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Microarray analysis in sperm from fertile and infertile men without basic sperm analysis abnormalities reveals a significantly different transcriptome

2008; Elsevier BV; Volume: 91; Issue: 4 Linguagem: Inglês

10.1016/j.fertnstert.2008.01.078

1556-5653

Nicolás Garrido, José Antonio Martínez-Conejero, Juan C. Jáuregui, J.A. Horcajadas, Carlos Simón, J. Remohı́, Marcos Meseguer,

Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities

Sperm analysis following World Health Organization guidelines is unable to explain the molecular causes of male infertility when basic sperm parameters are within a normal range and women do not present gynecologic pathology. Consequently, there is a need for accurate diagnostic tools in this area, and microarray technology emerges as promising. We present, for the first time, preliminary results of a comparison of sperm mRNA expression profiles between fertile and infertile men with normal semen parameters, discovering profound discrepancies between groups, with potential diagnostic and therapeutic possibilities. Sperm analysis following World Health Organization guidelines is unable to explain the molecular causes of male infertility when basic sperm parameters are within a normal range and women do not present gynecologic pathology. Consequently, there is a need for accurate diagnostic tools in this area, and microarray technology emerges as promising. We present, for the first time, preliminary results of a comparison of sperm mRNA expression profiles between fertile and infertile men with normal semen parameters, discovering profound discrepancies between groups, with potential diagnostic and therapeutic possibilities. Sperm analysis based on sperm count and motility has been used for the diagnosis of male fertility for several decades. It is an easy, inexpensive, and useful tool to determine the fertile status of a male. But a significant number of men with normal sperm features as determined by the basic sperm analysis remain unable to reach a full-term pregnancy (1Garrido N. Meseguer M. Alvarez J. Simon C. Pellicer A. Remohi J. Relationship among standard semen parameters, glutathione peroxidase/glutathione reductase activity, and mRNA expression and reduced glutathione content in ejaculated spermatozoa from fertile and infertile men.Fertil Steril. 2004; 82: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This fact clearly indicates the need to develop new male infertility tests to accurately diagnose the sperm samples from these individuals.Recent investigations about sperm mRNA contents have described the relevance of the sperm mRNA stock in fertilization and early embryo development in several species. (2Krawetz S.A. Paternal contribution: new insights and future challenges.Nat Rev Genet. 2005; 6: 633-642Crossref PubMed Scopus (300) Google Scholar) Sperm is a quiescent cell from the translational point of view, but several functional mRNAs are delivered into the oocyte after fertilization which were synthesized in an earlier phase of the spermatogenesis process and conserved throughout the process (3Kramer J.A. Krawetz S.A. RNA in spermatozoa: implications for the alternative haploid genome.Mol Hum Reprod. 1997; 3: 473-478Crossref PubMed Scopus (83) Google Scholar, 4Wykes S.M. Visscher D.W. Krawetz S.A. Haploid transcripts persist in mature human spermatozoa.Mol Hum Reprod. 1997; 3: 15-19Crossref PubMed Scopus (111) Google Scholar). The fertile male transcriptome (stock of mRNAs within the sperm of a male able to have progeny) has been described (5Ostermeier G.C. Dix D.J. Miller D. Khatri P. Krawetz S.A. Spermatozoal RNA profiles of normal fertile men.Lancet. 2002; 360: 772-777Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 6Zhao Y.X. Li Q.L. Wang Z.X. Wang Y.F. Wang L.Y. Qiao Z.D. Characterization of the mRNA profile in ejaculated spermatozoa from healthy fertile men.Zhonghua Nan Ke Xue. 2006; 12: 900-903PubMed Google Scholar), confirming the presence of thousands of sequence tags with different expression levels. Moreover, several investigations have demonstrated a differential expression of some key mRNAs in infertile men compared with fertile men (1Garrido N. Meseguer M. Alvarez J. Simon C. Pellicer A. Remohi J. Relationship among standard semen parameters, glutathione peroxidase/glutathione reductase activity, and mRNA expression and reduced glutathione content in ejaculated spermatozoa from fertile and infertile men.Fertil Steril. 2004; 82: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Meseguer M. de los Santos M.J. Simon C. Pellicer A. Remohi J. Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.Fertil Steril. 