Comprehensive analysis of chromosomal breakpoints and candidate genes associated with male infertility: insights from cytogenetic studies and expression analyses.

The study aimed to investigate prevalent chromosomal breakpoints identified in balanced structural chromosomal anomalies and to pinpoint potential candidate genes linked with male infertility. This was acchieved through a comprehensive approach combining RNA-seq and microarray data analysis, enabling precise identification of candidate genes. The Cytogenetics data from 2,500 infertile males referred to Royan Research Institute between 2009 and 2022 were analyzed, with 391 cases meeting the inclusion criteria of balanced chromosomal rearrangement. Of these, 193 cases exhibited normal variations and were excluded from the analysis. By examining the breakpoints, potential candidate genes were suggested. Among the remaining 198 cases, reciprocal translocations were the most frequent anomaly (129 cases), followed by Robertsonian translocations (43 cases), inversions (34 cases), and insertions (3 cases).Some patients had more than one chromosomal abnormality. Chromosomal anomalies were most frequently observed in chromosomes 13 (21.1%), 14 (20.1%), and 1 (16.3%) with 13q12, 14q12, and 1p36.3 being the most prevalent breakpoints, respectively. Chromosome 1 contributed the most to reciprocal translocations (20.2%) and inversions (17.6%), while chromosome 14 was the most involved in the Robertsonian translocations (82.2%). The findings suggested that breakpoints at 1p36.3 and 14q12 might be associated with pregestational infertility, whereas breakpoints at 13q12 could be linked to both gestational and pregestational infertility. Several candidate genes located on common breakpoints were proposed as potentially involved in male infertility. Bioinformatics analyses utilizing three databases were conducted to examine the expression patterns of 78 candidate genes implicated in various causes of infertility. In azoospermic individuals, significant differential expression was observed in 19 genes: 15 were downregulated (TSSK2, SPINK2, TSSK4, CDY1, CFAP70, BPY2, BTG4, FKBP6, PPP2R1B, SPECC1L, CENPJ, SKA3, FGF9, NODAL, CLOCK), while four genes were upregulated ( HSPB1, MIF, PRF1, ENTPD6). In the case of Asthenozoospermia, seven genes showed significant upregulation (PRF1, DDX21, KIT, SRD5A3, MTCH1, DDX50, NODAL). Though RNA-seq data for Teratozoospermia were unavailable, microarray data revealed differential expression insix genes: three downregulated (BUB1, KLK4, PIWIL2) and three upregulated (AURKC, NPM2, RANBP2). These findings enhance our understanding of the molecular basis of male infertility and could provide valuable insights for future diagnostic and therapeutic strategies.

A review of recent developments in the application of nanostructures for sperm cryopreservation.

In the 1970s, sperm cryopreservation was presented as a unique route to fertility preservation. The ability to cryopreserve sperm from all species is challenging. The sperm cryopreservation process encompasses various cellular stresses such as increased osmotic pressure, ice crystal formation, and thermal shock, therefore decreasing the quality of sperm. The nanostructures due to their inherent features such as reactivity, high uptake, active surface area, and antioxidant activity, have contributed to modifying freezing protocols. In this review, the current state of the art with regards to emerging applications of nanotechnology in sperm cryopreservation are reviewed, some of the most promising advances are summarized, and the limitations and advantages are comprehensively discussed.

KIF3B gene silent variant leading to sperm morphology and motility defects and male infertility†.

Sperm structural and functi onal defects are leading causes of male infertility. Patients with immotile sperm disorders suffer from axoneme failure and show a significant reduction in sperm count. The kinesin family member 3B (KIF3B) is one of the genes involved in the proper formation of sperm with a critical role in intraflagellar and intramanchette transport. A part of exon 2 and exons 3-5 of the KIF3B encodes a protein coiled-coil domain that interacts with intraflagellar transport 20 (IFT20) from the intraflagellar transport protein complex. In the present study, the coding region of KIF3B coiled-coil domain was assessed in 88 oligoasthenoteratozoospermic (OAT) patients, and the protein expression was evaluated in the mature spermatozoa of the case and control groups using immunocytochemistry and western blotting. According to the results, there was no genetic variation in the exons 3-5 of the KIF3B, but a new A>T variant was identified within the exon 2 in 30 patients, where nothing was detected in the control group. In contrast to healthy individuals, significantly reduced protein expression was observable in oligoasthenoteratozoospermic patients carrying variation where protein organization was disarranged, especially in the principal piece and midpiece of the sperm tail. Besides, the protein expression level was lower in the patients' samples compared to that of the control group. According to the results of the present study the KIF3B gene variation as well as lower protein expression leads to defects in sperm morphology and motility and consequently to male infertility.

