: NZ, normozoospermia; AS, asthenozoospermia; OZ, oligozoospermia; AZ, azoospermia; CASA, computer assisted semen analysis; WHO, World Health Organization; BCA, bicinchoninic acid; TMT, tandem mass tag; TEAB, triethyl ammonium bicarbonate; TCEP, tris (2-carboxyethyl) phosphine; IAA, iodoacetamide; RT, room temperature; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; MS/MS, tandem mass spectrometry; HCD, higher energy collision dissociation; FDR, false discovery rate; PSMs, peptide spectrum matches; ANOVA, one-way analysis of variance; HPA, Human Protein Atlas; TBST, TBS with 0.1% (v/v) Tween 20; PAS, periodic acid and schiff's reagent; SEMGs, semenogelins; PTMs, post-translational modifications.
      1The abbreviations used are: NZ, normozoospermia; AS, asthenozoospermia; OZ, oligozoospermia; AZ, azoospermia; CASA, computer assisted semen analysis; WHO, World Health Organization; BCA, bicinchoninic acid; TMT, tandem mass tag; TEAB, triethyl ammonium bicarbonate; TCEP, tris (2-carboxyethyl) phosphine; IAA, iodoacetamide; RT, room temperature; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; MS/MS, tandem mass spectrometry; HCD, higher energy collision dissociation; FDR, false discovery rate; PSMs, peptide spectrum matches; ANOVA, one-way analysis of variance; HPA, Human Protein Atlas; TBST, TBS with 0.1% (v/v) Tween 20; PAS, periodic acid and schiff's reagent; SEMGs, semenogelins; PTMs, post-translational modifications.
      ; asthenozoospermia, AS; oligozoospermia, OZ; azoospermia, AZ) by applying conventional and novel approaches for the analysis of quantitative proteomic data to try to better stratify the different subgroups of infertile patients. The results derived from this study suggest that the combination of conventional and novel analytical approaches may be useful toward the identification of pathogenic mechanisms of male infertility and, furthermore, to provide the bases for future studies to design new therapies to improve male fertility and move toward the application of an individual and personalized diagnostic of male infertility.

