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Objective: Activator of CREM in the testis (ACT) is a tissue specific transcription factor which activates cAMP responsive element modulator (CREM), a key transcription factor in differentiation of round spermatids into mature spermatozoa. They bind to CRE region in the promoters of transition protein genes (TNP1, TNP2) and protamine genes (PRM1 and PRM2), which are essential for sperm chromatin compaction, and regulates their transcription. This study was conducted to consider the expression of ACT and CREM and their regulatory roles on the expression of PRM1, PRM2, TNP1 and TNP2 genes in testis tissues of infertile men. Materials and Methods: In this case-control study, testicular biopsies were collected from 40 infertile men and classified into three groups: obstructive azoospermia (OA, n=10, positive control), round spermatid maturation arrest (SMA, n=20), Sertoli cell-only syndrome (SCOS, n=10, negative control group). Using quantitative real-time polymerase chain reaction (PCR), the expression profile of ACT, CREM, TNP1, TNP2, PRM1 and PRM2 genes were assessed in testicular samples and incorporation of ACT and CREM proteins on the promoters of PRM1, PRM2, TNP1 and TNP2 genes were also evaluated by ChIP-real time PCR. Results: Our results demonstrated significant decrease in the expression levels of ACT, CREM and in their incorporations on their target genes in SMA group in comparison to control groups (P≤0.05). Conclusion: These data confirm that there is low expression and incorporation of ACT and CREM and of their target genes in infertilities which are associated with post-meiotic arrest.

To assess the role of three testis-specific genes including ZPBP2, PGK2, and ACRV1 in the prediction of sperm retrieval result and quality of retrieved sperm by microdissection testicular sperm extraction (micro-TESE) in non-obstructive azoospermia (NOA) patients. This was a case-control study including 57 testicular samples of NOA patients including 32 patients with successful sperm retrieval (NOA+) and 25 patients with failed sperm retrieval (NOA-), and 9 samples of men with normal spermatogenesis in the testes as the positive control (OA). We investigated the expression of candidate genes by RT-qPCR and germ cell population patterns by DNA flow cytometry in testicular biopsy samples. The association between PGK2 expressions with the quality of retrieved spermatozoa was also evaluated. The RT-qPCR data revealed a significantly higher expression of ZPBP2 and PGK2 in the NOA+ in comparison to NOA- group (P = 0.002, and P = 0.002, respectively). Flow cytometry results revealed that the haploid cell percentage was significantly higher in NOA+ vs. NOA- group (P = 0.0001). In samples with a higher percentage of haploid cells, expression levels of ZPBP2 and PGK2 were higher (P = 0.001). The PGK2 expression was significantly associated with retrieved sperm quality (P = 0.01). Our results contribute to the search for the biomarkers for predicting the presence of testicular sperm and would be useful to avoid unnecessary multiple micro-TESE. Overall, the expression pattern of the ZPBP2 and PGK2 may be useful in predicting sperm recovery success and quality of retrieved sperm in NOA patients.

Introduction: MiRNA expression alterations are closely related to growth, proliferation, and cell development. In addition, miR-187 has been found to regulate cellular proliferation. Further, spermatogenesis is a highly complicated process which is regulated through several genes targeted by miRNAs. However, no study has completely addressed the etiology of spermatogenic impairments. Accordingly, the present study examined the expression pattern of Homo sapiens (human) microRNA (hsa-mir-187) in the testicular biopsies of infertile men with non-obstructive azoospermia (NOA) and Sertoli cell-only syndrome (SCOS) subtype in comparison with those with obstructive azoospermia (OA). Methods: Quantitative reverse‐transcription polymerase chain reaction analysis was performed to assess the expression level of hsa-mir-187 using the ΔΔCt method. Results: Our data indicated that hsa-mir-187 was significantly up-regulated in the testis of the SCOS group (P˂ 0.05) in comparison with the OA group. Conclusion: In general, the findings suggest that the differential expression of hsa-mir-187 is linked with male infertility and shed more light on molecular defects that underlie spermatogenic impairments.

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.