Sperm-oviduct interaction: Differential gene expression of growth factors induced by sperm DNA fragmentation.

The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.

Observational retrospective study of Intracytoplasmic sperm injection outcomes of 117 infertile men with severe short tail sperm defect.

Intracytoplasmic sperm injection (ICSI) is increasingly used to treat male-factor infertility when sperm parameters are not proper for intrauterine insemination (IUI) or in vitro fertilization (IVF). Among sperm abnormalities, short tail sperm defect is a rare kind of teratozoospermia, which is a severe cause of male infertility. In this study, we evaluated the ICSI outcomes of infertile men with severely short tail sperm defect. 117 infertile men with primary infertility were included in this study. We evaluated the impact of short tail sperm defect on large ICSI series (228 cycles) outcomes. The fertilisation rate (FR) was 49.0%, the clinical pregnancy rate (PR) was 21.7%, and the delivery rate (DR) was 17.5%. The results of statistical analysis show that there is no relationship between short tail sperm defect and clinical pregnancy. According to the present study, there were patients with successful ICSI outcomes despite the severe defect in their spermatozoa flagella. Our results can be considered in two main aspects: (a) it seems that ICSI could be a proper therapy for infertile men with short-tailed sperm defect and (b) the abnormal sperm morphology (especially in sperm flagellum) is not a reliable predictor for the ICSI outcomes. In conclusion, our study suggests that ICSI should be considered as a proper treatment way for infertile men with severe short tail sperm defect and probably other sperm flagella abnormalities.

Single-nucleotide polymorphism c.474G>A in the SEPT12 gene is a predisposing factor in male infertility.

SEPT12 is a testis-specific gene involved in the terminal differentiation of male germ cells. SEPT12 protein is required for sperm head-tail formation and acts as a fundamental constituent of sperm tail annulus. In this study, we screened genetic variations in exons 5, 6, 7 of the SEPT12 and assessed the annulus status in teratozoospermic, globozoospermic, and patients with immotile short tail sperm. DNA sequencing was performed for 90 teratozoospermic and 30 normozoospermic individuals. Immunocytochemistry, transmission electron microscopy and western blotting were conducted to evaluate annulus status and the expression level of SEPT12 in patients with a distinct exonic variation (c.474G>A), respectively. Five polymorphisms identified within the desired regions of the SEPT12, among them c.474G>A had the potential to induce aberrant splicing results in the expression of a truncated protein. The annulus was detected in most of the spermatozoa from teratozoospermic and normozoospermic men with c.474G>A. In contrast, in the patient with short tail sperm defect carrying c.474G>A, 99% of spermatozoa were devoid of the annulus. Based on our findings there would be no association between exons 5, 6, 7 polymorphisms of the SEPT12 gene and the occurrence of mentioned disease but c.474G>A would be a predisposing factor in male infertility.

Deletion of dpy-19 like 2 (DPY19L2) gene is associated with total but not partial globozoospermia.

The dpy-19 like 2 (DPY19L2) gene is the most common genetic cause of globozoospermia characterised by the production of round-headed spermatozoa without an acrosome. The present study was performed on 63 men with globozoospermia and 41 normozoospermic individuals to evaluate the frequency of the DPY19L2 gene and exons; deletion and genetic changes in exons 1, 5, 7-11, 19, 21 and interval introns; and some epidemiological factors (e.g. varicocele, smoking, drug use, alcohol consumption and a family history of infertility). Homozygous deletion of DPY19L2 was identified in 35% of men with globozoospermia. Exon 7 was deleted in 4.8% of men with globozoospermia in which DPY19L2 was not deleted. No genetic variations were observed within the DPY19L2 exons examined, but five intronic polymorphisms were detected: 1054-77T>C in intron 9, 1131+65T>C and 1131+53A>G in intron 10 and 1218+22T>C and 1218+73T>C in intron 11. There were significant differences in the frequency of 1054-77T>C and 1218+22T>C polymorphisms between the globozoospermic and normozoospermic groups. In addition, there were significant differences between the two groups in sperm count, sperm motility, a history of infertility in the family and varicocele. Based on these findings, DPY19L2 deletion is the major cause of total globozoospermia and there is no association between exons 1, 5, 8-11, 19 and 21 polymorphisms of the DPY19L2 gene in the occurrence of this defect.

