Investigation of seminal plasma chitotriosidase-1 and leukocyte elastase as potential markers for 'silent' inflammation of the reproductive tract of the infertile male - a pilot study.

Inflammatory mediators - chitotriosidase-1 (CHIT1) and leukocyte elastase (LE) - were analyzed in human seminal plasma in relation to total antioxidative status (TAS) and pro-inflammatory markers IL-1ß and IL-6. Samples collected from 34 males who were part of infertile couples were divided into normozoospermic (N; n = 12, without symptoms of inflammation), oligozoospermic (O; n = 11) and teratozoospermic (T; n = 11) groups. significant differences were observed only in CHIT1 concentration between N and O samples. However, a higher mean LE concentration was also observed in O and T patients (3.7-times and 900-times, respectively) compared with the N group. in IL-1ß and IL-6 concentrations, an upward trend was observed from N, through O, up to the T group. The positive correlation between the concentration of IL-1ß and the activity and specific activity of CHIT11 as well as the moderate negative correlation between concentrations of IL-1ß and CHIT1 may suggest that elevated CHIT11 levels appeared in early stages of inflammation before the increase in IL-1ß concentrations, or remained stable even after the levels of cytokine decreased. The above seem to confirm the role of CHIT1 in the manifestation of 'silent' inflammation at a very early stage. To conclude, CHIT1 concentration appears to be an interesting biomarker that signals the presence of possible 'silent' inflammation accompanying oligozoospermia. We cannot draw such conclusions regarding LE concentration, because, although we observed differences in the mean values and medians between analyzed groups, they were not significant. The utility of CHIT1 in the follow-up of oligozoospermia-associated 'silent' subclinical inflammation is promising, but further studies on a larger patient test set are required.

Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of concanavalin A- and wheat germ agglutinin-reactive glycans mark seminal prostasome populations from normozoospermic and oligozoospermic men.

Background: Human seminal prostasomes are intrinsically heterogeneous extracellular vesicles (EVs) whose composition is, additionally, influenced by different physiological conditions. Aiming at the molecular properties of the prostasomal surface exemplified by glycan compositions as a possible distinction factor, we applied lectin-affinity chromatography (LAC) as a new tool for their separation. Since glycans, generally, exhibit various biological activities, introduction of glyco-parameters as reference could upgrade standardization of EVs isolated by different methods and intended for use in biomedicine.Methods: Preparations of seminal prostasomes from normozoospermic (sPro-N) and oligozoospermic (sPro-O) men were subjected to LAC on concanavalin A (Con A) and wheat germ agglutinin (WGA) columns. Prostasomes recovered in LAC-separated fractions were characterized according to the distribution of selected markers: gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), tetraspanin CD63, and total protein/glycoprotein composition.Results: Two CD63-immunoreactive populations exhibiting prostasome signature bands but differing in GGT activity and surface glycans were separated on the WGA column. Additional populations having distinct profiles of total glycoproteins and which can be tracked down by ALP activity were enriched on the Con A column. WGA-separated populations were similar in sPro-N and sPro-O, whereas Con A-separated ones were strikingly different.Conclusions: Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of Con A- and WGA-reactive glycans mark seminal prostasomes populations from normozoospermic and oligozoospermic men.

[Clinical report of testicular hypoplasia combined with 21-hydroxylase deficiency].

