Partial-AZFc deletions in Chilean men with primary spermatogenic impairment: gene dosage and Y-chromosome haplogroups.

PURPOSE: To investigate the association of partial-AZFc deletions in Chilean men with primary spermatogenic failure and their testicular histopathological phenotypes, analyzing the contribution of DAZ dosage, CDY1 copies, and Y-chromosome haplogroups. SUBJECTS AND METHODS: We studied 479 Chilean men: 334 infertile patients with histological examination (233 cases with spermatogenic defects and 101 normal spermatogenesis, obstructive controls, OC), and 145 normozoospermic controls (NC). AZFc subdeletions were detected by single-tagged sequences and single nucleotide variants analysis. DAZ-copy number was quantified by real-time qPCR. Y-chromosome haplogroups (Y-hg) were hierarchically genotyped through 16 biallelic-markers. RESULTS: The prevalence of AZFc-partial deletions was increased in cases (6%) compared with NC (1.4%) (P = 0.035). There was no difference between 143 Sertoli-cell only syndrome, 35 maturation arrest, or 35 mix atrophy patients and controls. However, gr/gr deletions were more frequent in 16 subjects with hypospermatogenesis compared with NC (P = 0.003) and OC (P = 0.013). Y-hg R was the most prevalent (~ 50%), but decreased among gr/gr deletions (21%, P = 0.03). The prevalence of Y-hg M increased in cases versus controls, both in total and non-deleted men (3.9 and 3.7% versus 0.4%, P = 0.009 and P = 0.016, respectively). Among gr/gr deletions, Y-hg H increased compared with non-deleted men (14.3% versus 0.4%, P = 0.0047). CONCLUSION: Partial-AZFc deletions in a Chilean admixed population are associated with secretory azo/oligozoospermia and might have a role in the development of hypospermatogenesis. Low represented haplogroups, Y-hg M and Y-hg H, show an association with the occurrence of spermatogenic failure and gr/gr deletions respectively; however, additional studies are required.

Dynamic and regulated TAF gene expression during mouse embryonic germ cell development.

Germ cells undergo many developmental transitions before ultimately becoming either eggs or sperm, and during embryonic development these transitions include epigenetic reprogramming, quiescence, and meiosis. To begin understanding the transcriptional regulation underlying these complex processes, we examined the spatial and temporal expression of TAF4b, a variant TFIID subunit required for fertility, during embryonic germ cell development. By analyzing published datasets and using our own experimental system to validate these expression studies, we determined that both Taf4b mRNA and protein are highly germ cell-enriched and that Taf4b mRNA levels dramatically increase from embryonic day 12.5-18.5. Surprisingly, additional mRNAs encoding other TFIID subunits are coordinately upregulated through this time course, including Taf7l and Taf9b. The expression of several of these germ cell-enriched TFIID genes is dependent upon Dazl and/or Stra8, known regulators of germ cell development and meiosis. Together, these data suggest that germ cells employ a highly specialized and dynamic form of TFIID to drive the transcriptional programs that underlie mammalian germ cell development.

Evaluation of chromosomal abnormalities and Y chromosome microdeletion in infertile males of 10 families.

This study was designed to investigate the hormonal, seminal changes and chromosomal aberrations in cases of male infertility. A total of ten infertile families from Khyber Pakhtunkhwa of Pakistan were included in the study. The families were clinically evaluated by standard criteria; diagnosis of azoospermic and oligospermic males was confirmed. Seminal, hormonal, ultra sonographic and histopathological examinations were carried out for all the affected participants of the study. Karyotyping was performed on peripheral blood lymphocytes according to standard methods. Hormones were altered in six families. Ultrasonographic abnormal finding was observed in six families. Karyotyping analysis revealed numerical aberration in family G (0X) and family I (XXY). The remainingfamilies had no structural or numerical aberration. Y chromosome microdeletion analysis revealed AZFc deletion in both the affected participants of the family C. The remaining families were found normal for microdeletion. The occurrence of chromosomal anomalies and Y chromosome microdeletions among infertile males strongly suggests the need to include these two tests in routine investigations of male in fertility cases.