2006; 86: 1376-1385Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Actually, there is no information available about either the characteristics of the stock sperm mRNAs on infertile men or the differences between fertile and infertile men, although several authors have hypothesized that sperm microarray analysis will be the future in the male infertility diagnosis (2Krawetz S.A. Paternal contribution: new insights and future challenges.Nat Rev Genet. 2005; 6: 633-642Crossref PubMed Scopus (300) Google Scholar, 8Krawetz S.A. Kramer J.A. McCarrey J.R. Reprogramming the male gamete genome: a window to successful gene therapy.Gene. 1999; 234: 1-9Crossref PubMed Scopus (16) Google Scholar, 9Miller D. Ostermeier G.C. Spermatozoal RNA: why is it there and what does it do?.Gynecol Obstet Fertil. 2006; 34: 840-846Crossref PubMed Scopus (56) Google Scholar, 10Miller D. Ostermeier G.C. Krawetz S.A. The controversy, potential and roles of spermatozoal RNA.Trends Mol Med. 2005; 11: 156-163Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 11Ostermeier G.C. Goodrich R.J. Diamond M.P. Dix D.J. Krawetz S.A. Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility.Fertil Steril. 2005; 83: 1687-1694Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar).Microarray technology can yield information about thousands of mRNAs' expression in a single experiment, enabling the analysis and comparison of complete sperm expression profiles (SEP). Bioinformatics can help in the organization of the results by following logical processes of gene expression grouping and statistically analyzing the findings.Our aim with the present work was to compare the SEP in spermatozoa obtained from men with idiopathic infertility with those from sperm donors of proven fertility by using microarray technology followed by a functional analysis, to determine the genes, sequences, and biologic processes involved in the sperm physiology that are different in infertile versus fertile men.This project was approved by the Institutional Review Board on the use of human subjects in research at the Instituto Valenciano de Infertilidad.All men involved in this work were identified from our database to meet the inclusion criteria and asked to participate.After obtaining written consent from each man, ten sperm samples were obtained from strictly selected infertile men (five samples, one per man) from couples attending our infertility clinic after 1 year of unprotected regular intercourses (mean infertility length was 1.5 years), presenting normal sperm count and motility (World Health Organization criteria) parameters, where no infertility cause was observed in their partners' (ages <35 years) routine work-ups (12Ballesteros A. Idrobo J. Landeras J. Cano F. Sagastegui C. Pellicer A. et al.Estudio de la pareja estéril.in: Remohí J. Pellicer A. Simon C. Navarro J. Reproducción humana. 2nd ed. McGraw-Hill, Madrid, Spain2002: 51Google Scholar), and sperm donors (five samples, one per donor) of proven fertility, demonstrated by their declaration of having their own children and having had healthy newborns in our sperm donation program.Those men with fever episodes, genital tract inflammation, varicocele, recurrent infections, exposure to toxins, or other similar situations leading to confounding results were not accepted in the study. These data were obtained by a directed questionnaire.Sperm samples were obtained in our facilities, and directly transported to the andrology laboratory. After liquefaction, a basic sperm analysis was done as described elsewhere (7Meseguer M. de los Santos M.J. Simon C. Pellicer A. Remohi J. Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.Fertil Steril. 2006; 86: 1376-1385Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). No differences were found in any of the sperm parameters between groups, i.e., sperm concentration, motility, and morphology. Also, mean age was similar between groups.The total number of sperm collected for the experiments was standardized to 50 million to provide enough spermatozoa for all tests, concentrating by centrifugation for 10 min at 400 g and eliminating the supernatant. Then sperm samples were immediately frozen by direct immersion in liquid nitrogen until the total numbers of samples programmed for this study were obtained and experiments were performed (less than 1 week).Sperm mRNA was extracted using Trizol protocol (TelTest, Friendswood, TX), suspended in DEPC-treated water, and frozen at −80°C until the microarray experiments were performed in duplicate. The total amount of RNA was quantified by spectrophotometry on a BioRad (Durviz, Valencia, Spain) spectrophotometer.Equal amounts of RNAs from the same groups were pooled before the analysis, as recommended for short series (13Peng X. Wood C.L. Blalock E.M. Chen K.C. Landfield P.W. Stromberg A.J. Statistical implications of pooling RNA samples for microarray experiments.BMC Bioinformatics. 