Background Microdeletions of the Yq chromosome are among the most frequent genetic etiological factor of male infertility which spans the azoospermia factor regions (AZFa, AZFb and AZFc). Microdeletions are mostly seen in the AZFc region and usually cover genes actively involved in spermatogenesis. Partial AZFc microdeletions may also occur with various spans, namely gr/gr, b2/b3 and b1/b3. It is known that the outcome of microtesticular sperm extraction (TESE), the surgical process for sperm retrieval from the testis in infertile azoospermic men, may be pre- dicted based on the type of AZF microdeletion. We therefore aimed to evaluate the correlation between partial AZFc microdeletions and microTESE results. Materials and Methods In this cross-sectional study, 200 infertile azoospermic men referred to the Royan Institute were examined for the presence of partial AZFc microdeletions before undergoing microTESE. Partial AZFc micro- deletions were detected by multiplex polymerase chain reaction (PCR) of seven different sequence-tagged site (STS) markers. The data were analyzed with the Chi-square test. Results Among the 90 patients (45%) with a positive microTESE outcome, 9 (10%) showed a partial microdeletion in AZFc region. Of the 110 (55%) patients with a negative microTESE outcome, 7 (6.3%) had an AZFc partial micro- deletion. With respect to the span of the microdeletions, among the 200 patients, 11 (5.5%) were gr/gr and 5 (2.5%) were b2/b3. Statistical analysis showed no significant difference between the patients with and without partial AZFc microdeletions with respect to microTESE outcome. Conclusion Partial AZFc microdeletions is not a predictor of microTESE outcome in azoospermic men.

Research question Membrane lipid replacement (MLR) of oxidized membrane lipids can restore sperm cellular membrane functionality and help improve surface protein stability during cryopreservation. What are the effects of MLR with nano-micelles made from a glycerophospholipid (GPL) mixture and cholesterol-loaded cyclodextrin (CLC), on the cryosurvival and expression of acrosome-related proteins in thawed human spermatozoa? Design Twenty samples were used to determine the optimum level of nano-micelles by incubation of semen with different concentrations of GPL (0.1 and 1%) and CLC (1 and 2 mg/ml) (including GPL-0.1, GPL-1, CLC-1, CLC-2, CLC-1/GPL-0.1, CLC-2/GPL-0.1, CLC-1/GPL-1 and CLC-2/GPL-1) before cryopreservation. Then, 30 semen samples were collected, and each sample was divided into the following three aliquots: fresh, frozen control and frozen incubated with optimum level of nano-micelles (0.1% GPL and 1 mg/ml CLC). Results CLC-1/GPL-0.1 and GPL-0.1 significantly increased motility parameters. CLC-1, GPL-0.1 and CLC-1/GPL-0.1 significantly improved viability rate compared with frozen control group. Significantly higher mitochondrial activity and acrosome integrity, and a lower rate of apoptosis, were observed in the CLC-1/GPL-0.1 compared with the frozen control group. The expression ratios of arylsulfatase A (ARSA), serine protease 37 (PRSS37), serine protease inhibitor Kazal-type 2 (SPINK2) and equatorin (EQTN) significantly increased compared with the frozen control group. Conclusions Modification of membrane cholesterol and GPL mixtures in spermatozoa enhances their acrosome protein integrity by inhibiting early apoptotic changes and spontaneous acrosome reactions.

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.

Varicocele is associated with excessive production of reactive oxygen species (ROS). Although the harmful effects of ROS on sperm DNA, proteins and lipids are well documented, its impact on the expression of miRNAs in spermatozoa has not been fully understood. In this study, the expression patterns of microRNAs (miRNAs), miR-21, miR-34a and miR-122a as well as the level of ROS in the fertile control (FC; proven fertility without varicocele, n = 15) and grade III varicocele patients with normal (VN; n = 15) and abnormal (VA; n = 15) spermogram were investigated. The real-time PCR was performed to analyse the expression of the miRNAs, while oxidative stress was evaluated by measuring the concentrations of MDA. Our results showed that the expression levels of miR-21 (p = .001), miR-34a, (p = .007) and miR-122a (p