OBJECTIVE To investigate the correlation of 21-hydroxylase deficiency (21-OHD) with male testicular dysplasia. METHODS Clinical data of 8 infertile males with congenital adrenal hyperplasia due to 21-OHD was retrospectively analyzed. In addition, potential mutations of the CYP21A2 gene was detected. RESULTS All patients were referred because of azoospermia or severe oligospermia and had small testis with averaged testicular volume of 6.1 mL. Three patients had testicular adrenal rest tumors. Endocrinologic examinations revealed low levels of leutinizing hormone and follicular stimulating hormone, normal or elevated testosterone, elevated progesterone, elevated or normal adrenocoticotropic hormone, and low or normal cortisol. All patients had adrenal cortical hyperplasia, 5 with adrenal adenoma, 1 case associated with bilateral adrenal myelolipoma. All patients were given glucocorticoid replacement therapy for 3 to 6 months, which successfully improved the seminal status of 6 patient and resulted pregnancies in 5 couples. Seven pathogenic mutations of the CYP21A2 gene among the 8 patients. CONCLUSION 21-OHD can cause testicular hypoplasia and spermatogenic failure. Glucocorticoids and operations can obtain good result and improve spermatogenesis. Our results have shown a good genotype/phenotype correlation in these cases. All patients have carried the p.Ile172Asn mutation, which is associated with simple virilizing form.

Oligoasthenoteratozoospermic (OAT) men display altered phospholipase C ζ (PLCζ) localization and a lower percentage of sperm cells expressing PLCζ and post-acrosomal sheath WW domain-binding protein (PAWP).

Oligoasthenoteratozoospermia (OAT) is demonstrated to be one of the most common causes of male subfertility. Phospholipase C ζ (PLCζ), a sperm-specific protein, is considered to be one of the sperm-borne oocyte activating factors (SOAFs), which play a vital role in fertilization. The post-acrosomal sheath WW domain-binding protein (PAWP) is another candidate for SOAF. The aim of this study was to compare the PLCζ localization patterns and percentage of PLCζ- and PAWP-positive sperm cells in patients with OAT and fertile men with normozoospermia. A total of 40 men included in this study were classified into two groups: OAT (n = 25) and control group (n = 15). Semen samples were collected and analyzed using conventional semen analysis according to the World Health Organization guidelines. The percentage of PLCζ- and PAWP-positive sperm cells and localization patterns of PLCζ were evaluated using immunofluorescence staining. The mean percentage of sperm cells expressing PAWP and PLCζ was significantly lower in OAT compared to control group (52.8 ± 4.2 vs. 76.8 ± 5 and 63.4 ± 3.5 vs. 86.7 ± 2.1, respectively). In addition, statistically significant differences were found with regard to the PLCζ localization patterns, including equatorial, acrosomal + equatorial, and equatorial + post-acrosomal pattern, between the two groups (p < 0.01). The present study showed a lower percentage of sperm cells expressing PLCζ and PAWP, as well as altered localization patterns of PLCζ in men with OAT. Given the role of PLCζ and PAWP in fertilization, as two major candidates for SOAFs, our findings indicate that PLCζ and PAWP impairments may be one of the possible etiologies of decreased fertility in OAT.

Ubiquitin Carboxy-Terminal HydrolaseL3 Correlates with Human Sperm Count, Motility and Fertilization.

Ubiquitin C-terminal hydrolase L3 (UCHL3) belongs to the group of deubiquitinating enzymes and plays a part in apoptosis of germ cells and the differentiation of spermatocytes into spermatids. However, the exact role of UCHL3 in human spermatogenesis and sperm function remains unknown. Here we examined the level and activity of UCHL3 in spermatozoa from men with asthenozoospermia (A), oligoasthenozoospermia (OA) or normozoospermia (N). Immunofluorescence indicated that UCHL3 was mainly localized in the acrosome and throughout the flagella, and western blotting revealed a lower level in A or OA compared with N (p < 0.05). The catalytic activity of UCHL3 was decreased in spermatozoa from A or OA (p < 0.05, p < 0.001, respectively). The level and activity of UCHL3 were positively correlated with sperm count, concentration and motility. The UCHL3 level was positively correlated with the normal fertilization rate (FR) and percentage of embryos suitable for transfer/cryopreservation of in vitro fertilization (IVF). The UCHL3 activity was also positively correlated with FR, the percentage of embryos suitable for transfer/cryopreservation and high-quality embryos rate of IVF. Aforementioned correlations were not manifested in intra-cytoplasmic sperm injection (ICSI). These findings suggest that UCHL3 may play a role in male infertility.