Effects of ten-eleven translocation 1 (Tet1) on DNA methylation and gene expression in chicken primordial germ cells.

Ten-eleven translocation 1 (Tet1) is involved in DNA demethylation in primordial germ cells (PGCs); however, the precise regulatory mechanism remains unclear. In the present study the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing PGCs and the role of Tet1 in PGC demethylation were analysed. Results show that 5mC levels dropped significantly after embryonic Day 4 (E4) and 5hmC levels increased reaching a peak at E5-E5.5. Interestingly, TET1 protein was highly expressed during E5 to E5.5, which showed a consistent trend with 5hmC. The expression of pluripotency-associated genes (Nanog, PouV and SRY-box 2 (Sox2)) and germ cell-specific genes (caveolin 1 (Cav1), piwi-like RNA-mediated gene silencing 1 (Piwi1) and deleted in azoospermia-like (Dazl)) was upregulated after E5, whereas the expression of genes from the DNA methyltransferase family was decreased. Moreover, the Dazl gene was highly methylated in early PGCs and then gradually hypomethylated. Knockdown of Tet1 showed impaired survival and proliferation of PGCs, as well as increased 5mC levels and reduced 5hmC levels. Further analysis showed that knockdown of Tet1 led to elevated DNA methylation levels of Dazl and downregulated gene expression including Dazl. Thus, this study reveals the dynamic epigenetic reprogramming of chicken PGCs invivo and the molecular mechanism of Tet1 in regulating genomic DNA demethylation and hypomethylation of Dazl during PGC development.

High frequency of de novo DAZ microdeletion in sperm nuclei of subfertile men: possible involvement of genome instability in idiopathic male infertility.

The occurrence and diagnosis of Y-chromosome microdeletions, specifically deletions of the DAZ (Deleted in Azoospermia) genes are an important issue in male infertility. Screening Y chromosome microdeletion is mainly done using polymerase chain reaction (PCR) on blood leukocytes. However, there is some evidence indicating that presence of DAZ in somatic cells might not be indicative of its presence in the germ cell lineage. Therefore, a total of 130 men with poor semen quality were examined for presence of DAZ microdeletion in their leukocytes. From these, sperm from 40 randomly selected men with no DAZ microdeletions in their leukocytes (n = 10 oligozoospermia; n = 10 asthenozoospermia; n = 10 oligoasthenozoospermia; and n = 10 near-azoospermia) were were compared to sperm from men of normal semen quality (n = 10) using combined primed in situ labelling and fluorescent in situ hybridization (PRINS-FISH) technique as well as screening for sex chromosome aneuploidy. There was an increased frequency of DAZ microdeletion in blood samples from oligozoospermic (5%) (p < 0.05) and near azoospermic patients (14%) (p < 0.01). A high frequency of DAZ microdeletion was observed in the sperm of patients with no DAZ microdeletion in their leukocytes compared to control (p < 0.01). The frequency of sex chromosome aneuploidy also increased, correlating with the severity of infertility in the studied groups. A similar result was observed for sex chromosome aneuploidy. The results might be indicative of DAZ microdeletion induction during spermatogenesis.

Immunolocalization of proteins in the spermatogenesis process of canine.

Spermatogenesis is a process in which differentiated cells are produced and the adult stem cell population-known as spermatogonial stem cells (SSCs)-is continuously replenished. However, the molecular mechanisms underlying these processes are not fully understood in the canine species. We addressed this in this study by analysing the expression of specific markers in spermatogonia of seminiferous tubules of canine testes. SSCs at different stages of reproductive development (prepubertal and adult) were examined by immunohistochemistry and flow cytometry. Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRA1), deleted in azoospermia-like (DAZL) and promyelocytic leukaemia zinc finger (PLZF) were expressed in SSCs, while stimulated by retinoic acid gene 8 (STRA8) was detected only in undifferentiated spermatogonia in prepubertal testis and differentiated spermatogonia and spermatocytes in adult canine. Octamer-binding transcription factor 4 (OCT4) showed an expression pattern, and the levels did not differ between the groups examined. However, C-kit expression varied as a function of reproductive developmental stage. Our results demonstrate that these proteins play critical roles in the self-renewal and differentiation of SSCs and can serve as markers to identify canine spermatogonia at specific stages of development.