Application of a three-dimensional (3D) culture system for in vitro proliferation and differentiation of human spermatogonial stem cells (SSCs) is a useful tool for the investigation of the spermatogenesis process and the management of male infertility particularly in prepubertal cancer patients. The main purpose of this study was to investigate the proliferation of human SSCs co-cultured with Sertoli cells in soft agar culture system (SACS) supplemented by Laminin and growth factors. Testicular cells were isolated from testes of brain-dead patients and cultured in two-dimensional (2D) culture system for 3 weeks. After 3 weeks, functional SSCs were evaluated by xenotransplantation and also identification of cells was assessed by immunocytochemistry, flow cytometry, and RT-PCR. Then, SSCs and Sertoli cells were transferred to the upper layer of SACS for 3 weeks. After 3 weeks, the number of colonies and the expression of specific SSCs and Sertoli cell markers, as well as apoptotic genes were evaluated. Our results showed that transplanted SSCs, migrated into the basement membrane of seminiferous tubules of recipient mice. The expression of PLZF, α6-Integrin, and Vimentin proteins in SSCs and Sertoli cells were observed in 2D and 3D culture systems. The expression rate of PLZF, α6-Integrin, Bcl2, and colony number in SACS supplemented by Laminin and growth factors group were significantly higher than non-supplemented groups (P ≤ 0.01), but the expression rate of c-kit and Bax in supplemented group were significantly lower than non-supplemented groups (P ≤ 0.05). This 3D co-culture system decreased apoptosis and increased propagation of human SSCs. Therefore, this designed system can be utilized to increase the proliferation of human SSCs in prepubertal male cancer and azoospermic men to obtain an adequate SSCs number to outotransplant success and in vitro spermatogenesis.

Background Assessing the net-results of microsurgical varicocelectomy in infertile men with non-obstructive azoosper- mic (NOA) and clinical varicocele in five years at Royan Institute. Materials and Methods This is a descriptive retrospective cohort study. A backward-looking review of patients treated for NOA and varicocele from march 2011 to march 2016 was performed. In addition, MDTESE results of 57 patients with NOA and clinical varicocele, with 537 NOA patients without varicocele were compared. Results Of 57 patients who underwent varicocelectomy, eight patients (14%) had sperm on sperm analysis post-opera- tively. One of the eight patients was single, and one of them had spontaneous pregnancy (1/7) 14%, and one had a child by microinjection (1/7) 14%. Out of these 8 patients, 6 had hypospermatogenesis pathology. Of 38 patients who under- went MDTESE, 14 patients (36%) had sperm on their testis tissues, but one of them had no egg fertilization. Therefore, the fertilization rate was (92%). Of the remaining 13 patients, 3 had live child birth (3/13) 23%. Sperm retrieval rate (SRR) in NOA men without clinical varicocele was lower from those who had varicocele and NOA (22 vs. 36%). Also live birth rate in NOA men with varicocelectomy was higher than NOA men without varicocele (23 vs. 11%). Conclusion Microsurgical varicocelectomy in NOA men may have positive effects on post-operative sperm in ejacu- late and natural or assisted pregnancies, but it seems that the effect is more significant on MDTESE results and follow- ing successful microinjection. Meanwhile, SRR and live birth rate was higher in our patients compare to NOA men without clinical varicocele.

To elucidate the association between asymptomatic infections caused by Mycoplasma genitalium and male infertility, and evaluate the role of antibiotic therapy in treatment of this failure. A total of 165 infertile males having abnormal semen parameters (study group) and 165 healthy fertile men (control group) were included. Semen samples were taken from all participants and after analyzing for semen parameters, undergone real-time PCR, microbial culture, and reactive oxygen species (ROS), as well as total antioxidant capacity (TAC) assays. Infected individuals of study group were treated with antibiotic. One month after the treatment completion, second semen samples were taken and subjected to all the tests mentioned. The data were analyzed using SPSS statistical software, version 22.0. The frequency of M. genitalium was significantly higher in the infertile men compared with the fertile ones (9.7% vs. 1.2%; p = 0.001). Mean cycle threshold (C t) value was lower in infected infertile than infected fertile men (p

Objective: Toll-like receptors (TLRs) on Sertoli cells are thought to have essential roles in sperm protection. This study was conducted to investigate the expression of TLR2 and TLR3 in Sertoli cells of men with azoospermia. Materials and Methods: In this experimental study, testicular biopsies were taken from ten azoospermic men. Following enzymatic dissociation, the samples were moved to lectin coated petri dishes. After a few passages, all cells were cultivated and Seroli cells were sorted by flow cytometry. To confirm Sertoli cell purification, alkaline phosphatase activity (ALP) and immunohistochemistry assays were employed. The expression of TLR2 and TLR3 at the transcript and protein levels was examined with real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) and western blot, respectively. Results: Isolation, purification and cultivation of human Sertoli cells were performed successfully. Efficacy of purification of Sertoli cells by fluorescence-activated cell sorting (FACS) sorter was ~97%. The type of cultured cells was confirmed by vimentin and follicle-stimulating hormone (FSH) receptor markers. Furthermore, the existence of anti- Müllerian hormone in culture was confirmed. RT-PCR showed that both genes were expressed in Sertoli cells. Consistently, proteins of both were also expressed in Sertoli cells. Moreover, QPCR showed that the relative expression of TLR3 transcripts was significantly higher than TLR2 in Sertoli cells. Although both genes are expressed in fibroblast cells, their level of expression was significantly lower than in Sertoli cells. Conclusion: This study confirmed expression of TLR2 and TLR3 in human Sertoli cells. This may be an indicator of their roles in developing immunity against pathogens as well as allo- and auto-antigens or viral antigens in seminiferous tubules.