Association of C677T transition of the human methylenetetrahydrofolate reductase (MTHFR) gene with male infertility.

The human methylenetetrahydrofolate reductase (MTHFR) gene encodes one of the key enzymes in folate metabolism. This gene is located on chromosome 1 (1p36.3), which has 12 exons. The aim of the present study was to investigate the possible association of the two (C677T and A1298C) polymorphisms of this gene with male infertility. In a case-control study, 250 blood samples were collected from IVF centres in Sari and Babol (Iran): 118 samples were from oligospermic men and 132 were from controls. Two single nucleotide polymorphisms of the MTHFR genotype were detected using polymerase chain reaction-restriction fragment length polymorphism. There was no association found between the A1298C variant and male infertility. However, carriers of the 677T allele (CT and TT genotypes) were at a higher risk of infertility than individuals with other genotypes (odds ratio 1.84; 95% confidence interval 1.11-3.04; P=0.0174). Structural analysis of human MTHFR flavoprotein showed that C677T transition played an important role in the change in affinity of the MTHFR-Flavin adenine dinucleotide binding site. Based on our results, we suggest that C677T transition in MTHFR may increase the risk of male infertility, and detection of the C677T polymorphism biomarker may be helpful in the screening of idiopathic male infertility.

Seminal cyclooxygenase relationship with oxidative stress in infertile oligoasthenoteratozoospermic men with varicocele.

This study aimed to assess the relation of seminal cyclooxygenase COX-1, COX-2 with oxidative stress in infertile oligoasthenoteratozoospermic (OAT) men with varicocele (Vx). In all, 128 men were allocated into fertile men, fertile men with Vx, infertile OAT men without Vx and infertile OAT men with Vx. They were subjected to history taking, clinical examination and semen analysis. Also, seminal COX-1, COX-2, malondialdehyde (MDA) and glutathione peroxidase (GPx) were estimated. Mean levels of seminal COX-1, COX-2 were over-expressed, the mean level of seminal MDA was significantly increased, and the mean level of seminal GPx was significantly decreased in infertile OAT men with Vx compared with other groups. Seminal COX-1 and COX-2 were over-expressed in cases with Vx grade III compared with Vx grades I, II cases and in cases with bilateral Vx compared with unilateral Vx. There was significant negative correlation between seminal COX-1 and COX-2 with sperm concentration, sperm motility, sperm normal morphology, seminal GPx and significant positive correlation with seminal MDA. It is concluded that seminal COX-1 and COX-2 are over-expressed in infertile OAT men with Vx compared with fertile men with/without and infertile OAT men without Vx being associated with oxidative stress, Vx grade and Vx laterality.

THE PECULIARITIES OF ARGINASE PATHWAY OF L-ARGININE IN SPERMATOZOA IN MEN WITH DIFFERENT FORMS OF PATHOSPERMIA.

The changes in arginase activity of spermatozoa and hormonal profile of peripheral blood of infertile men with various forms pathospermia have been studied. It has been found that arginase activity in the sperm cells of men with oligozoo-, antenozoo-, oligoastenozoo- and leucocytospermia is decreased in 2.1, 2.3, 2.4 and 3.3 times respectively. This indicates about inhibition of arginase pathway of L-arginine metabolism, which is not significantly dependent on the type of disruption of spermatogenesis. The most significant changes have been observed in infertile men with leucocytospermia since white blood cells stimulate the formation of reactive oxygen species, induction and development of oxidative and nitrative stress in spermatozoa. Inhibition of arginase pathway of L-arginine metabolism has adaptive role, which is to limit bioavailabil- ity of L-arginine and to prevent excessive formation of NO in cytotoxic concentrations to sperm cells. It has been noted changes in serum concentrations of gonadotropin and sex hormones in men with various forms of pathospermia. The most expressed significant changes were in levels of follicle stimulating hormone and testosterone. The concentration of follicle stimulating hormone in patients with oligozoospermia caused by hypogonadism is twice higher and in patients with leucocytospermia in 1.8 times higher than in fertile men. In patients with astenozoospermia this value is in 2.2 times lower than in normozoospermic samples but within the physiological norm. The testosterone level in men with oligozoospermia is in 1.6 times lower than in fertile men but within the physiological norm. It has been found that arginase inhibition of spermatozoa po6itively correlated with a decrease in their concentration in the ejaculate of infertile men with oligozoospermia (r =0.68).