2003; 4: 26Crossref PubMed Scopus (255) Google Scholar, 14Kendziorski C.M. Zhang Y. Lan H. Attie A.D. The efficiency of pooling mRNA in microarray experiments.Biostatistics. 2003; 4: 465-477Crossref PubMed Scopus (195) Google Scholar). CodeLink Expression Analysis System was used according to the manufacturer's instructions. Human Whole Genome Bioarray contains probes for more than 55,000 gene targets. Comparisons between the two groups were performed in duplicate. Spot intensities were normalized and analyzed using CodeLink Expression Analysis v4.1 software (15Horcajadas J.A. Sharkey A.M. Catalano R.D. Sherwin J.R. Dominguez F. Burgos L.A. et al.Effect of an intrauterine device on the gene expression profile of the endometrium.J Clin Endocrinol Metab. 2006; 91: 3199-3207Crossref PubMed Scopus (60) Google Scholar).No ejaculate showed significant levels of leukocytes. To confirm this, specific amplification of the leukocyte-specific antigen CD45 was carried out (7Meseguer M. de los Santos M.J. Simon C. Pellicer A. Remohi J. Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.Fertil Steril. 2006; 86: 1376-1385Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Quantitative (fluorescent) polymerase chain reaction confirmation of the expression of TRY1 (trypsin X3), GGT1 (gamma-glutamyltransferase 1) transcript variant 3, and CAB39L (calcium-binding protein 39–like) was performed successfully. As shown in Figure 1A, the data validation by means of this technique was similar to the data provided by the microarrays.The results were analyzed in terms of single-gene comparative expression between fertile and infertile men as well as by using bioinformatic tools helping to interpret them as a function of their biologic implications. To this end, the GEPAS (Gene Expression Profile Analysis Suite, available at http://www.gepas.org) platform was used, including different possibilities of gene clustering and selection (16Montaner D. Tarraga J. Huerta-Cepas J. Burguet J. Vaquerizas J.M. Conde L. et al.Next station in microarray data analysis: GEPAS.Nucleic Acids Res. 2006; 34: W486-W491Crossref PubMed Scopus (97) Google Scholar). This analysis was performed in blind: the team in charge of the data management did not know the samples' origins, tissue from which mRNA was obtained, or the aim of the experiment.The results are summarized in Figure 1B. Hundreds of gene sequences (targets) were found to be differentially expressed (DE) at least two times, but only the most DE genes in the sperm donors compared with the study group were listed. When we considered just those genes DE ten times or more, our preliminary results confirmed that there are few genes that are overexpressed (n = 3), whereas all others are underexpressed (n = 136) in infertile men. The results suggest that failure to impregnate a woman with normal sperm production in her partner may be due to a lack of factors involved in correct sperm function.Among all of the gene sequences found to be DE, we noted the presence of several ribosomal proteins and factors involved in spermatogenesis. Interestingly, these spermatogenesis factors are not related to sperm production in terms of number of ejaculated spermatozoa, given that all of the samples obtained for this analysis were within the normal range of sperm count and similar in both groups.When we use bioinformatic tools to analyze genes grouped by their functions, several clusters exhibit a significantly different proportion over- or underexpressed in infertile men versus fertile sperm donors. Depending on the grouping depth, different biochemical pathways or processes are implicated. Interestingly, when over- or underexpressed genes in infertile males were grouped by their function, those involving spermatozoa differentiation were present in all comparisons. Spermatid development, gametogenesis, spermatid differentiation, and male gamete generation processes present between 80% and 100% of their genes with a statistically significant overexpression in donors compared with infertile men showing normal sperm count. This clearly suggests that infertility markers may not be related to sperm production in terms of sperm count but are probably related to sperm function.To our knowledge, this is the first time that a significant difference in the spermatozoa transcriptomes has been obtained in a comparison between infertile and fertile men with similar sperm counts. These differences imply several molecules in a broad spectrum of biochemical and physiologic pathways, at different points, thus confirming the complexity of the events involved in sperm function.When the sequences analyzed are organized into functionally similar clusters, several biochemical or functional