The role of asymptomatic infections caused by Ureaplasma species in male infertility and the efficacy of antibiotics in treatment of this failure is not yet definitely determined. A total of 165 infertile males having abnormal semen parameters (study group) as well as 165 healthy fertile men (control group) were included in this study. Semen samples were taken from all participants and after analyzing, undergone real-time PCR, microbial culture, and reactive oxygen species (ROS) as well as total antioxidant capacity (TAC) assays. Infected individuals of study group were treated with antibiotic. One month after the treatment completion, second semen samples were taken and undergone all the tests mentioned. The frequency of Ureaplasma spp. was significantly higher in the infertile men compared with the fertile ones (36.4% versus 11.5%; p < 0.001). Most of semen parameters were improved (p < 0.05) and reached their normal range, the level of TAC elevated (p < 0.001), and ROS level (p = 0.003) as well as ROS/TAC ratio (p = 0.003) reduced after antibiotic treatment. Moreover, wives of 37 infertile men (61.7%) became pregnant six months after the treatment completion. These findings indicate that Ureaplasma species are correlated with male infertility and that antibiotic therapy can improve the semen parameters and treat the male infertility.

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. 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. 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

Background: Although the effectiveness of ginger as an antioxidant agent has been exploited, little human research has been conducted on its activity on male reproductive functions. Objective: This study was designed to investigate the effects of ginger (Zingiber officinale) on sperm DNA fragmentation (SDF) in infertile men. Materials and Methods: This randomized double-blind, placebo-controlled trial with a 1:1 allocation was performed on 100 infertility treatment candidates who were admitted to Royan Institute for Reproductive Biomedicine, Tehran, Iran. Patients were randomly assigned to receive one of two treatments: ginger and placebo. Patients were given a 3-month oral treatment (members received capsules containing 250 mg of ginger powder twice a day in ginger and a placebo in other group). Before and after treatment, standardized semen samples were obtained to determine sperm concentration, motility, and SDF according to World Health Organization. Results: There was no significant difference between two groups regarding SDF at baseline (53.48. 95%CI: 37.95-69.02) in cases and (56.75, 95%CI: 40.01-73.5) in controls. The average positive percentage of SDF in patients receiving ginger (17.77, 95%CI: 6.16-29.39) was lower compared with placebo (40.54, 95%CI: 23.94-57.13) after three month of treatment (p=0.02). In multivariate analysis, SDF was significantly lower in patients receiving ginger compared with placebo (mean difference: 3.21, 95%CI: 0.78-5.63, p=0.009). There were no significant differences between two groups regarding to semen parameters. Conclusion: The present study has demonstrated that ginger in a controlled study of efficacy was effective in decreasing SDF in infertile men.

Background: The availability of testis specific genes will be of help in choosing the most promising biomarkers for the detection of testicular sperm retrieval in patients with non-obstructive azoospermia (NOA). CDY1 histone acetyltransferase is a histone modifier which concentrates in the nucleus of round spermatids, where histone hyperacetylation occurs and causes the replacement of histones by the sperm-specific DNA packaging proteins, TNPs and PRMs. Objective: The aim of the present study was to evaluate CDY1 gene as a marker for predicting of successful sperm retrieval in NOA patients. Materials and Methods : This research was conducted on 29 patients with non-obstructive azoospermia who had undergone testicular sperm extraction (TESE) procedure. NOA patients were subdivided into patients with successful sperm retrieval (NOA+, n=12) and patients with unsuccessful sperm retrieval (NOA−, n=17). Relative expression of CDY1 gene and chromatin incorporation of CDY1 protein were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and ELISA assay, respectively. Results: Quantification of mRNA relative expression and incorporation of CDY1 protein in chromatin showed significant lower expressions and protein levels of CDY1 in testis tissues of NOA− group in comparison to NOA+ group. Conclusions: The findings in this study demonstrated a correlation between the low levels of CDY1 function and unsuccessful sperm recovery in the testicular tissues of NOA− compared to NOA+ patients. Therefore, it can be reasonable to consider CDY1 as a potential biomarker for predicting the presence of spermatozoa, although the claim needs. more samples to be confirmed