Corrigendum to: Deletion of dpy-19 like 2 (DPY19L2) gene is associated with total but not partial globozoospermia.

The dpy-19 like 2 (DPY19L2) gene is the most common genetic cause of globozoospermia characterised by the production of round-headed spermatozoa without an acrosome. The present study was performed on 63 men with globozoospermia and 41 normozoospermic individuals to evaluate the frequency of the DPY19L2 gene and exons; deletion and genetic changes in exons 1, 5, 7-11, 19, 21 and interval introns; and some epidemiological factors (e.g. varicocele, smoking, drug use, alcohol consumption and a family history of infertility). Homozygous deletion of DPY19L2 was identified in 35% of men with globozoospermia. Exon 7 was deleted in 4.8% of men with globozoospermia in which DPY19L2 was not deleted. No genetic variations were observed within the DPY19L2 exons examined, but five intronic polymorphisms were detected: 1054-77T>C in intron 9, 1131+65T>C and 1131+53A>G in intron 10 and 1218+22T>C and 1218+73T>C in intron 11. There were significant differences in the frequency of 1054-77T>C and 1218+22T>C polymorphisms between the globozoospermic and normozoospermic groups. In addition, there were significant differences between the two groups in sperm count, sperm motility, a history of infertility in the family and varicocele. Based on these findings, DPY19L2 deletion is the major cause of total globozoospermia and there is no association between exons 1, 5, 8-11, 19 and 21 polymorphisms of the DPY19L2 gene in the occurrence of this defect.

Evaluation of sperm DNA fragmentation and chromatin structure in infertile men with immotile short-tail sperm defect.

Teratozoospermia is characterised by the presence of spermatozoa with abnormal morphology. One of the morphological disorders that lead to male infertility is immotile short-tail sperm (ISTS) defect. In this study, we evaluated the levels of chromatin packing and DNA fragmentation in patients with immotile short-tail sperm defect. Semen samples were obtained from 31 infertile men with ISTS as case group and 31 normozoospermic men as a control group. Protamine status was evaluated using chromomycin A3 (CMA3) staining and sperm DNA fragmentation assessed by sperm chromatin structure assay (SCSA) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick-end labelling (TUNEL). The percentage of positive CMA3 spermatozoa was significantly higher in patients' samples (22.6 ± 6.9) compared with controls (16.3 ± 4.2) (p < .05) and also mean (±SD) of sperm DNA fragmentation was significantly higher in patients compared with controls, as measured by TUNEL assay (10.45 ± 4.60 vs. 7.03 ± 2.86, p < .05) and SCSA (24.80 ± 13.1 vs. 15.2 ± 7.2, p < .05). According to our study, sperm DNA fragmentation and chromatin packing abnormality are significantly higher in the ISTS samples compared with normal samples. A possible explanation for this relationship is that sperm chromatin condensation and sperm flagellum formation occur during the same phase of spermatogenesis.

The role of DEFB126 variation in male infertility and medically assisted reproduction technique outcome.