      DISCUSSION

      The heterogeneous composition of the seminal plasma together with the rapid changes that occur in its molecular composition after ejaculation, such as the proteolytic cascade associated to the coagulation-liquefaction process, introduce further complexity to seminal plasma proteomic studies (
      • Jodar M.
      • Soler-Ventura A.
      • Oliva R.
      Semen proteomics and male infertility.
      ,
      • Camargo M.
      • Intasqui P.
      • Bertolla R.P.
      Understanding the seminal plasma proteome and its role in male fertility.
      ). In the present study, a total of 349 proteins were identified in the 16 seminal plasma samples analyzed (supplemental Table S2), which are functionally related to metabolism, response to stress, proteolysis, immune system and energy production. Also, with less extent, these identified human seminal plasma proteins seemed to be involved in processes related to fertilization and embryogenesis (
      • Castillo J.
      • Jodar M.
      • Oliva R.
      The contribution of human sperm proteins to the development and epigenome of the preimplantation embryo.
      ). This apparently low number of identified seminal plasma proteins could be explained by the detection of the semenogelins I and II (SEMG1 and SEMG2) as the most abundant proteins of the seminal plasma. Specifically, around 40% of the PSMs identified in our proteomic study corresponded to SEMG1 and SEMG2, thus hindering the detection of low abundant proteins. This low number of protein identifications is also observed in other studies assessing the human seminal plasma proteome using MS methods and identification criteria comparable to ours (
      • Rolland A.D.
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      • Calvel P.
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      • Freour T.
      • Evrard B.
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      • Auger J.
      • Pineau C.
      Identification of genital tract markers in the human seminal plasma using an integrative genomics approach.
      ,
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      • Camargo M.
      • Antoniassi M.P.
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      • Carvalho V.M.
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      Association between the seminal plasma proteome and sperm functional traits.
      ,
      • Antoniassi M.P.
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      • Carvalho V.M.
      • Cardozo K.H.M.
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      Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.
      ,
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      Human spermatozoa quantitative proteomic signature classifies normo- and asthenozoospermia.
      ,
      • Herwig R.
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      • Bennett K.L.
      Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
      ,
      • Intasqui P.
      • Antoniassi M.P.
      • Camargo M.
      • Nichi M.
      • Carvalho V.M.
      • Cardozo K.H.M.
      • Zylbersztejn D.S.
      • Bertolla R.P.
      Differences in the seminal plasma proteome are associated with oxidative stress levels in men with normal semen parameters.
      ,
      • Sharma R.
      • Agarwal A.
      • Mohanty G.
      • Jesudasan R.
      • Gopalan B.
      • Willard B.
      • Yadav S.P.
      • Sabanegh E.
      Functional proteomic analysis of seminal plasma proteins in men with various semen parameters.
      ,
      • Sharma R.
      • Agarwal A.
      • Mohanty G.
      • Du Plessis S.S.
      • Gopalan B.
      • Willard B.
      • Yadav S.P.
      • Sabanegh E.
      Proteomic analysis of seminal fluid from men exhibiting oxidative stress.
      ,
      • Milardi D.
      • Grande G.
      • Vincenzoni F.
      • Messana I.
      • Pontecorvi A.
      • De Marinis L.
      • Castagnola M.
      • Marana R.
      Proteomic approach in the identification of fertility pattern in seminal plasma of fertile men.
      ,
      • Del Giudice P.T.
      • Belardin L.B.
      • Camargo M.
      • Zylbersztejn D.S.
      • Carvalho V.M.
      • Cardozo K.H.M.
      • Bertolla R.P.
      • Cedenho A.P.
      Determination of testicular function in adolescents with varicocoele - a proteomics approach.
      ). For this reason, future studies should consider the incorporation of strategies to deplete SEMGs prior the proteomic characterization of seminal plasma, as for example the use of HPLC columns containing antibodies against SEMGs (
      • Jodar M.
      • Soler-Ventura A.
      • Oliva R.
      Semen proteomics and male infertility.
      ,
      • Rolland A.D.
      • Lavigne R.
      • Dauly C.
      • Calvel P.
      • Kervarrec C.
      • Freour T.
      • Evrard B.
      • Rioux-Leclercq N.
      • Auger J.
      • Pineau C.
      Identification of genital tract markers in the human seminal plasma using an integrative genomics approach.
      ).