Testicular ischemia is the main consequence of testicular torsion, in both clinical and experimental aspects. Preservation and auto-transplantation of spermatogonial stem cells (SSCs) could be a new treatment for infertility in testicular ischemia following testicular torsion. To apply the idea in this study, animals were randomly divided into four groups of control, sham, with torsion, and with torsion followed by transplantation (TT). Isolated SSCs from neonatal mice were cultured and identified by flow cytometry (C-KIT−, INTEGRIN β1 +) and RT-PCR (Reverse transcription polymerase chain reaction) for specific spermatogonial cell markers (Oct4, Gfrα-1, Plzf, Vasa, Itgα 6 , and Itgβ 1 ). SSCs were transplanted upon a 2-h testicular torsion in the TT group. Cultured cells were transplanted into ischemia reperfusion testicle 2 weeks post-testicular torsion. Eight weeks after SSCs transplantation, the SSCs-transplanted testes and epididymis were removed for sperm analysis, weight and histopathological evaluation, and pre- and post-meiotic gene expression assessment by qRT-PCR. Our findings indicated that all evaluated parameters (epididymal sperm profile, Johnsen score, Plzf, Gfrα-1, Scp-1, Tekt-1 expressions, and histopathological profile) were significantly decreased following testicular torsion (group 3) when compared to the control group (p ≤ 0.05). However, all abovementioned parameters showed a significant increase/improvement in torsion-transplantation group compared to torsion group. However, these parameters in the TT group were significantly lower in the sham and control groups (p ≤ 0.05). SSCs transplantation could up-regulate the expression of pre- and post-meiotic genes in testicular ischemia, which resulted in improvement of both testicular function and structure after testicular torsion.

Cysteine-Rich Secretory Protein 2 (CRISP2) plays an important role in the morphology and motion of male ejaculated spermatozoa. The association of its expression with some miRNAs is also well known. The aim of this study was to determine the expression of CRISP2 and mir-582 in the seminal plasma fluid and spermatozoa of three groups of infertile men and the possible association of their expressions. In this experimental study, the expression of CRISP2 in seminal plasma fluid and spermatozoa of 17 men with asthenozoospermia, 15 men with teratozoospermia, 17 men with teratoasthenozoospermia, and 18 infertile individuals with normozoospermia were measured using western blotting. Then by using bioinformatics studies, miR-582-5p was nominated as a CRISP2-associated miRNA, and its expression was evaluated by means of Real-Time PCR. Comparison of expression of CRISP2 and miRNA-582 in the studied groups was analyzed by t-test and Mann-Whitney U test. The expression of CRISP2 showed a significant reduction in the spermatozoa and seminal plasma fluid of all three groups, (p 0.05). MiR-582-5p expression significantly increased in teratozoospermia patients (0.05), and significantly decreased in teratoasthenozoospermia patients (p 0.05). Meanwhile, changes in the expression of miR-582-5p in teratoasthenozoospermia individuals was associated with a decrease in the expression of CRISP2, which could represent the potential role of miR-582-5p in regulation of CRISP2 expression in teratoasthenozoospermia individuals.

OBJECTIVE Spermatogenesis is a complex process controlled by a plethora of genes. Changes in expression and function of these genes may thus lead to spermatogenic deficiency and male infertility. TEX11, TEX12, TEX14 and TEX15 are germ cell-specific genes expressed in the testis. TEX11, involved in the initiation and maintenance of chromosome synapses in meiotic chromosomes, has been shown to be essential for meiosis and fertility in males. TEX14, a component of intercellular bridges in germ cells, is required for spermatogenesis and fertility. TEX12 and TEX15 are essential for correct assembly of the synaptonemal complex and thus meiosis progression. METHODS In order to examine whether changes in expression of these genes is associated with impaired spermatogenesis, expression levels of these genes were quantified by RT-qPCR on samples retrieved from infertile patients submitted to diagnostic testicular biopsy at Royan institute. Samples were divided into two groups of 18 patients with non-obstructive azoospermia considered as case; nine patients with obstructive azoospermia were included in the control group. RESULTS A significant down-regulation of these genes was observed in the SCOS group when compared to the control group. CONCLUSION This result suggests that regular expression of TEX11, TEX12, TEX14 and TEX15 is essential for the early stages of spermatogenesis.

