BACKGROUND
microdeletions of the y chromosome long arm  represent the main molecular determinants of male infertility and account for about 10% of cases of non obstructive azoospermia or severe hypospermatogenesis  <cit> .
yq microdeletions involve three azoospermia factors  loci, azfa, azfb and azfc  <cit>  and remove many genes likely involved in male germ cell development and maintenance  <cit> .
the most frequent deletion of the y chromosome  spans  <dig>  mb and eliminates  <dig> genes and transcription units of the azfc region.
among these, the main candidate for spermatogenesis failure is the deleted in azoospermia  gene, a testis specific gene present in four copies within azfc and encoding for an rna binding protein  <cit> .
the azfc deletion has been associated with wide range of phenotypes ranging from the complete absence of germ cells in the testes  to a relevant reduction of germ cells still including mature sperm , to a maturation spermatogenesis arrest.
despite the large number of studies investigating the prevalence and the molecular basis of these rearrangements, the testicular gene expression of patients carrying yq deletions is still largely unknown and so are the molecular mechanisms leading to spermatogenesis disruption.
in recent years, expression profiling of human testis has been widely used for the identification of genes that are involved in different key steps of testis development and function  <cit> .
however, at the moment, not a single study has investigated the testicular transcriptome of patients who carry yq microdeletions.
in this study, we analyzed testis expression profiles of  <dig> infertile patients with different testicular phenotypes, including men carrying azfc deletions as well as subjects with idiopathic infertility.
RESULTS
in order to discriminate the genes specifically involved in the different pathological phenotypes, we performed a hierarchical clustering of the data set originated from  <dig> experiments  .
all the azfc samples clustered together independently from their testis phenotype  .
the same cluster also contained  <dig> patients without yq deletions .
the remaining three patients without yq deletions showed a different gene expression profile which was not clustered together with azfc deleted patients.
all the testes with normal spermatogenesis clustered appropriately into their group.
the cluster containing azfc-deleted samples was characterized by the presence of  <dig> transcripts showing at least a  <dig> -fold change in expression as compared to testis with normal spermatogenesis .
semiquantitative rt-pcr, carried out on selected genes , confirmed microarray results .
ingenuity pathway analysis  was carried out to investigate the main functions played by genes that were found to be downregulated  and showed that "cellular development" and "reproductive system development and function" were the main functions correlated with these genes .
these functional categories contained several genes playing a role in spermatogenesis, fertilization, and determination of the testicular mass, some of which are involved in human and murine male infertility   <cit>    <cit>  .
when we analyzed the upregulated genes , ipa showed that "cellular growth and proliferation" and "cell death" were the functions to be involved, whereas "reproductive system development and function" was less represented .
network analysis of the dowregulated genes that are linked with spermatogenesis showed an interesting network centered on the ybx <dig>  gene, a member of the y-box gene family that is selectively expressed in male and female germ cells   <cit> .
we also found an additional network that is centered on the arnt <dig> gene and contains other genes critically involved in the spermatogenetic process  .
a specific analysis of the daz gene signal in the samples represented in the cluster showed that daz was not expressed in all the four patients who were not carrying the azfc deletion .
these results were confirmed by a rt-pcr analysis carried out as previously described  <cit> .
the same kind of analysis was carried out also for cdy <dig> and vcy <dig> genes, mapped in multiple copies within azfc locus close to the daz gene cluster  <cit> .
this analysis evidenced cdy <dig> expression in patients with idiopathic infertility but not in those with azfc deletion.
on the other hand, an overexpression of vcy <dig> gene was detected in  <dig> out of  <dig> patients with azfc deletion.
a blast search using the vcy <dig> oligonucleotide sequence spotted on the array discovered a 60% homology with the sequence mapped in yp, corresponding to a portion of the vcy <dig> exon  <dig>  in addition to the expected 100% homology with vcy <dig> coding region.
discussion
in the present study, we carried out a microarray analysis to investigate testicular gene expression profiles in patients affected by different forms of infertility.
due to the absence or reduced number of germ cells in these patients, a downregulation of genes with prominent or exclusive germ cell expression was expected in all samples, whereas genes expressed mainly in somatic cells were expected to show increased expression.
however, the main purpose of this study was to analyze the inter-individual differences among infertile patients, in order to identify specific expression profiles for each different conditions.