Genetic polymorphisms of CYP2D6*10 and the effectiveness of combined tamoxifen citrate and testosterone undecanoate treatment in infertile men with idiopathic oligozoospermia.

Tamoxifen citrate, as the first line of treatment for infertile men with idiopathic oligozoospermia, was proposed by the World Health Organization (WHO), and testosterone undecanoate has shown benefits in semen values. Our objective was to assess the effectiveness of treatment with tamoxifen citrate and testosterone undecanoate in infertile men with idiopathic oligozoospermia, and whether the results would be affected by polymorphisms of CYP2D6*10. A total of 230 infertile men and 147 controls were included in the study. Patients were treated with tamoxifen citrate and testosterone undecanoate. Sex hormone, sperm parameters, and incidence of spontaneous pregnancy were detected. There were no significant differences between the control and patient groups with respect to CYP2D6*10 genotype frequencies (P>0.05). The follicle-stimulation hormone (FSH), luteinizing hormone (LH), and testosterone (T) levels were raised, and sperm concentration and motility were increased at 3 months and became significant at 6 months, and they were higher in the wild-type allele (C/C) than in the heterozygous variant allele (C/T) or homozygous variant allele (T/T) subgroups (P<0.05). In addition, the percentage of normal morphology was raised at 6 months, and represented the highest percentage in the C/C subgroup (P<0.05). The incidence of spontaneous pregnancy in the C/C subgroup was higher than that in the C/T or T/T subgroups (P<0.01). This study showed that the CYP2D6*10 variant genotype demonstrated worse clinical effects in infertile men with idiopathic oligozoospermia.

Cytotoxic and genotoxic effects of titanium dioxide nanoparticles in testicular cells of male wistar rat.

Serious concerns have been expressed about potential risks of engineered nanoparticles. Regulatory health risk assessment of such particles has become mandatory for the safe use in consumer products and medicines; also, the potential effects on reproduction and fertility are relevant for this risk evaluation. In the present study, we examined the effects of intravenously injected titanium dioxide nanoparticles (TiO2-NPs; 21 nm), with special emphasis on reproductive system. Antioxidant enzymes such as catalase, glutathione peroxidase, and superoxide dismutase showed a significant decrease, while significant increase in lipid peroxidase was observed. Our results confirmed the bioaccumulation of TiO2-NPs in testicular cells. In TiO2-NPs-treated animals, various functional and pathological disorders, such as reduced sperm count, increase in caspase-3 (a biomarker of apoptosis), creatine kinase activity, DNA damage, and cell apoptosis were observed. Moreover, the testosterone activity was decreased significantly in a dose-dependent manner in the animals treated with TiO2-NPs as compared with control group animals. It is concluded that TiO2-NPs induce oxidative stress, which produce cytotoxic and genotoxic changes in sperms which may affect the fertilizing potential of spermatozoa.

Nucleoside triphosphate diphosphohydrolase-1 ectonucleotidase is required for normal vas deferens contraction and male fertility through maintaining P2X1 receptor function.