Analysis of partial azoospermia factor c deletion and DAZ copy number in azoospermia and severe oligozoospermia.

Microdeletions of the azoospermia factor (AZF) regions in the Y chromosome are a well-known genetic cause of male infertility, resulting in impairment of spermatogenesis. However, the partial deletions of AZFc region related to spermatogenetic impairment are controversial. We investigated partial deletion of AZFc region and DAZ copy number in a population of Iranian infertile men and normozoospermic controls. In total, 154 infertile men (113 patients with azoospermia, 41 with oligozoospermia) and 111 normozoospermic controls were analysed using PCR. Gene dosage analysis of the DAZ genes was performed by fragment analysis. Our results showed that the frequencies of gr/gr deletion in the azoospermic, severe oligozoospermic and normozoospermic men were 4.4% (5/113), 7.3% (3/41) and 1.8% (2/111) respectively. In the azoospermic patients, the frequency of b2/b3 was 1.8% (2/113). Partial AZFc deletions were not significantly different between the infertile and normozoospermic men. The frequencies of gr/gr deletions and b2/b3 were not significantly different between the azoospermic/severe oligozoospermic men and normozoospermic controls. Our data suggested that gr/gr deletion was not associated with azoospermia/oligozoospermia in an Iranian population.

Discrimination of Deletion and Duplication Subtypes of the Deleted in Azoospermia Gene Family in the Context of Frequent Interloci Gene Conversion.

Due to its palindromic setup, AZFc (Azoospermia Factor c) region of chromosome Y is one of the most unstable regions of the human genome. It contains eight gene families expressed mainly in the testes. Several types of rearrangement resulting in changes in the cumulative copy number of the gene families were reported to be associated with diseases such as male infertility and testicular germ cell tumors. The best studied AZFc rearrangement is gr/gr deletion. Its carriers show widespread phenotypic variation from azoospermia to normospermia. This phenomenon was initially attributed to different gr/gr subtypes that would eliminate distinct members of the affected gene families. However, studies conducted to confirm this hypothesis have brought controversial results, perhaps, in part, due to the shortcomings of the utilized subtyping methodology. This proof-of-concept paper is meant to introduce here a novel method aimed at subtyping AZFc rearrangements. It is able to differentiate the partial deletion and partial duplication subtypes of the Deleted in Azoospermia (DAZ) gene family. The keystone of the method is the determination of the copy number of the gene family member-specific variant(s) in a series of sequence family variant (SFV) positions. Most importantly, we present a novel approach for the correct interpretation of the variant copy number data to determine the copy number of the individual DAZ family members in the context of frequent interloci gene conversion.Besides DAZ1/DAZ2 and DAZ3/DAZ4 deletions, not yet described rearrangements such as DAZ2/DAZ4 deletion and three duplication subtypes were also found by the utilization of the novel approach. A striking feature is the extremely high concordance among the individual data pointing to a certain type of rearrangement. In addition to being able to identify DAZ deletion subtypes more reliably than the methods used previously, this approach is the first that can discriminate DAZ duplication subtypes as well.

[Association of the deleted DAZ gene copy related to gr/gr and b2/b3 deletions with spermatogenic impairment].