using this approach we found a similar gene expression profile in all the samples originated from individuals carrying the azfc deletion as well as in four patients with idiopathic infertility.
all these case showed a common gene cluster that contained  <dig> transcripts .
since all hs patients had the same johnsen score, the difference in the expression profiles of samples present in the cluster and patients outside the cluster cannot be due to a different number of germ cells in their testis, but likely indicates a real difference in the transcription levels of specific genes.
among the downregulated transcripts, we detected several genes involved in spermatogenesis, fertilization, and male infertility.
to clarify a potential pathogenic link between the downregulation of these transcripts and spermatogenesis failure, we carried out an ipa network analysis that revealed two interesting networks.
the first network is centered on the ybx <dig>  gene, a member of the y-box gene family that is specifically expressed in male and female germ cells.
ybx <dig> marks specific mrnas for cytoplasmic storage, stabilization, and suppression of translation  <cit> .
rna storage is a crucial step during spermatogenesis.
rna synthesis terminates during mid-spermiogenesis and stabilization and storage of mrnas are key for the encoding of proteins needed during the terminal phases of spermatogenesis  <cit> .
quite surprisingly, network analysis of the cluster of patients carrying azfc deletions showed that ybx <dig> was not present in the cluster, being not significantly downregulated in the samples within the cluster as compared to the other samples not present in the cluster.
however, several genes whose activity is regulated by ybx <dig> were found to be downexpressed in the network.
among these, we found odf <dig>  tnp <dig> and prm <dig>  all genes that play a key role in the postmeiotic phase of the spermatogenesis.
odf <dig> encodes for proteins located on the outside of the axoneme in the mid piece and principal piece of the mammalian sperm tail, thereby maintaining the passive elastic structures and elastic recoil of the sperm tail.
tnp <dig> is a spermatid-specific product of the haploid genome which replaces histones and is itself replaced in the mature sperm by the protamines.
prm <dig> encodes for proteins playing a key role in dna packaging during sperm differentiation.
the combined downregulation of all these genes suggests reduced storage of these transcripts during spermatogenesis, via a ybx2-independent process as the ybx <dig> transcript was not found within the cluster.
a possible intriguing hypothesis is that the loss of function of the daz gene, the most important gene disrupted by azfc deletions  <cit> , leads to a pathogenic defective mrna storage.
substantiating this idea, rna-binding proteins encoded by daz are known to be required for the translational regulation of gene expression that is essential for gametogenesis  <cit> .
furthermore, it has been showed that dazl, the murine homologous of daz, plays a role in the transport of specific mrnas via a dynein motor complex, and that the dazl-bound mrnas is stored at specific sites to be available and used for later developmental processes  <cit> .
therefore we hypothesize that daz disruption may represent a possible cause of the reduced storage of important testis transcripts encoded in the first steps of the spermatogenesis.
interestingly, the second network we analyzed is centered on the arnt <dig> gene and contains several genes involved in the spermatogenesis process .
crisp <dig> is a testis-enriched protein localized to the sperm acrosome and tail.
tssk <dig> encodes a testis-specific serine/threonine kinase and is bound by the human and murine forms of dazl.
egr <dig> is a member of the egr family of zinc-finger transcription factors, and regulates genes critically involved in the early stages of meiosis.
thus, it is intriguing to conceive that also arnt <dig> may play an important role in the pathogenesis of infertility in azfc deleted patients.
the microarray analysis of samples from patients with azfc deletion also showed the presence of  <dig> upregulated transcripts.
it is interesting to note that only a few of these genes have been previously linked to infertility.
a2m, a gene that is encoding for a protease inhibitor, is up-regulated in the testis of the abp transgenic mouse that shows an impairment of spermatogenesis  <cit> .
increased expression of hif1a, an hypoxia-inducible transcription factor, is present in the internal spermatic vein of patients with varicocele  <cit> .
finally, hmgb <dig>  encoding for a high mobility group protein, works as a pro-inflammatory and antibacterial factor in human testis  <cit> .
these data suggest that the overexpression of genes related to cell growth and proliferation as well as to apoptosis in the testis of patients with azfc deletions may represent an unspecific mechanism of reaction to testis damage, rather than the cause of defective spermatogenesis.
an unexpected finding of the present study is that the four patients without azfc deletion are included in the same cluster containing samples from azfc deleted patients.