      Conventional Approach to Analyze Quantitative Proteomics Data

      Protein quantification of TMT-labeled peptides using conventional approaches is obtained from the average of relative ion abundance ratios for all peptides encompassing the same protein (
      • Amaral A.
      • Paiva C.
      • Attardo Parrinello C.
      • Estanyol J.M.
      • Ballescà J.L.
      • Ramalho-Santos J.
      • Oliva R.
      Identification of proteins involved in human sperm motility using high-throughput differential proteomics.
      ). The conventional statistical analyses conducted in this study showed: (i) The underexpression of the glycoprotein ANPEP in patients with altered sperm motility (Fig. 3), as previously reported by others (
      • Wang J.
      • Wang J.
      • Zhang H.-R.
      • Shi H.-J.
      • Ma D.
      • Zhao H.-X.
      • Lin B.
      • Li R.-S.
      Proteomic analysis of seminal plasma from asthenozoospermia patients reveals proteins that affect oxidative stress responses and semen quality.
      ); and (ii) a gradual decline of CRISP1, NPC2, and SPINT3 abundance in infertile patients, ranging from high to low sperm concentration (in decreasing order: NZ-AS, OZ, and AZ) (Table I, Fig. 3). Of note, this gradual declined abundance was also observed for the protein levels of SPINT3, NPC2, ECM1, and CRISP1, independently of sperm motility parameter (Table I). The low abundance of those proteins in seminal plasma from patients with low or absence of sperm cells could reflect either proteomic alterations in the accessory sex glands secretions, or the presence of low amounts of male germ cells remnants coming from apoptotic sperm or from sperm cytoplasmic droplets. This specific question may be elucidated by deciphering the potential tissue origin of these altered seminal plasma proteins. According to the HPA Database, the testicular or extra-testicular origin of NPC2 could not be assessed, because, although NPC2 is a major component of epididymal secretions, it is also expressed in testis (
      • Giacomini E.
      • Ura B.
      • Giolo E.
      • Luppi S.
      • Martinelli M.
      • Garcia R.C.
      • Ricci G.
      Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men.
      ) (Fig. 5). In contrast, ECM1, SPINT3, and CRISP1 are mainly expressed in the epididymis whereas they are not detected in testis (Fig. 5), suggesting that epididymal secretions could be regulated by the presence of sperm themselves in the epididymis. Interestingly, this potential cross-talk between the spermatozoa and epididymis has also been observed in rat and bovine species (
      • Reyes-Moreno C.
      • Laflamme J.
      • Frenette G.
      • Sirard M.A.
      • Sullivan R.
      Spermatozoa modulate epididymal cell proliferation and protein secretion in vitro.
      ,
      • Garrett J.E.
      • Garrett S.H.
      • Douglass J.
      A spermatozoa-associated factor regulates proenkephalin gene expression in the rat epididymis.
      ). Drabovich and colleagues, in contrast, detected decreased levels of SPINT3, NPC2, ECM1 and CRISP1 only in obstructive azoospermic patients but not in patients with non-obstructive azoospermia (
      • Drabovich A.P.
      • Saraon P.
      • Jarvi K.
      • Diamandis E.P.
      Seminal plasma as a diagnostic fluid for male reproductive system disorders.
      ). Other groups assessing potential seminal biomarkers to discern the different subtypes of azoospermia found other distinct differential proteins, such as LGALS3BP (
      • Freour T.
      • Com E.
      • Barriere P.
      • Bouchot O.
      • Jean M.
      • Masson D.
      • Pineau C.
      Comparative proteomic analysis coupled with conventional protein assay as a strategy to identify predictors of successful testicular sperm extraction in patients with non-obstructive azoospermia.
      ) and STAB2, CP135, GNRP, and PIP (
      • Yamakawa K.
      • Yoshida K.
      • Nishikawa H.
      • Kato T.
      • Iwamoto T.
      Comparative analysis of interindividual variations in the seminal plasma proteome of fertile men with identification of potential markers for azoospermia in infertile patients.
      ). Altogether, it indicates the presence of some differences between seminal plasma proteomic data from different studies. Of note, a similar lack of concordance has also been observed in quantitative proteomic data of sperm samples from the same type of patients. This is exemplified by the detection of only 17 proteins out the 179 reported as differentially expressed in the sperm cells in at least 2 of the 7 comparative proteomic studies conducted so far for the study of asthenozoospermia (
      • Jodar M.
      • Barrachina F.
      • Oliva R.
      The use of sperm proteomics in the assisted reproduction laboratory.
      ). Differences in sample collection, handling and storage, proteomic strategies, and the biological intra- and inter-individual variance may be important causes contributing to this lack of reproducibility between studies (
      • Camargo M.
      • Intasqui P.
      • Bertolla R.P.
      Understanding the seminal plasma proteome and its role in male fertility.
      ,
      • Gilany K.
      • Minai-Tehrani A.
      • Savadi-Shiraz E.
      • Rezadoost H.
      • Lakpour N.
      Exploring the human seminal plasma proteome: An unexplored gold mine of biomarker for male Infertility and male reproduction disorder.
      ,
      • Codina M.
      • Estanyol J.M.
      • Fidalgo M.J.
      • Ballescà J.L.
      • Oliva R.
      Advances in sperm proteomics: best-practise methodology and clinical potential.
      ). Moreover, the high and fast protease activity in seminal plasma after ejaculation could introduce even more heterogeneity to the results because of the presence of distinct proteolytic fragments, in addition to the protein PTMs not detected in standard proteomic procedures (
      • Laflamme B.A.
      • Wolfner M.F.
      Identification and function of proteolysis regulators in seminal fluid.
      ). In order to evaluate this putative heterogeneity of seminal plasma proteome we also assessed the correlation of peptides encompassing the proteins SPINT3, NPC2, ECM1, CRISP1 and IGHG2 with sperm concentration (Table II). Of note, NPC2 is the only protein showing most of its corresponding peptides correlated with the sperm concentration (Table II). In agreement with our findings, Giacomini and colleagues, by using nano LC-electrospray ionization-MS/MS, found decreased levels of NPC2 in seminal plasma from idiopathic oligoasthenozoospermic patients (
      • Giacomini E.
      • Ura B.
      • Giolo E.
      • Luppi S.
      • Martinelli M.
      • Garcia R.C.
      • Ricci G.
      Comparative analysis of the seminal plasma proteomes of oligoasthenozoospermic and normozoospermic men.
      ). As a summary, there is a need of novel approaches to analyze the results from quantitative shotgun proteomic studies, in order to overcome the limitations produced by the heterogeneity of seminal plasma in the proteolytic fragments, as well as by its conjunction with other variations such as the protein PTMs not detectable by standard proteomic procedures.