RESEARCH QUESTION: Human DEFB126 is an important component of the glycocalyx of human spermatozoa. Beta-defensins play a primary role in male infertility due to their involvement in maturation and capacitation of spermatozoa. A 2-nt deletion of DEFB126 affects sperm function and so this study investigated the possible association between DEFB126 variants and its protein expression on medically assisted reproduction (MAR) technique outcome in Iranian infertile males. DESIGN: The presence of a 2-nt deletion of DEFB126, and its protein expression in spermatozoa, were investigated by standard polymerase chain reaction (PCR) sequencing and immunocytochemistry, respectively. MAR technique outcome according to clinical pregnancy rates was assessed in 277 Iranian males with unexplained infertility, including 139 patients who underwent intrauterine insemination (IUI) and 103 patients who underwent IVF/intracytoplasmic sperm injection (ICSI), as well as 35 infertile males who declined to use any MAR treatment. As the control group, 100 fertile males with a normal spermiogram were enrolled. RESULTS: The 2-nt deletion of DEFB126 was significantly higher in infertile patients than controls (P ≤ 0.05). The presence of this deletion resulted in significantly lower clinical pregnancy rates following IUI (P ≤ 0.05); however, there were no differences in IVF/ICSI outcomes according to genotype. The protein expression in del/del males was also remarkably lower than that of the other genotypes. CONCLUSIONS: This sequence variation of DEFB126 may impair male reproductive function and can be related to male infertility. Interestingly, males with the del/del genotype have a normal spermiogram; however, their spermatozoa are evidently functionally impaired, which can affect IUI treatment outcome, but not treatment by IVF/ICSI.

Spermatogenesis disorder is associated with mutations in the ligand-binding domain of an androgen receptor.

Androgens play a key role in spermatogenesis, and their functions are mediated by the androgen receptor (AR). Some mutations in the AR gene have the potential to alter the primary structure and function of the protein. The aim of this study was to investigate the AR gene mutations in a cohort of males with idiopathic azoospermia referred to Royan Institute. Fifty-one biopsy samples were obtained for routine clinical purposes from 15 men with hypospermatogenesis (HS), 17 patients with maturation arrest (MA) and 19 patients with Sertoli cell-only syndrome (SCOS). The AR cDNAs were prepared from tissue mRNAs and were sequenced. One synonymous variant and three nonsynonymous protein coding single nucleotide polymorphisms (nsSNPs) were detected. Protein structure prediction demonstrated that the S815I and M746T nonsynonymous variants would affect protein structure and its normal function. Our study suggests that mutations in the AR gene would change or disturb the receptor's normal activity. Although these variations may influence spermatogenesis, it is difficult to say that they lead to a lack of spermatogenesis.

Gene alterations and expression spectrum of SPATA33 in nonobstructive azoospermic Iranian men.

Genetic abnormalities have been considered a significant cause of male infertility. Increased expression of SPATA33 during the first wave of spermatogenesis indicates its possible association with the meiotic process. The aim of the current study was to investigate the genetic variations in the SPATA33 gene and its expression in patients with nonobstructive azoospermia (NOA). A total of 100 Iranian NOA men with idiopathic infertility were taken as the case group. The control group comprised 100 fertile men who had at least one child. The presence of nucleotide variations was analyzed in both groups using the standard polymerase chain reaction (PCR) sequencing technique. For mRNA and protein expression studies, testicular biopsy specimens from 27 patients were subdivided into three groups: nine obstructive azoospermic patients with hypospermatogenesis as control; nine maturation arrest (MA) and nine Sertoli cell-only syndromes (SCOS) as case groups. The expression of SPATA33 at both mRNA and protein levels was compared among these three groups using the reverse transcription PCR technique, the realtime-PCR technique, and immunohistochemistry. Mutation analysis of the SPATA33 gene revealed five nucleotide changes among the population studied. All but one showed no significant differences between the groups. The genotype distributions of rs112536073A > T in the transcription factor binding site region with heterozygote and homozygote genotypes were significantly different ( p < 0.05) between the two groups. More heterozygotes of this polymorphism were observed in patients, whereas more homozygotes were detected in controls. Accordingly, the current study illustrated that alterations in SPATA33 gene, at least those found in this study, may not impair spermatogenesis in patients with NOA. Reduction of gene expression at the level of mRNA in patients with SCOS can be interpreted by the absence of germ cells in the testicular tissue of these patients.

Sperm retrieval rate and reproductive outcome of infertile men with azoospermia factor c deletion.