OBJECTIVE: To investigate the correlation of the deleted azoospermia (DAZ) gene copy related to gr/gr and b2/b3 deletions in the AZFc region with male spermatogenic impairment. METHODS: This study included 121 infertile men with different de- grees of spermatogenic impairment and 95 healthy donors from the sperm bank. Using PCR, PCR-RFLP, and Y chromosome specific sequence tagged sites (STS) , we analyzed the association of DAZ gene copy deletions related to gr/gr and b2/b3 deletions in the AZFc region with spermatogenic impairment. RESULTS: There were 15 cases of gr/gr deletion (12. 40% ) and 6 cases of b2/b3 deletion (4.96%) in the infertility group as compared with 13 cases of gr/gr deletion (13.68%) and 1 case of b2/b3 deletion (1.05%) in the control. Analysis of the DAZ-specific single nucleotide variant (SNV) loci revealed 11 gr/gr-DAZI/DAZ2 deletions (9.09%), 4 gr/gr-DAZ3/DAZ4 deletions (3.31%), and 6 b2/b3-DAZ1/DAZ2 deletions (4.96%) in the infertile men in comparison with 3 gr/ gr-DAZ1/DAZ2 deletions (3.16%), 10 gr/gr-DAZ3/DAZ4 deletions (10.53%), and 1 b2/b3- DAZ3/DAZ4 deletion (1.05%) in the control. CONCLUSION: Partial deletions of gr/gr and b2/b3 exist in both healthy men and male patients with different degrees of spermatogenic impairment and cannot be considered as a risk factor for spermatogenesis impairment. However, deletions of different DAZ duplicons in gr/gr and b2/b3 deletions have different effects on spermatogenesis. DAZ1/DAZ2 instead of DAZ3/DAZ4 deletions might be associated with spermatogenesis impairment.

Association of the deleted DAZ gene copy related to gr/gr and b2/b3 deletions with spermatogenic impairment / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the correlation of the deleted azoospermia (DAZ) gene copy related to gr/gr and b2/b3 deletions in the AZFc region with male spermatogenic impairment.</p><p><b>METHODS</b>This study included 121 infertile men with different de- grees of spermatogenic impairment and 95 healthy donors from the sperm bank. Using PCR, PCR-RFLP, and Y chromosome specific sequence tagged sites (STS) , we analyzed the association of DAZ gene copy deletions related to gr/gr and b2/b3 deletions in the AZFc region with spermatogenic impairment.</p><p><b>RESULTS</b>There were 15 cases of gr/gr deletion (12. 40% ) and 6 cases of b2/b3 deletion (4.96%) in the infertility group as compared with 13 cases of gr/gr deletion (13.68%) and 1 case of b2/b3 deletion (1.05%) in the control. Analysis of the DAZ-specific single nucleotide variant (SNV) loci revealed 11 gr/gr-DAZI/DAZ2 deletions (9.09%), 4 gr/gr-DAZ3/DAZ4 deletions (3.31%), and 6 b2/b3-DAZ1/DAZ2 deletions (4.96%) in the infertile men in comparison with 3 gr/ gr-DAZ1/DAZ2 deletions (3.16%), 10 gr/gr-DAZ3/DAZ4 deletions (10.53%), and 1 b2/b3- DAZ3/DAZ4 deletion (1.05%) in the control.</p><p><b>CONCLUSION</b>Partial deletions of gr/gr and b2/b3 exist in both healthy men and male patients with different degrees of spermatogenic impairment and cannot be considered as a risk factor for spermatogenesis impairment. However, deletions of different DAZ duplicons in gr/gr and b2/b3 deletions have different effects on spermatogenesis. DAZ1/DAZ2 instead of DAZ3/DAZ4 deletions might be associated with spermatogenesis impairment.</p>

DAZ Family Proteins, Key Players for Germ Cell Development.

DAZ family proteins are found almost exclusively in germ cells in distant animal species. Deletion or mutations of their encoding genes usually severely impair either oogenesis or spermatogenesis or both. The family includes Boule (or Boll), Dazl (or Dazla) and DAZ genes. Boule and Dazl are situated on autosomes while DAZ, exclusive of higher primates, is located on the Y chromosome. Deletion of DAZ gene is the most common causes of infertility in humans. These genes, encoding for RNA binding proteins, contain a highly conserved RNA recognition motif and at least one DAZ repeat encoding for a 24 amino acids sequence able to bind other mRNA binding proteins. Basically, Daz family proteins function as adaptors for target mRNA transport and activators of their translation. In some invertebrate species, BOULE protein play a pivotal role in germline specification and a conserved regulatory role in meiosis. Depending on the species, DAZL is expressed in primordial germ cells (PGCs) and/or pre-meiotic and meiotic germ cells of both sexes. Daz is found in fetal gonocytes, spermatogonia and spermatocytes of adult testes. Here we discuss DAZ family genes in a phylogenic perspective, focusing on the common and distinct features of these genes, and their pivotal roles during gametogenesis evolved during evolution.