pathways are seen to be altered, but mainly those involved in male gamete formation or maturation. This is unequivocal evidence of the existence of several molecular infertility factors in sperm independent from the testes' capability for sperm production, given that all sperm samples were similar in sperm density, motility, and percentage of normal forms.Nevertheless, we must acknowledge the limitations of pooling samples for microarray analysis, and the results must be confirmed in single individuals before a diagnostic test is offered.Further clinical investigations and different experimental designs are on the way to determine the sperm expression profile analysis by microarrays ability to diagnose male infertility and characterize the most relevant male infertility factors to find therapeutic options. Sperm analysis based on sperm count and motility has been used for the diagnosis of male fertility for several decades. It is an easy, inexpensive, and useful tool to determine the fertile status of a male. But a significant number of men with normal sperm features as determined by the basic sperm analysis remain unable to reach a full-term pregnancy (1Garrido N. Meseguer M. Alvarez J. Simon C. Pellicer A. Remohi J. Relationship among standard semen parameters, glutathione peroxidase/glutathione reductase activity, and mRNA expression and reduced glutathione content in ejaculated spermatozoa from fertile and infertile men.Fertil Steril. 2004; 82: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). This fact clearly indicates the need to develop new male infertility tests to accurately diagnose the sperm samples from these individuals. Recent investigations about sperm mRNA contents have described the relevance of the sperm mRNA stock in fertilization and early embryo development in several species. (2Krawetz S.A. Paternal contribution: new insights and future challenges.Nat Rev Genet. 2005; 6: 633-642Crossref PubMed Scopus (300) Google Scholar) Sperm is a quiescent cell from the translational point of view, but several functional mRNAs are delivered into the oocyte after fertilization which were synthesized in an earlier phase of the spermatogenesis process and conserved throughout the process (3Kramer J.A. Krawetz S.A. RNA in spermatozoa: implications for the alternative haploid genome.Mol Hum Reprod. 1997; 3: 473-478Crossref PubMed Scopus (83) Google Scholar, 4Wykes S.M. Visscher D.W. Krawetz S.A. Haploid transcripts persist in mature human spermatozoa.Mol Hum Reprod. 1997; 3: 15-19Crossref PubMed Scopus (111) Google Scholar). The fertile male transcriptome (stock of mRNAs within the sperm of a male able to have progeny) has been described (5Ostermeier G.C. Dix D.J. Miller D. Khatri P. Krawetz S.A. Spermatozoal RNA profiles of normal fertile men.Lancet. 2002; 360: 772-777Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar, 6Zhao Y.X. Li Q.L. Wang Z.X. Wang Y.F. Wang L.Y. Qiao Z.D. Characterization of the mRNA profile in ejaculated spermatozoa from healthy fertile men.Zhonghua Nan Ke Xue. 2006; 12: 900-903PubMed Google Scholar), confirming the presence of thousands of sequence tags with different expression levels. Moreover, several investigations have demonstrated a differential expression of some key mRNAs in infertile men compared with fertile men (1Garrido N. Meseguer M. Alvarez J. Simon C. Pellicer A. Remohi J. Relationship among standard semen parameters, glutathione peroxidase/glutathione reductase activity, and mRNA expression and reduced glutathione content in ejaculated spermatozoa from fertile and infertile men.Fertil Steril. 2004; 82: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 7Meseguer M. de los Santos M.J. Simon C. Pellicer A. Remohi J. Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.Fertil Steril. 2006; 86: 1376-1385Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Actually, there is no information available about either the characteristics of the stock sperm mRNAs on infertile men or the differences between fertile and infertile men, although several authors have hypothesized that sperm microarray analysis will be the future in the male infertility diagnosis (2Krawetz S.A. Paternal contribution: new insights and future challenges.Nat Rev Genet. 2005; 6: 633-642Crossref PubMed Scopus (300) Google Scholar, 8Krawetz S.A. Kramer J.A. McCarrey J.R. Reprogramming the male gamete genome: a window to successful gene therapy.Gene. 1999; 234: 1-9Crossref PubMed Scopus (16) Google Scholar, 9Miller D. Ostermeier G.C. Spermatozoal RNA: why is it there and what does it do?.Gynecol Obstet Fertil. 2006; 34: 840-846Crossref PubMed Scopus (56) Google Scholar, 10Miller D. Ostermeier G.C. Krawetz S.A. The controversy, potential and roles of spermatozoal RNA.Trends Mol Med. 2005; 11: 156-163Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 11Ostermeier G.C. Goodrich R.J. Diamond M.P. Dix D.J. Krawetz S.A. Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility.Fertil Steril. 2005; 83: 1687-1694Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Microarray technology can yield information about thousands of mRNAs' expression in a single experiment, enabling the analysis and comparison of complete sperm expression profiles (SEP). Bioinformatics can help in the organization of the results by following logical processes of gene expression grouping and statistically analyzing the findings. Our aim with the present work was to compare the SEP in spermatozoa obtained from men with idiopathic infertility with those from sperm donors of proven fertility by using microarray technology followed by a functional analysis, to determine the genes, sequences, and biologic processes involved in the sperm physiology that are different in infertile versus fertile men. This project was approved by the Institutional Review Board on the use of human subjects in research at the Instituto Valenciano de Infertilidad. All men involved in this work were identified from our database to meet the inclusion criteria and asked to participate. After obtaining written consent from each man, ten sperm samples were obtained from strictly selected infertile men (five samples, one per man) from couples attending our infertility clinic after 1 year of unprotected regular intercourses (mean infertility length was 1.5 years), presenting normal sperm count and motility (World Health Organization criteria) parameters, where no infertility cause was observed in their partners' (ages <35 years) routine work-ups (12Ballesteros A. Idrobo J. Landeras J. Cano F. Sagastegui C. Pellicer A. et al.Estudio de la pareja estéril.in: Remohí J. Pellicer A. Simon C. Navarro J. Reproducción humana. 2nd ed. McGraw-Hill, Madrid, Spain2002: 51Google Scholar), and sperm donors (five samples, one per donor) of proven fertility, demonstrated by their declaration of having their own children and having had healthy newborns in our sperm donation program. Those men with fever episodes, genital tract inflammation, varicocele, recurrent infections, exposure to toxins, or other similar situations leading to confounding results were not accepted in the study. These data were obtained by a directed questionnaire. Sperm samples were obtained in our facilities, and directly transported to the andrology laboratory. After liquefaction, a basic sperm analysis was done as described elsewhere (7Meseguer M. de los Santos M.J. Simon C. Pellicer A. Remohi J. Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.Fertil Steril. 2006; 86: 1376-1385Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). No differences were found in any of the sperm parameters between groups, i.e., sperm concentration, motility, and morphology. Also, mean age was similar between groups. The total number of sperm collected for the experiments was standardized to 50 million to provide enough spermatozoa for all tests, concentrating by centrifugation for 10 min at 400 g and eliminating the supernatant. Then sperm samples were immediately frozen by direct immersion in liquid nitrogen until the total numbers of samples programmed for this study were obtained and experiments were performed (less than 1 week). Sperm mRNA was extracted using Trizol protocol (TelTest, Friendswood, TX), suspended in DEPC-treated water, and frozen at −80°C until the microarray experiments were performed in duplicate. The total amount of RNA was quantified by spectrophotometry on a BioRad (Durviz, Valencia, Spain) spectrophotometer. Equal amounts of RNAs from the same groups were pooled before the analysis, as recommended for short series (13Peng X. Wood C.L. Blalock E.M. Chen K.C. Landfield P.W. Stromberg A.J. Statistical implications of pooling RNA samples for microarray experiments.BMC Bioinformatics. 2003; 4: 26Crossref PubMed Scopus (255) Google Scholar, 14Kendziorski C.M. Zhang Y. Lan H. Attie A.D. The efficiency of pooling mRNA in microarray experiments.Biostatistics. 2003; 4: 465-477Crossref PubMed Scopus (195) Google Scholar). CodeLink Expression Analysis System was used according to the manufacturer's instructions. Human Whole Genome Bioarray contains probes for more than 55,000 gene targets. Comparisons between the two groups were performed in duplicate. Spot intensities were normalized and analyzed using CodeLink Expression Analysis v4.1 software (15Horcajadas J.A. Sharkey A.M. Catalano R.D. Sherwin J.R. Dominguez F. Burgos L.A. et al.Effect of an intrauterine device on the gene expression profile of the endometrium.J Clin Endocrinol Metab. 