in order to verify if the presence of an intact azfc region in peripheral blood actually correlates with a normal testicular daz expression, we focused our attention to the presence/absence of the specific signals for this gene on the array in all cases contained in the cluster.
interestingly, no daz signal was detected in any of the four patients without azfc deletion.
lack of daz expression was expected in the samples from patients affected by idiopathic scos, due to the absence of germ cells, but it was surprisingly found in the  <dig> patients affected by idiopathic hs.
the lack of daz expression in the testis of infertile patients who are not carrying the azfc deletion in their peripheral blood has been previously reported and interpreted as the result of somatic azfc rearrangements producing a mosaic in which yq deletion is present only in testicular cells  <cit> .
our results strongly confirm this finding, corroborating the hypothesis that a portion of patients classified as affected by idiopathic infertility actually lack daz gene expression in their testis, thus showing a pathogenic mechanism similar to the one responsible of infertility in azfc deleted patients.
in order to investigate also other azfc genes, we analyzed the specific spots for cdy <dig> and vcy <dig>  in order to verify their expression in patients with and without azfc deletion.
as expected, cdy <dig> was expressed in infertile patients without azfc deletion independently fromtheir collocation with or without the cluster, but not in patients with azfc deletion.
on the other hand, expression levels showed by vcy <dig> gene were inconsistent with those of daz and cdy <dig> genes, being this gene overexpressed in  <dig> out of the  <dig> patients with azfc deletion.
in order todisclose the cause of this unexpected result, we performed a blast search using the vcy <dig> oligonucleotide sequence spotted on the array, discovering a 60% homology with a sequence mapped within yp, in addition of the expected 100% homology with vcy <dig> gene.
this yp sequence matched with the vcy <dig> exon  <dig>  target of the vcy <dig> probe present in the array.
on the other hand, daz and cdy <dig> probes showed homology only with their target genes.
this suggests that the vcy <dig> oligonucleotide on the array is not specific enough to consider the expression levels of this gene in the context of the present study.
CONCLUSIONS
in summary, our study provides the following novel information: i) all patients with azfc deletions show a similar testis gene expression profile, independently from their scos or hs phenotype, suggesting that all azfc deletions produce a similar transcriptional pattern and that variability in phenotypes is related to environmental or other genetic mechanisms.
ii) one half of the patients with idiopathic infertility cluster together with patients with azfc deletions, suggesting a common pathogenic mechanism.
we hypothesize that a crucial link that can explain this phenomenon is the lack of expression of the daz gene in the testes these cases.
a potential alternative explanation may be related to the presence of an azfc deletion in mosaic and/or the loss of function of azfc genes in absence of yq deletion.
if our finding will be confirmed on larger series of patients, daz gene dysfunctions may be proved to account for a larger portion of cases of infertility than one could expect by simply analyzing the prevalence of yq deletions among these subjects; iii) we found that several genes that are mainly related to the postmeiotic phase of the spermatogenesis and have been previously reported as involved in male infertility are dowregulated in patients with azfc deletions.
given the crucial role played by rna storage during spermatogenesis, the loss of function of daz can translate to a defective mrna storage in testis with great pathogenic implications.
the identification of the molecular mechanisms underlying the spermatogenesis failure in cases characterized by daz loss of function will provide useful information for the disclosure of key molecules which could represent in the future the target for personalized drug therapy.