Impact of partial DAZ1/2 deletion and partial DAZ3/4 deletion on male infertility.

This study aims to investigate the effect of the partial DAZ1/2 deletion and partial DAZ3/4 deletion on male infertility through a comprehensive literature search. All case-control studies related to partial DAZ1/2 and DAZ3/4 deletions and male infertility risk were included in our study. Odd ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association and its precision, respectively. Eleven partial DAZ1/2 deletion and nine partial DAZ3/4 deletion studies were included. Partial DAZ1/2 deletion was significantly associated with male infertility risk in the overall analysis (ORs=2.58, 95%CI: 1.60-4.18, I(2)=62.1%). Moreover, in the subgroup analysis stratified by ethnicity, partial DAZ1/2 deletion was significantly associated with male infertility risk in the East Asian populations under the random effect model (ORs=2.96, 95%CI: 1.87-4.71, I(2)=51.3%). Meanwhile, the analysis suggested that partial DAZ3/4 deletion was not associated with male infertility risk in East-Asian ethnicity (ORs=1.02, 95%CI: 0.54-1.92, I(2)=71.3%), but not in Non-East Asian under the random effect model (ORs=3.56, 95%CI: 1.13-11.23, I(2)=0.0%,). More interestingly, partial DAZ1/2 deletion was associated with azoospermia (ORs=2.63, 95%CI: 1.19-5.81, I(2)=64.7%) and oligozoospermia (ORs=2.53, 95%CI: 1.40-4.57, I(2)=51.8%), but partial DAZ3/4 deletion was not associated with azoospermia (ORs=0.71, 95%CI: 0.23-2.22, I(2)=71.7%,) and oligozoospermia (ORs=1.21, 95%CI: 0.65-2.24, I(2)=55.5%). In our meta-analysis, partial DAZ1/2 deletion is a risk factor for male infertility and different ethnicities have different influences, whereas partial DAZ3/4 deletion has no effect on fertility but partial DAZ3/4 deletion might have an impact on Non-East Asian male.

Susceptibility of gr/gr rearrangements to azoospermia or oligozoospermia is dependent on DAZ and CDY1 gene copy deletions.

PURPOSE: The purpose of this study was to determine the association of AZFc subdeletions (gr/gr, b1/b3 and b2/b3) and deletion of DAZ and CDY1 gene copies with male infertility METHODS: Three hundred twelve controls, 172 azoospermic and 343 oligozoospermic subjects were subjected to AZFc subdeletion typing by STS PCR. Deletion of DAZ and CDY1 gene copies was done using sequence family variant analysis. Sperm concentration and motility were compared between men with and without AZFc subdeletions. Effect of the AZFc subdeletions on ICSI outcome was evaluated. RESULTS: Amongst the three AZFc subdeletions, the frequency of gr/gr was higher in oligozoospermic (10.5 %) and azoospermic (11.6 %) men as compared to controls (5.1 %). In men with AZFc subdeltions, loss of two DAZ and one CDY1 gene copy made them highly susceptible to azoospermia and severe oligozoospermia with OR of 29.7 and 26, respectively. These subdeletions had no effect on ICSI outcome, albeit there were an increased number of poor quality embryos in AZFc subdeleted group. CONCLUSION: AZFc subdeletions are a major risk factor for male infertility in the Indian population. In the subjects with AZFc subdeletions, the deletion of DAZ and CDY1 gene copies increases its susceptibility to azoospermia or severe oligozoospermia. Since these deletions can be vertically transmitted to the future male offspring by ICSI, it will be essential to counsel the couples for the transmission of the genetic defect in the male offspring born after assisted reproduction and the risk of perpetuating infertility in future generation.

Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination.