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 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.

Background The main purpose of this study is to evaluate the effects of varicocelectomy on serum testoster- one levels and semen quality in infertile men who suffer from varicocele. Materials and Methods This prospective study enrolled 115 subjects with clinical varicocele grades II and III and 240 fertile men as the control group. Total volume of testosterone serum level (ng/dl) and semen quality were com- pared before and after microscopic varicocelectomy. We normalized testosterone serum levels for age, grade, and testis size basis. SPSS 20 software was used to analyze the data. All results of continuous variables were reported as mean ± SD. Statistical significance was set at a P

To find a relationship between absence of annulus and asthenozoospermia in Iranian men.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.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.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.

Background: Spermatogonial stem cells (SSCs) are considered in fertility management approaches of prepubertal boys facing cancer therapies. However, in vitro propagation has become an important issue due to a small number of SSCs in testicular tissue. The present study aimed to investigate a modified soft agar culture system by using a nanofibrous scaffold as a new approach to mimic in vivo conditions of SSCs development. Materials and Methods: The SSCs were isolated from neonate mouse testes, cultured on polycaprolactone scaffold, and covered by a layer of soft agar for 2 weeks. Then, the number and diameter of colonies formed in experimental groups were measured and spermatogonial markers (i.e., Plzf, Gfrα1, Id4, and c-Kit) in SSCs colonies were evaluated by a real-time polymerase chain reaction and immunostaining. Results: Our results indicated that the colonization rate of SSCs was significantly higher in the present modified soft agar culture system (P

To study the sperm deoxyribonucleic acid (DNA) fragmentation index (DFI), testicular volume, semen parameters, and hormone profile in human chorionic gonadotropin (hCG)- and human menopausal gonadotrophin (hMG)-treated patients with hypogonadotropic hypogonadism (HH) with and without a successful pregnancy.This is a cross sectional study. The study initially included 81 patients with HH and azoospermia at the Infertility Unit of Royan Institute between 2010 and 2012. Fifty-eight of 81 patients achieved1 × 10(6) sperm/mL during hCG and hMG therapy. These 58 patients were divided into the following 2 groups: 20 patients with HH who achieved pregnancy in response to hCG/hMG (responders, 16 naturally and 4 by intrauterine insemination) and 38 gonadotropin-treated patients with HH with failed pregnancy (nonresponders, 29 naturally, 5 by intrauterine insemination, 1 by in vitro fertilization, and 3 by intracytoplasmic sperm injection). Sperm DNA fragmentation was visualized by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.Average of DFI (responders: 13.45 ± 0.64; nonresponders: 21.92 ± 0.86), age, body mass index, testis volume semen parameters, and follicle-stimulating hormone, luteinizing hormone, and testosterone levels in the 2 groups were calculated. Cut-off point for DFI was determined by receiver operating curve analysis (17.5%).It was shown that DFI in responders is significantly lower than DFI in nonresponders (P.001), and duration of hCG and hMG therapy in responders is significantly higher than those of nonresponders (P.05). DFI could be predictive of conception (P.001; odds ratio 0.57; 95% confidence interval 0.417-0.778). It can be concluded that despite low sperm quality, especially sperm concentration in these patients, decreasing sperm DNA damage may result in successful fertilization.

Identification of molecular markers which can predict the outcome of sperm retrieval non-invasively in patients with non-obstructive azoospermia (NOA) are valuable in clinical andrology. Jumonji domain-containing 1a (JMJD1A) is a significant epigenetic regulator during spermatogenesis, which plays an important role in the differentiation of post-meiotic germ cells into mature spermatozoa. We therefore aimed to examine the potential association between JMJD1A expression and the outcome of sperm retrieval in patients with NOA. Testicular biopsy specimens from 50 NOA patients with either successful sperm retrieval (sperm+, n=22) or failed sperm retrieval (sperm-, n=28) were collected and then examined for JMJD1A expression by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, conventional clinical parameters including luteinizing hormone, follicle-stimulating hormone, testosterone, age, and testicular volume were compared between the two NOA groups. The expression of JMJD1A in the sperm+ group was significantly higher than in the sperm- group (P