 microdeletions of the y chromosome long arm represent the main molecular determinants of male infertility and account for about 10% of cases of non obstructive azoospermia or severe hypospermatogenesis. yq microdeletions involve three azoospermia factors loci, azfa, azfb and azfc and remove many genes likely involved in male germ cell development and maintenance. the most frequent deletion of the y chromosome spans mb and eliminates genes and transcription units of the azfc region. among these, the main candidate for spermatogenesis failure is the deleted in azoospermia gene, a testis specific gene present in four copies within azfc and encoding for an rna binding protein. the azfc deletion has been associated with wide range of phenotypes ranging from the complete absence of germ cells in the testes to a relevant reduction of germ cells still including mature sperm, to a maturation spermatogenesis arrest. despite the large number of studies investigating the prevalence and the molecular basis of these rearrangements, the testicular gene expression of patients carrying yq deletions is still largely unknown and so are the molecular mechanisms leading to spermatogenesis disruption. in recent years, expression profiling of human testis has been widely used for the identification of genes that are involved in different key steps of testis development and function. however, at the moment, not a single study has investigated the testicular transcriptome of patients who carry yq microdeletions. in this study, we analyzed testis expression profiles of infertile patients with different testicular phenotypes, including men carrying azfc deletions as well as subjects with idiopathic infertility.  in order to discriminate the genes specifically involved in the different pathological phenotypes, we performed a hierarchical clustering of the data set originated from experiments. all the azfc samples clustered together independently from their testis phenotype. the same cluster also contained patients without yq deletions. the remaining three patients without yq deletions showed a different gene expression profile which was not clustered together with azfc deleted patients. all the testes with normal spermatogenesis clustered appropriately into their group. the cluster containing azfc-deleted samples was characterized by the presence of transcripts showing at least a -fold change in expression as compared to testis with normal spermatogenesis. semiquantitative rt-pcr, carried out on selected genes, confirmed microarray results. ingenuity pathway analysis was carried out to investigate the main functions played by genes that were found to be downregulated and showed that "cellular development" and "reproductive system development and function" were the main functions correlated with these genes. these functional categories contained several genes playing a role in spermatogenesis, fertilization, and determination of the testicular mass, some of which are involved in human and murine male infertility. when we analyzed the upregulated genes, ipa showed that "cellular growth and proliferation" and "cell death" were the functions to be involved, whereas "reproductive system development and function" was less represented. network analysis of the dowregulated genes that are linked with spermatogenesis showed an interesting network centered on the ybx gene, a member of the y-box gene family that is selectively expressed in male and female germ cells. we also found an additional network that is centered on the arnt gene and contains other genes critically involved in the spermatogenetic process. a specific analysis of the daz gene signal in the samples represented in the cluster showed that daz was not expressed in all the four patients who were not carrying the azfc deletion. these results were confirmed by a rt-pcr analysis carried out as previously described. the same kind of analysis was carried out also for cdy and vcy genes, mapped in multiple copies within azfc locus close to the daz gene cluster. this analysis evidenced cdy expression in patients with idiopathic infertility but not in those with azfc deletion. on the other hand, an overexpression of vcy gene was detected in out of patients with azfc deletion. a blast search using the vcy oligonucleotide sequence spotted on the array discovered a 60% homology with the sequence mapped in yp, corresponding to a portion of the vcy exon in addition to the expected 100% homology with vcy coding region. discussion in the present study, we carried out a microarray analysis to investigate testicular gene expression profiles in patients affected by different forms of infertility. due to the absence or reduced number of germ cells in these patients, a downregulation of genes with prominent or exclusive germ cell expression was expected in all samples, whereas genes expressed mainly in somatic cells were expected to show increased expression. however, the main purpose of this study was to analyze the inter-individual differences among infertile patients, in order to identify specific expression profiles for each different conditions. using this approach we found a similar gene expression profile in all the samples originated from individuals carrying the azfc deletion as well as in four patients with idiopathic infertility. all these case showed a common gene cluster that contained transcripts. since all hs patients had the same johnsen score, the difference in the expression profiles of samples present in the cluster and patients outside the cluster cannot be due to a different number of germ cells in their testis, but likely indicates a real difference in the transcription levels of specific genes. among the downregulated transcripts, we detected several genes involved in spermatogenesis, fertilization, and male infertility. to clarify a potential pathogenic link between the downregulation of these transcripts and spermatogenesis failure, we carried out an ipa network analysis that