The azoopsermia factor c (AZFc) region of human Y-chromosome is an essential genomic segment for spermatogenesis with frequent non-allele homologous recombination (NAHR). Recent case-control studies on the association of the NAHR-based AZFc structural mutations with spermatogenic failure produced inconsistent results. To more precisely evaluate their spermatogenesis effects, we investigated the correlation between the subdivided AZFc mutations and sperm production in 3,439 Han Chinese males. Our results showed that both partial AZFc deletion-only and primary duplication mutation presented a significant risk for decreased sperm production. Restoration of the reduced dosage of the AZFc content to the normal level had a milder effect, whereas an overdose of the AZFc content arising from multiple duplications of a partial AZFc-deleted structure produced a more serious consequence compared to the partial deletion-only mutation. Additionally, the AZFc-mutated structures with excessive NAHR-substrate showed a notably negative effect on spermatogenesis. These results suggest that the recurrent NAHR-based AZFc mutations may be associated with decreased spermatogenesis efficiency in present population. More significantly, our finding implies that the overdose of AZFc NAHR-substrate may produce an additional risk for the massive AZFbc deletions during the multi-stage division process of germ cells and thus impair the global spermatogenesis efficiency in the carriers.

Transcriptome analysis of the cancer/testis genes, DAZ1, AURKC, and TEX101, in breast tumors and six breast cancer cell lines.

Breast cancer is the most frequent cancer with second mortality rate in women worldwide. Lack of validated biomarkers for early detection of breast cancer to warranty the diagnosis and effective treatments in early stages has directed to the new therapeutic approach. Cancer/testis antigens which have restricted normal expression in testis and aberrant expression in different cancers are promising targets for generating cancer vaccines, monoclonal antibodies, or dendritic cell-based immunotherapy. In this context, we investigated the expression of two known cancer testis genes, Aurora kinase C (AURKC) and testis expressed 101 (TEX101), and one new candidate, deleted in azoospermia 1 (DAZ1), in six breast cancer cell lines including two ductal carcinomas, T47D and BT-474, and four adenocarcinomas, MDA-MB-231, MDA-MB-468, MCF7, and SKBR3 as well as 50 breast cancer tumors in comparison to normal mammary epithelial cells using quantitative real-time reverse transcription PCR (RT-PCR). Results showed significant overexpression (p = 0.000) of all three genes in BT474, DAZ1 in MDA-MB-231, and AURKC and DAZ1 in SKBR3 and significant downregulation (p = 0.000) of AURKC in MCF7 cell line relative to normal breast epithelial cells. Breast tumors showed significant overexpression of AURKC in comparison to normal breast tissues (p = 0.016). The results are noticeable especially in the case of AURKC; however, there is a little knowledge about the nature, causes, consequences, and effects of cancer/testis antigens activation in different cancers. It is suggested that AURKC has effects on cell division via its serin/threonin kinases activity and organizing microtubules in relation to centrosome/spindle function during mitosis.

AZFc deletions do not affect the function of human spermatogonia in vitro.

Azoospermic factor c (AZFc) deletions are the underlying cause in 10% of azoo- or severe oligozoospermia. Through extensive molecular analysis the precise genetic content of the AZFc region and the origin of its deletion have been determined. However, little is known about the effect of AZFc deletions on the functionality of germ cells at various developmental steps. The presence of normal, fertilization-competent sperm in the ejaculate and/or testis of the majority of men with AZFc deletions suggests that the process of differentiation from spermatogonial stem cells (SSCs) to mature spermatozoa can take place in the absence of the AZFc region. To determine the functionality of AZFc-deleted spermatogonia, we compared in vitro propagated spermatogonia from six men with complete AZFc deletions with spermatogonia from three normozoospermic controls. We found that spermatogonia of AZFc-deleted men behave similar to controls during culture. Short-term (18 days) and long-term (48 days) culture of AZFc-deleted spermatogonia showed the same characteristics as non-deleted spermatogonia. This similarity was revealed by the same number of passages, the same germ cell clusters formation and similar level of genes expression of spermatogonial markers including ubiquitin carboxyl-terminal esterase L1 (UCHL1), zinc finger and BTB domain containing 16 (ZBTB16) and glial cell line-derived neurotrophic factor family receptor alpha 1 (GFRA1), as well as germ cell differentiation markers including signal transducer and activator of transcription 3 (STAT3), spermatogenesis and oogenesis specific basic helix-loophelix 2 (SOHLH2), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and synaptonemal complex protein 3 (SYCP3). The only exception was melanoma antigen family A4 (MAGEA4) which showed significantly lower expression in AZFc-deleted samples than controls in short-term culture while in long-term culture it was hardly detected in both AZFc-deleted and control spermatogonia. These data suggest that, at least in vitro, spermatogonia of AZFc-deleted men are functionally similar to spermatogonia from non-deleted men. Potentially, this enables treatment of men with AZFc deletions by propagating their SSCs in vitro and autotransplanting these SSCs back to the testes to increase sperm counts and restore fertility.