2006; 91: 3199-3207Crossref PubMed Scopus (60) Google Scholar). No ejaculate showed significant levels of leukocytes. To confirm this, specific amplification of the leukocyte-specific antigen CD45 was carried out (7Meseguer M. de los Santos M.J. Simon C. Pellicer A. Remohi J. Garrido N. Effect of sperm glutathione peroxidases 1 and 4 on embryo asymmetry and blastocyst quality in oocyte donation cycles.Fertil Steril. 2006; 86: 1376-1385Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Quantitative (fluorescent) polymerase chain reaction confirmation of the expression of TRY1 (trypsin X3), GGT1 (gamma-glutamyltransferase 1) transcript variant 3, and CAB39L (calcium-binding protein 39–like) was performed successfully. As shown in Figure 1A, the data validation by means of this technique was similar to the data provided by the microarrays. The results were analyzed in terms of single-gene comparative expression between fertile and infertile men as well as by using bioinformatic tools helping to interpret them as a function of their biologic implications. To this end, the GEPAS (Gene Expression Profile Analysis Suite, available at http://www.gepas.org) platform was used, including different possibilities of gene clustering and selection (16Montaner D. Tarraga J. Huerta-Cepas J. Burguet J. Vaquerizas J.M. Conde L. et al.Next station in microarray data analysis: GEPAS.Nucleic Acids Res. 2006; 34: W486-W491Crossref PubMed Scopus (97) Google Scholar). This analysis was performed in blind: the team in charge of the data management did not know the samples' origins, tissue from which mRNA was obtained, or the aim of the experiment. The results are summarized in Figure 1B. Hundreds of gene sequences (targets) were found to be differentially expressed (DE) at least two times, but only the most DE genes in the sperm donors compared with the study group were listed. When we considered just those genes DE ten times or more, our preliminary results confirmed that there are few genes that are overexpressed (n = 3), whereas all others are underexpressed (n = 136) in infertile men. The results suggest that failure to impregnate a woman with normal sperm production in her partner may be due to a lack of factors involved in correct sperm function. Among all of the gene sequences found to be DE, we noted the presence of several ribosomal proteins and factors involved in spermatogenesis. Interestingly, these spermatogenesis factors are not related to sperm production in terms of number of ejaculated spermatozoa, given that all of the samples obtained for this analysis were within the normal range of sperm count and similar in both groups. When we use bioinformatic tools to analyze genes grouped by their functions, several clusters exhibit a significantly different proportion over- or underexpressed in infertile men versus fertile sperm donors. Depending on the grouping depth, different biochemical pathways or processes are implicated. Interestingly, when over- or underexpressed genes in infertile males were grouped by their function, those involving spermatozoa differentiation were present in all comparisons. Spermatid development, gametogenesis, spermatid differentiation, and male gamete generation processes present between 80% and 100% of their genes with a statistically significant overexpression in donors compared with infertile men showing normal sperm count. This clearly suggests that infertility markers may not be related to sperm production in terms of sperm count but are probably related to sperm function. To our knowledge, this is the first time that a significant difference in the spermatozoa transcriptomes has been obtained in a comparison between infertile and fertile men with similar sperm counts. These differences imply several molecules in a broad spectrum of biochemical and physiologic pathways, at different points, thus confirming the complexity of the events involved in sperm function. When the sequences analyzed are organized into functionally similar clusters, several biochemical or functional pathways are seen to be altered, but mainly those involved in male gamete formation or maturation. This is unequivocal evidence of the existence of several molecular infertility factors in sperm independent from the testes' capability for sperm production, given that all sperm samples were similar in sperm density, motility, and percentage of normal forms. Nevertheless, we must acknowledge the limitations of pooling samples for microarray analysis, and the results must be confirmed in single individuals before a diagnostic test is offered. Further clinical investigations and different experimental designs are on the way to determine the sperm expression profile analysis by microarrays ability to diagnose male infertility and characterize the most relevant male infertility factors to find therapeutic options.