revealed two interesting networks. the first network is centered on the ybx gene, a member of the y-box gene family that is specifically expressed in male and female germ cells. ybx marks specific mrnas for cytoplasmic storage, stabilization, and suppression of translation. rna storage is a crucial step during spermatogenesis. rna synthesis terminates during mid-spermiogenesis and stabilization and storage of mrnas are key for the encoding of proteins needed during the terminal phases of spermatogenesis. quite surprisingly, network analysis of the cluster of patients carrying azfc deletions showed that ybx was not present in the cluster, being not significantly downregulated in the samples within the cluster as compared to the other samples not present in the cluster. however, several genes whose activity is regulated by ybx were found to be downexpressed in the network. among these, we found odf tnp and prm all genes that play a key role in the postmeiotic phase of the spermatogenesis. odf encodes for proteins located on the outside of the axoneme in the mid piece and principal piece of the mammalian sperm tail, thereby maintaining the passive elastic structures and elastic recoil of the sperm tail. tnp is a spermatid-specific product of the haploid genome which replaces histones and is itself replaced in the mature sperm by the protamines. prm encodes for proteins playing a key role in dna packaging during sperm differentiation. the combined downregulation of all these genes suggests reduced storage of these transcripts during spermatogenesis, via a ybx2-independent process as the ybx transcript was not found within the cluster. a possible intriguing hypothesis is that the loss of function of the daz gene, the most important gene disrupted by azfc deletions, leads to a pathogenic defective mrna storage. substantiating this idea, rna-binding proteins encoded by daz are known to be required for the translational regulation of gene expression that is essential for gametogenesis. furthermore, it has been showed that dazl, the murine homologous of daz, plays a role in the transport of specific mrnas via a dynein motor complex, and that the dazl-bound mrnas is stored at specific sites to be available and used for later developmental processes. therefore we hypothesize that daz disruption may represent a possible cause of the reduced storage of important testis transcripts encoded in the first steps of the spermatogenesis. interestingly, the second network we analyzed is centered on the arnt gene and contains several genes involved in the spermatogenesis process. crisp is a testis-enriched protein localized to the sperm acrosome and tail. tssk encodes a testis-specific serine/threonine kinase and is bound by the human and murine forms of dazl. egr is a member of the egr family of zinc-finger transcription factors, and regulates genes critically involved in the early stages of meiosis. thus, it is intriguing to conceive that also arnt may play an important role in the pathogenesis of infertility in azfc deleted patients. the microarray analysis of samples from patients with azfc deletion also showed the presence of upregulated transcripts. it is interesting to note that only a few of these genes have been previously linked to infertility. a2m, a gene that is encoding for a protease inhibitor, is up-regulated in the testis of the abp transgenic mouse that shows an impairment of spermatogenesis. increased expression of hif1a, an hypoxia-inducible transcription factor, is present in the internal spermatic vein of patients with varicocele. finally, hmgb encoding for a high mobility group protein, works as a pro-inflammatory and antibacterial factor in human testis. these data suggest that the overexpression of genes related to cell growth and proliferation as well as to apoptosis in the testis of patients with azfc deletions may represent an unspecific mechanism of reaction to testis damage, rather than the cause of defective spermatogenesis. an unexpected finding of the present study is that the four patients without azfc deletion are included in the same cluster containing samples from azfc deleted patients. in order to verify if the presence of an intact azfc region in peripheral blood actually correlates with a normal testicular daz expression, we focused our attention to the presence/absence of the specific signals for this gene on the array in all cases contained in the cluster. interestingly, no daz signal was detected in any of the four patients without azfc deletion. lack of daz expression was expected in the samples from patients affected by idiopathic scos, due to the absence of germ cells, but it was surprisingly found in the patients affected by idiopathic hs. the lack of daz expression in the testis of infertile patients who are not carrying the azfc deletion in their peripheral blood has been previously reported and interpreted as the result of somatic azfc rearrangements producing a mosaic in which yq deletion is present only in testicular cells. our results strongly confirm this finding, corroborating the hypothesis that a portion of patients classified as affected by idiopathic infertility actually lack daz gene expression in their testis, thus showing a pathogenic mechanism similar to the one responsible of infertility in azfc deleted patients. in order to investigate also other azfc genes, we analyzed the specific spots for cdy and vcy in order to verify their expression in patients with and without azfc deletion. as expected, cdy was expressed in infertile patients without azfc deletion independently fromtheir collocation with or without the cluster, but not in patients with azfc deletion. on the other hand, expression levels showed by vcy gene were inconsistent with those of daz and cdy genes, being this gene overexpressed in out of the patients with azfc deletion. in order todisclose the cause of this unexpected result, we performed a blast search using the vcy oligonucleotide sequence spotted on the array, discovering a 60% homology with a sequence mapped within yp, in addition of the expected 100% homology with vcy gene. this yp sequence matched with the vcy exon target of the vcy probe present in the array. on the other hand, daz and cdy probes showed homology only with their target genes. this suggests that the vcy oligonucleotide on the array is not specific enough to consider the expression levels of this gene in the context of the present study.  