      Novel Approaches to Analyze Quantitative Proteomics and Identify Patient-specific Alterations

      A set of seminal plasma strictly co-regulated proteins was established by following a new approach based on the correlation of the intensities of all unique peptides comprising one specific protein with all the unique peptides quantified for all the other detected proteins in the sample (Fig. 2). This strategy, called stable-protein pairs analysis herein, may contribute to reduce the heterogeneity observed in the seminal plasma proteomic data, because only those proteins displaying a consistent pattern in a specific phenotype are obtained. A total of 182 stable-protein pairs comprising 24 proteins were detected in patients with normal semen parameters (Table III), reflecting the strict co-regulation of these proteins in NZ individuals. These stable-protein pairs include gene products involved in: (i) Sperm function, such as CKB that plays a critical role in the demand of energy necessary for sperm motility (
      • Hallak J.
      • Sharma R.K.
      • Pasqualotto F.F.
      • Ranganathan P.
      • Thomas A.J.
      • Agarwal A.
      Creatine kinase as an indicator of sperm quality and maturity in men with oligospermia.
      ), CST3, PAEP, and CLU that regulate sperm capacitation (
      • Chiu P.C.N.
      • Chung M.K.
      • Tsang H.Y.
      • Koistinen R.
      • Koistinen H.
      • Seppala M.
      • Lee K.F.
      • Yeung W.S.B.
      Glycodelin-S in human seminal plasma reduces cholesterol efflux and inhibits capacitation of spermatozoa.
      ,
      • Lee R.K.-K.
      • Tseng H.-C.
      • Hwu Y.-M.
      • Fan C.-C.
      • Lin M.-H.
      • Yu J.-J.
      • Yeh L.-Y.
      • Li S.-H.
      Expression of cystatin C in the female reproductive tract and its effect on human sperm capacitation.
      ,
      • Saewu A.
      • Kadunganattil S.
      • Raghupathy R.
      • Kongmanas K.
      • Astudillo P.D.
      • Hermo L.
      • Tanphaichitr N.
      Clusterin in the mouse epididymis: possible roles in sperm maturation and capacitation.
      ) and CRISP1 and NPC2, which are necessary for sperm-oocyte binding and fertilization (
      • Maldera J.A.
      • Muñoz M.W.
      • Chirinos M.
      • Busso D.
      • Raffo F.G.E.
      • Battistone M.A.
      • Blaquier J.A.
      • Larrea F.
      • Cuasnicu P.S.
      Human fertilization: Epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3.
      ,
      • Busso D.
      • Oñate-Alvarado M.J.
      • Balboa E.
      • Castro J.
      • Lizama C.
      • Morales G.
      • Vargas S.
      • Härtel S.
      • Moreno R.D.
      • Zanlungo S.
      Spermatozoa from mice deficient in Niemann-Pick disease type C2 (NPC2) protein have defective cholesterol content and reduced in vitro fertilising ability.
      ); (ii) the regulation of semen clotting-liquefaction processes, such as ACCP, KLK3, and MME, which are directly involved in the proteolysis of the SEMGs or other proteins (
      • Brillard-Bourdet M.
      • Réhault S.
      • Juliano L.
      • Ferrer M.
      • Moreau T.
      • Gauthier F.
      Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates.
      ,
      • Lilja H.
      A kallikrein-like serine protease in prostatic fluid cleaves the predominant seminal vesicle protein.
      ), or as WFDC2, ALB, and TGM4, which regulate other components required for clotting-liquefaction process such as proteases, zinc ions and polyamines (
      • Chhikara N.
      • Saraswat M.
      • Tomar A.K.
      • Dey S.
      • Singh S.
      • Yadav S.
      Human Epididymis Protein-4 (HE-4): A novel cross-class protease inhibitor.
      ,
      • Frenette G.
      • Tremblay R.R.
      • Dubé J.Y.
      Zinc binding to major human seminal coagulum proteins.
      ,
      • Romijn J.C.
      Polyamines and transglutaminase actions: Polyamine und Transglutaminase-Wirkungen.
      ); (iii) immunology, including proteins that could participate in the leukocyte-mediated immune response, such as IGKC, IGHG2, ANPEP, LGALSBP3, ECM1, and B2M (
      • Le T.M.
      • Le Q.V.C.
      • Truong D.M.
      • Lee H.J.
      • Choi M.K.
      • Cho H.
      • Chung H.J.
      • Kim J.H.
      • Do J.T.
      • Song H.
      • Park C.
      β2-microglobulin gene duplication in cetartiodactyla remains intact only in pigs and possibly confers selective advantage to the species.
      ,
      • Hannan N.J.
      • Salamonsen L.A.