Background The most common chromosomal abnormality due to non-obstructive azoospermia (NOA) is Klinefelter syndrome (KS) which occurs in 1-1.72 out of 500-1000 male infants. The probability of retrieving sperm as the outcome could be asymmetrically different between patients with and without KS, therefore logistic regression analysis is not a well-qualified test for this type of data. This study has been designed to evaluate skewed regression model analysis for data collected from microsurgical testicular sperm extraction (micro-TESE) among azoospermic patients with and without non-mosaic KS syndrome. Materials and Methods This cohort study compared the micro-TESE outcome between 134 men with classic KS and 537 men with NOA and normal karyotype who were referred to Royan Institute between 2009 and 2011. In addition to our main outcome, which was sperm retrieval, we also used logistic and skewed regression analyses to compare the following demographic and hormonal factors: age, level of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone between the two groups. Results A comparison of the micro-TESE between the KS and control groups showed a success rate of 28.4% (38/134) for the KS group and 22.2% (119/537) for the control group. In the KS group, a significantly difference (P

Background Infertility can be a major concern for couples trying to conceive, and occupational hazards may constitute a main cause of infertility in men. Studies conducted throughout the world indicate that physical and chemical hazards in the workplace can have a negative impact on male fertility. The main objective of this study was to determine the frequency of occupational categories of men who attended an infertility clinic, and to evaluate the differences in the semen quality parameters among occupational categories. Materials and Methods This cross-sectional study was conducted on 1164 males who were referred to the Infertility Research Center in Tehran for treatment of infertility in order to evaluate the effects of certain occupations on infertility. The participants were divided into several categories according to their occupations and evaluated by means of a questionnaire for duration of infertility, BMI, sperm count, percentage of normal sperm morphology and percentages of sperm with class A and class B motilities. Descriptive statistics, analysis of variance, and correlations were conducted using SPSS 16.0 for Windows. Results There were no statistically significant differences in the mean sperm count or sperm morphology between occupational categories. Assessment of the differences in the frequency of sperm motility classes between occupational categories revealed a significant difference only in the frequency of sperm with class B motility. The lowest mean percentages of sperm with class B motility were seen in those involved in the transportation industry, a finding in agreement with a number of other researches. Conclusion Our findings revealed an association between occupation and sperm motility. Since our study population was relatively small and in many cases exposures to work hazards were brief, a larger study group must be evaluated in order to support the preliminary results of this study.

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

Purpose Isolating spermatogonia cells with high purity and viability and achieving better survival rate following cryopreservation Methods Isolating the cells by Magnetic Activating Cell Sorting (MACS) method using anti CD49f (α6 integrin) antibody and Dynabeads and freezing in DMSO-based freezing mediums containing three different FBS concentrations of 50%, 60% and 70%. Results The mean (±SD) purity of the isolated cells was 92.52±3.57 (range 92.43–98.25). The cells frozen in group I, II and III had mean 39.60±1.48 (range 37.98–41.62), 89.05±3.83 (range 80.83–90.33) and 90.52±1.71 (range 89.07–92.52) viability, respectively. Conclusion Higher viable cell counts and purity can be attained by the use of α6 integrin and magnetic beads. After the thawing of spermatogonial cells, optimum viability was achieved in freezing media containing 60% FBS.

Sperm motility is known as an effective parameter in male fertility, and it depends on energy consumption. Low-level laser irradiation could increase energy supply to the cell by producing adenosine triphosphate. The purpose of this study is to evaluate how the low-level laser irradiation affects the human sperm motility. Fresh human semen specimens of asthenospermic patients were divided into four equal portions and irradiated by 830-nm GaAlAs laser irradiation with varying doses as: 0 (control), 4, 6 and 10 J/cm(2). At the times of 0, 30, 45 and 60 min following irradiation, sperm motilities are assessed by means of computer-aided sperm analysis in all samples. Two additional tests [HOS and sperm chromatin dispersion (SCD) tests] were also performed on the control and high irradiated groups as well. Sperm motility of the control groups significantly decreased after 30, 45 and 60 min of irradiation, while those of irradiated groups remained constant or slightly increased by passing of time. Significant increases have been observed in doses of 4 and 6 J/cm(2) at the times of 60 and 45 min, respectively. SCD test also revealed a non-significant difference. Our results showed that irradiating human sperms with low-level 830-nm diode laser can improve their progressive motility depending on both laser density and post-exposure time.