in summary, our study provides the following novel information: i) all patients with azfc deletions show a similar testis gene expression profile, independently from their scos or hs phenotype, suggesting that all azfc deletions produce a similar transcriptional pattern and that variability in phenotypes is related to environmental or other genetic mechanisms. ii) one half of the patients with idiopathic infertility cluster together with patients with azfc deletions, suggesting a common pathogenic mechanism. we hypothesize that a crucial link that can explain this phenomenon is the lack of expression of the daz gene in the testes these cases. a potential alternative explanation may be related to the presence of an azfc deletion in mosaic and/or the loss of function of azfc genes in absence of yq deletion. if our finding will be confirmed on larger series of patients, daz gene dysfunctions may be proved to account for a larger portion of cases of infertility than one could expect by simply analyzing the prevalence of yq deletions among these subjects; iii) we found that several genes that are mainly related to the postmeiotic phase of the spermatogenesis and have been previously reported as involved in male infertility are dowregulated in patients with azfc deletions. given the crucial role played by rna storage during spermatogenesis, the loss of function of daz can translate to a defective mrna storage in testis with great pathogenic implications. the identification of the molecular mechanisms underlying the spermatogenesis failure in cases characterized by daz loss of function will provide useful information for the disclosure of key molecules which could represent in the future the target for personalized drug therapy. 
interestingly, the four idiopathic patients present in the cluster showed no testicular expression of daz despite the absence of azfc deletion in the peripheral blood. finally, based on our data, it is intriguing to hypothesize that daz gene dysfunctions can account for a larger number of previously thought "idiopathic" infertility cases and investigation of such testicular gene dysfunction can be important to reveal the molecular determinant of infertility than are undetected when only testing yq deletions in peripheral blood. about 10% of cases of male infertility are due to the presence of microdeletions within the long arm of the y chromosome. furthermore, the presence of azfc deletions in mosaic or the loss of function of azfc genes in absence of yq deletion can perhaps explain these findings. despite the large literature covering this critical issue, very little is known about the pathogenic mechanism leading to spermatogenesis disruption in patients carrying these microdeletions. an intriguing and unexpected finding is that all the samples showing the azfc deletion cluster together irrespectively of their testicular phenotypes. in order to identify the presence of specific molecular pathways leading to spermatogenic damage, testicular gene expression profiling was carried out by employing a microarray assay in patients carrying an azfc microdeletion or affected by idiopathic infertility. our expression profiles analysis indicates that several forms of infertility can be triggered by a common pathogenic mechanism that is likely related to alterations in testicular mrna storage. this cluster, including also four patients affected by idiopathic infertility, showed a downregulation of several genes related to spermatogenesis that are mainly involved in testicular mrna storage.
 about 10% of cases of male infertility are due to the presence of microdeletions within the long arm of the y chromosome. despite the large literature covering this critical issue, very little is known about the pathogenic mechanism leading to spermatogenesis disruption in patients carrying these microdeletions. in order to identify the presence of specific molecular pathways leading to spermatogenic damage, testicular gene expression profiling was carried out by employing a microarray assay in patients carrying an azfc microdeletion or affected by idiopathic infertility. hierarchical clustering was performed pooling the data set from experiments.  an intriguing and unexpected finding is that all the samples showing the azfc deletion cluster together irrespectively of their testicular phenotypes. this cluster, including also four patients affected by idiopathic infertility, showed a downregulation of several genes related to spermatogenesis that are mainly involved in testicular mrna storage. interestingly, the four idiopathic patients present in the cluster showed no testicular expression of daz despite the absence of azfc deletion in the peripheral blood.  our expression profiles analysis indicates that several forms of infertility can be triggered by a common pathogenic mechanism that is likely related to alterations in testicular mrna storage. our data suggest that a lack of testicular daz gene expression may be the trigger of such mechanism. furthermore, the presence of azfc deletions in mosaic or the loss of function of azfc genes in absence of yq deletion can perhaps explain these findings. finally, based on our data, it is intriguing to hypothesize that daz gene dysfunctions can account for a larger number of previously thought "idiopathic" infertility cases and investigation of such testicular gene dysfunction can be important to reveal the molecular determinant of infertility than are undetected when only testing yq deletions in peripheral blood. 