      CX3CL1 and CCL14 regulate extracellular matrix and adhesion molecules in the trophoblast: potential roles in human embryo implantation.
      ) or in antimicrobial activity, as for example CPE and QSOX1 (
      • Kumar S.
      • Tomar A.K.
      • Singh S.
      • Gill K.
      • Dey S.
      • Singh S.
      • Yadav S.
      Heparin binding carboxypeptidase E protein exhibits antibacterial activity in human semen.
      ,
      • Michael C.O.
      • Kistler W.S.
      Properties of a Flavoprotein Sulfhydryl Oxidase from Rat Seminal Vesicle Secretion.
      ); and finally, (iv) other functions such as lipid metabolism (AZGP1, TF and IDH1) and matrix assembly (VWA1). In contrast to the high number of stable-protein pairs identified in NZ individuals, the stable correlations drastically decreased in the different groups of patients with altered semen parameters (Table III). Indeed, just 18, 3 and 0 stable-protein pairs were detected in AS, AZ, and OZ patients, respectively. The low number of stable-protein pairs observed in AZ patients was not surprising, because this group contains patients indistinctly diagnosed with obstructive and non-obstructive azoospermia, which probably results in different semen protein profiles as previously reported by others (
      • Drabovich A.P.
      • Saraon P.
      • Jarvi K.
      • Diamandis E.P.
      Seminal plasma as a diagnostic fluid for male reproductive system disorders.
      ). However, a high heterogeneity was also observed in the proteomic profile of seminal plasma from AS and OZ patients. The few stable-protein pairs detected in infertile patients with altered seminal parameters indicate that alterations in different proteins may result or be a consequence of the same altered phenotype. Although some hints for the presence of protein-pair correlations was already reported in the sperm proteome using ancillary proteomic methods (
      • de Mateo S.
      • Martínez-Heredia J.
      • Estanyol J.M.
      • Domínguez-Fandos D.
      • Domíguez-Fandos D.
      • Vidal-Taboada J.M.
      • Ballescà J.L.
      • Oliva R.
      Marked correlations in protein expression identified by proteomic analysis of human spermatozoa.
      ), the present study clearly demonstrates the potential of this approach using proteomics data at peptide level (Table III, Fig. 2).
      To assess which protein pairs might be associated to the alterations of the seminal parameters in individual samples, we repeated the analysis of the stable-protein pairs in the NZ population but adding data from one patient with altered parameters at a time. First, we observed that the patient AS2 had a very similar seminal plasma protein signature to the NZ population, because it maintained more than 75% of the NZ stable-protein pairs (Table IV). Of note, one of the proteins that loses more correlations in patient AS2 is the TF, a protein involved in lipid metabolism and sperm protection against oxidative stress (
      • Wakabayashi H.
      • Matsumoto H.
      • Hashimoto K.
      • Teraguchi S.
      • Takase M.
      • Hayasawa H.
      Inhibition of iron / ascorbate-induced lipid peroxidation by an N-terminal peptide of bovine lactoferrin and its acylated derivatives.
      ) (Table IV). It is interesting to note that TF loses more than 75% of the correlations in another asthenozoospermic patient (AS1) (Table IV), suggestive that oxidative stress may be related to the impairment of the sperm motility in both patients AS1 and AS2.
      We also observed that proteins involved in the induction of proteolysis of SEMGs and other regulators of the semen clotting-liquefaction process lose > 75% of the correlations in OZ and AZ patients (KLK3 in OZ2 and AZ2; ACPP in OZ2, AZ1, AZ2, and AZ3; and MME in AZ1; Table IV), suggesting that the sperm also contribute with regulators for the semen clot proteolysis (
      • Jodar M.
      • Sendler E.
      • Krawetz S.A.
      The protein and transcript profiles of human semen.
      ). Whereas some of the protein correlations were lost in the majority of the individual patients, as observed for CLU, PAEP, and WFDC2, suggesting that the correlations established for these proteins in NZ samples are weak, some seminal plasma proteins seem to be altered only in one unique sample such as TGM4 in patient AS4, IDH1 in OZ1, CST3 and LGALS3BP in OZ4, CPE in Z1, B2M in Z3 and IGKC in Z4, although these alterations could not clearly explain the observed phenotype.