Combined deletion of DAZ2 and DAZ4 copies of Y chromosome DAZ gene is associated with male infertility in Tunisian men.

The relationship between male infertility and AZFc micro-deletions that remove multiple genes of the Y chromosome varies among countries and populations. The purpose of this study was to analyze the prevalence and the characteristics of different Deleted in azoospermia (DAZ) gene copy deletions and their association with spermatogenic failure and male infertility in Tunisian men. 241 infertile men (30.7% azoospermic (n=74), 31.5% oligozoospermic (n=76) and 37.7% normozoospermic (n=91)) and 115 fertile healthy males who fathered at least one child were included in the study. Three DAZ-specific single nucleotide variant loci and six bi-allelic DAZ-SNVs (I-VI) were analyzed using polymerase chain reaction (PCR)-restriction fragment length polymorphism and PCR. Our findings showed high frequencies of infertile men (73.85%) and controls (78.26%) having only three DAZ gene copies (DAZ1/DAZ2/DAZ3 or DAZ1/DAZ3/DAZ4 variants); so deletion of DAZ2 or DAZ4 were frequent both in infertile (36.5% and 37.3%, respectively) and fertile groups (33.9% and 44.3%, respectively) and removing DAZ4 copy was significantly more frequent in oligospermic than in normospermic men (p=0.04) in infertile group. We also report for the first time that simultaneous deletion of both DAZ2 and DAZ4 copies was significantly more common in infertile men (12.4%) than in fertile men (4.3%) (p=0.01). However, deletions of DAZ1/DAZ2 and DAZ3/DAZ4 clusters were very rare. Analysis of DAZ gene copies in Tunisian population, suggested that the simultaneous deletion of DAZ2 and DAZ4 gene copies is associated with male infertility, and that oligospermia seems to be promoted by removing DAZ4 copy.

Gene copy number alterations in the azoospermia-associated AZFc region and their effect on spermatogenic impairment.

The azoospermia factor c (AZFc) region in the long arm of human Y chromosome is characterized by massive palindromes. It harbors eight multi-copy gene families that are expressed exclusively or predominantly in testis. To assess systematically the role of the AZFc region and these eight gene families in spermatogenesis, we conducted a comprehensive molecular analysis (including Y chromosome haplogrouping, AZFc deletion typing and gene copy quantification) in 654 idiopathic infertile men and 781 healthy controls in a Han Chinese population. The b2/b3 partial deletion (including both deletion-only and deletion-duplication) was consistently associated with spermatogenic impairment. In the subjects without partial AZFc deletions, a notable finding was that the frequency of DAZ and/or BPY2 copy number alterations in the infertile group was significantly higher than in the controls. Combined patterns of DAZ and/or BPY2 copy number abnormality were associated with spermatogenic impairment when compared with the pattern of all AZFc genes with common level copies. In addition, in Y chromosome haplogroup O1 (Y-hg O1), the frequency of copy number alterations of all eight gene families was significantly higher in the case group than that in the control group. Our findings indicate that the DAZ, BPY2 genes may be prominent players in spermatogenesis, and genomic rearrangements may be enriched in individuals belonging to Y-hg O1. Our findings emphasize the necessity of routine molecular analysis of AZFc structural variation during the workup of azoospermia and/or oligozoospermia, which may diminish the genetic risk of assisted reproduction.