To evaluate the success rate in sperm retrieval (SR) through microdissection testicular sperm extraction (micro-TESE) in infertile azoospermia factor c (AZFc)-deleted men and determining their reproductive outcomes following ICSI, medical records of couples with AZFc-deleted male partners were reviewed on patient's age, serum hormone levels, karyotype, testicular pathology and pregnancy outcomes. A comparison on age and serum hormone level was conducted between groups with positive and negative sperm retrieval in both azoospermic and oligozoospermic AZFc-deleted men. Of 225 who had AZFc deletion, 195 cases followed clinical treatments. From 195 cases, 116 were azoospermic, 79 were oligozoospermic. Pathology profile was available in 103 of 195 subjects which the predominant trait was SCOS and was seen in 66.9% of cases (69 of 103). Success rate of sperm retrieval in azoospermic patients who underwent micro-TESE was 36.3% (28/77). Forty-three oligozoospermic and 17 azoospermic patients started ART cycle. Pregnancy rate in oligozoospermic group was 35.4% (17 cases), whilst there was no clinical pregnancy in azoospermic group. In conclusion, the pregnancy and delivery in oligozoospermic patients with AZFc deletion are comparable with other studies, but despite of sperm retrieval in azoospermic patients with AZFc deletion, the chance of pregnancy or delivery in these patients was very low.

Study of trinucleotide expansions and expression of androgen receptor in infertile men with abnormal spermogram referred to Royan institute.

Androgen receptor (AR) mediates androgen activities such as the growth of accessory sex organs, and initiation and promotion of spermatogenesis. There are two trinucleotide polymorphisms (CAG and GGN repeats) in the first exon of AR gene that their association with infertility is still controversial. The variants of both polymorphic repeats were investigated by PCR-Sequencing in 220 infertile men (80 azoospermic, 60 oligospermic and 80 asthenospermic) and 80 healthy fertile controls. AR Expression level was quantified by RT-qPCR on 30 patients (20 patients with nonobstructive azoospermia (NOA) and 10 obstructive azoospermia patients as controls). Our results demonstrated that the medians of CAG and GGN repeats length in infertile group were significantly higher than fertile men (p < 0.05). AR expression results showed a significant increase in SCOS group compared to control (p < 0.05). Long stretches of tandem repeats of AR gene may negatively affect the function of the gene and consequently lead to male infertility. In patients with SCOS, AR expression increases because of the lack of germ cells. Therefore, with increasing AR expression, the probability of SCOS occurrence is also increased. It can be concluded that increasing AR expression in testes tissue decreases the probability of sperm presence.

The impact of RABL2B gene (rs144944885) on human male infertility in patients with oligoasthenoteratozoospermia and immotile short tail sperm defects.

PURPOSE: Male infertility is a multifactorial disorder with impressively genetic basis; besides, sperm abnormalities are the cause of numerous cases of male infertility. In this study, we evaluated the genetic variants in exons 4 and 5 and their intron-exon boundaries in RABL2B gene in infertile men with oligoasthenoteratozoospermia (OAT) and immotile short tail sperm (ISTS) defects to define if there is any association between these variants and human male infertility. METHODS: To this purpose, DNA was extracted from peripheral blood and after PCR reaction and sequencing, the results of sequenced segments were analyzed. In the present study, 30 infertile men with ISTS defect and 30 oligoasthenoteratozoospermic infertile men were recruited. All men were of Iranian origin and it took 3 years to collect patient's samples with ISTS defect. RESULTS: As a result, the 50776482 delC intronic variant (rs144944885) was identified in five patients with oligoasthenoteratozoospermia defect and one patient with ISTS defect in heterozygote form. This variant was not identified in controls. The allelic frequency of the 50776482 delC variant was significantly statistically higher in oligoasthenoteratozoospermic infertile men (p < 0.05). Bioinformatics studies suggested that the 50776482 delC allele would modify the splicing of RABL2B pre-mRNA. In addition, we identified a new genetic variant in RABL2B gene. CONCLUSIONS: According to the present study, 50776482 delC allele in the RABL2B gene could be a risk factor in Iranian infertile men with oligoasthenoteratozoospermia defect, but more genetic studies are required to understand the accurate role of this variant in pathogenesis of human male infertility.

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.

STUDY QUESTION: Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene? SUMMARY ANSWER: WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified. WHAT IS KNOWN ALREADY: Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein. MAIN RESULTS AND THE ROLE OF CHANCE: WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G > A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G > A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients.

The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.