      Quantitative Proteomics as A Tool to Provide Insights in Seminal Plasma Proteome Signatures Of Infertile Patients

      So far, proteomics biomarker discovery experiments have shown a relatively low concordance among different studies. In fact, we demonstrated that the results from relative quantitative proteomics are different if the analyses are performed at protein or at peptide level. These differences could explain the apparent lack of reproducibility of some of the findings, a fact that should be considered also when using antibody-based techniques recognizing specific peptides (such as Western blotting or ELISA) or targeted proteomic approaches to selected specific peptides. Here, we propose introducing a novel complementary approach for the analysis of quantitative proteomic data, which is based on the establishment of stable-protein pairs. This strategy has been previously applied to the study of RNA-seq data (
      • Lalancette C.
      • Platts A.E.
      • Johnson G.D.
      • Emery B.R.
      • Carrell D.T.
      • Krawetz S.A.
      Identification of human sperm transcripts as candidate markers of male fertility.
      ,
      • Mao S.
      • Goodrich R.J.
      • Hauser R.
      • Schrader S.M.
      • Chen Z.
      • Krawetz S.A.
      Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling.
      ), but to the best of our knowledge this is the first time it is applied to the proteomic data. The use of this new approach in our seminal plasma proteome dataset has allowed determining highly stable seminal plasma proteome signatures of men presenting normal seminal parameters (NZ). In contrast, we demonstrated that the current classification of infertile patients based on altered semen parameters resulted in a high heterogeneous seminal plasma proteomic profile, thereby suggesting that the current male infertility stratification performed in fertility clinics is not enough to obtain a good diagnosis. Moreover, the stable-protein pairs approach has the potential to pinpoint proteins potentially related to pathogenic mechanisms in individual samples when this strategy is applied for the evaluation of the NZ stable-protein pair alterations in individual infertile patients. Although our study has limitations, the novel data analysis approach proposed herein could be valuable toward the identification of altered proteins and pathogenic mechanisms of male infertility and might open a window to the personalized diagnosis of male infertility in future studies.

      Data Availability

      The MS proteomics data generated have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Vizcaíno J.A.
      • Csordas A.
      • del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.-W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ) partner repository with the dataset identifier PXD010579. The data files can be downloaded at: https://www.ebi.ac.uk/pride/archive/.

      Acknowledgments

      We thank Dr Judit Castillo for critical revision of the manuscript. We also recognize Raquel Ferreti and Alicia Diez for their assistance in the routine seminograms and sample collection.

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