Relationship between absence of annulus and asthenozoospermia in Iranian men.

PURPOSE: To find a relationship between absence of annulus and asthenozoospermia in Iranian men. METHODS: In the present study, semen samples from 100 asthenozoospermic and 20 normozospermic patients were analyzed for sperm concentration and motility. Spermatozoa were immunostained for the two septin subunits Sept4 and Sept7. The absence of the annulus structure was confirmed by transmission electron microscopy and western blot analysis for septin 4. DNA sequencing for all coding exons of SEPT12 was performed for a patient using peripheral blood sample. RESULTS: Specific antibodies for SEPT4 and SEPT7 consistently labeled the annuli in spermatozoa from all of the 20 normozospermic men, while in one of 100 patients with asthenozoospermia, 75% of sperms lacking septin 4 or septin 7 proteins at the annulus. It was shown that the structural defect in annulus formation is not caused by point mutation of SEPT12 gene. CONCLUSIONS: In conclusion, the results of this study demonstrated that the frequency of the absence of annulus in asthenozoospermic sample of Iranian population has a low frequency and could not be assume as a diagnostic marker for classifying asthenozoospermic patients.

Study of the effect of varicocelectomy on sperm proteins expression in patients with varicocele and poor sperm quality by using two-dimensional gel electrophoresis.

PURPOSE: To compare proteomic profiles of spermatozoa from patients with varicocele and poor sperm quality before and after varicocelectomy. METHODS: This work was designed as a prospective and observational study. The study was based on 20 men with varicocele grade 3 and poor sperm quality undergoing varicocelectomy at the Fertility Unit of Royan institute in 2009. Two semen samples were collected, one before varicocelectomy and the other after surgery. Protein separation was done by two-dimensional protein electrophoresis, and analyzed by gel densitometry and mass spectrometry. Differential sperm protein expression levels were measured by gel densitometry. RESULTS: Comparison of the sperm parameters showed that sperm motility and concentration were increased after varicocelectomy. At the level of protein, a total of 3 protein spots were identified whose expression was significantly lower in sperm samples before varicocelectomy compared with after surgery including heat shock protein A5 (HSPA5), superoxide dismutase 1 (SOD1) and δ-subunit of the catalytic core of mitochondrial adenosine triphosphate synthase (ATP5D). CONCLUSIONS: High grade varicocoele affects sperm protein expression presumably because of increasing testicular temperature. These proteins play essential roles in sperm production, DNA integrity protection, and sperm motility. This novel study demonstrates that varicocelectomy can improve both sperm quality and proteins expression.

Role of genetic variations and protein expression of ß-Microsemino protein in intrauterine insemination outcome of unexplained infertile men: A case-control study.

Background: Intrauterine insemination (IUI) is often the first-line treatment for unexplained infertility. ß -Microsemino protein (MSMB) is an abundant protein in seminal plasma that has an inhibitory effect on spontaneous acrosome reaction. Objective: The present study aimed to evaluate MSMB gene variations and protein expression on IUI success rate in unexplained infertile men. Materials and Methods: A case-control study was performed on 100 unexplained infertile Iranian men referred to the Royan Institute, Tehran, Iran for IUI (50 men with IUI positive result [IUI+], and 50 men with IUI negative result [IUI-]). Couples with female infertility factors (such as hormonal disorders, infrequent menstrual period, abnormality in uterus, fallopian tubes, or ovaries) and men with infections of the male accessory glands, hypogonadotropic hypogonadism, clinical varicocele, retractile testis, genital trauma, drug use, or concurrent hormonal treatment Y chromosome microdeletions, and abnormal karyotype were excluded from the study. The polymerase chain reaction sequencing was performed for the promoter and the coding regions of MSMB functional domains. To study the protein expression, the total protein of sperm was extracted, and sandwich enzyme-linked immunosorbent assay was performed. Results: 4 variations were detected (rs12770171, rs10993994, rs2075894, and rs4517463). None of them showed significant differences between the IUI+ and IUI- groups. The mean value of protein expression did not show any differences between the groups. Conclusion: In conclusion, there is no association between genetic variations of promoter and coding regions of MSMB functional domains as well as its expression with IUI success in unexplained infertile men.