In this work, we report that Entpd1(-/-) mice, deficient for the ectonucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), produce smaller litters (27% reduction) compared with wild-type C57BL6 animals. This deficit is linked to reduced in vivo oocyte fertilization by Entpd1(-/-) males (61 ± 11% versus 88 ± 7% for Entpd1(+/+)). Normal epididymal sperm count, spermatozoa morphology, capacitation, and motility and reduced ejaculated sperm number (2.4 ± 0.5 versus 3.7 ± 0.4 million for Entpd1(+/+)) pointed to vas deferens dysfunction. NTPDase1 was localized by immunofluorescence in the tunica muscularis of the vas deferens. Its absence resulted in a major ATP hydrolysis deficiency, as observed in situ by histochemistry and in primary smooth muscle cell cultures. In vitro, Entpd1(-/-) vas deferens displayed an exacerbated contraction to ATP, a diminished response to its non-hydrolysable analog αßMeATP, and a reduced contraction to electrical field stimulation, suggesting altered P2X1 receptor function with a propensity to desensitize. This functional alteration was accompanied by a 3-fold decrease in P2X1 protein expression in Entpd1(-/-) vas deferens with no variation in mRNA levels. Accordingly, exogenous nucleotidase activity was required to fully preserve P2X1 receptor activation by ATP in vitro. Our study demonstrates that NTPDase1 is required to maintain normal P2X1 receptor functionality in the vas deferens and that its absence leads to impaired peristalsis, reduced spermatozoa concentration in the semen, and, eventually, reduced fertility. This suggests that alteration of NTPDase1 activity affects ejaculation efficacy and male fertility. This work may contribute to unveil a cause of infertility and open new therapeutic potentials.

Genetic variants of eNOS gene may modify the susceptibility to idiopathic male infertility.

In testis, eNOS is responsible for synthesis of nitric oxide (NO) which is an essential gas message regulator in spermatogenesis, suggesting that eNOS gene plays a role in normal spermatogenesis and the genetic variants of eNOS gene may be potential genetic risk factors of spermatogenesis impairment. In this study, the polymorphic distributions of three common polymorphism loci including T-786C, 4A4B and G894T in eNOS gene were investigated in 355 Chinese infertile patients with azoospermia or oligozoospermia and 246 healthy fertile men and a meta-analysis was carried in order to explore the possible relationship between the three loci of eNOS gene and male infertility with spermatogenesis impairment. As a result, allele -786C of T-786C (11.4% versus 6.5%, p = 0.004) and 4A of 4A4B (11.0% versus 6.3%, p = 0.005) as well as genotype TC of T-786C (22.8% versus 13.0%, p = 0.002) and AB of 4A4B (18% versus 11%, p = 0.015) were significantly associated with idiopathic male infertility. The haplotypes T-4A-G (7.4% versus 4.1%, p = 0.015) and C-4B-G (7.6% versus 4.4%, p = 0.028) could increase the susceptibility to male infertility, whereas haplotype T-4B-G (67.0% versus 75.2%, p = 0.002) might be a protective factor for male infertility. The results of meta-analysis revealed that the polymorphism of T-786C was associated with male infertility. These findings suggested that the variants of eNOS gene may modify the susceptibility to male infertility with impaired spermatogenesis.

Caspase signalling pathways in human spermatogenesis.

PURPOSE: Little is known about the apoptotic mechanisms involved in abnormal spermatogenesis. In order to describe the significance of apoptosis in azoospermia, testicular tissue from abnormal spermatogenesis was analysed. METHODS: Testicular treatment biopsies were obtained from 27 men. Five presented oligozoospermia, 9 obstructive azoospermia (4 congenital bilateral absence of the vas deferens; 5 secondary azoospermia) and 13 non-obstructive azoospermia (5 hypospermatogenis; 3 maturation arrest; 5 Sertoli-cell-only syndrome). Immunohistochemical staining was performed for active caspases-3, -8 and -9. The presence of active caspases in Sertoli cells and germ cells was analyzed using stereological tools. RESULTS: Increased active caspase-3 was found in Sertoli-cell-only syndrome. No significant differences were found in maturation arrest. In hypospermatogenesis, primary spermatocytes were the germ cells with higher active caspases. Oligozoospermia and secondary obstruction showed significant differences among germ cells for the presence of all active caspases. In oligozoospermia, spermatogonia presented significant increased active caspase-9 in relation to active caspase-8. In primary obstruction and hypospermatogenesis, germ cells presented significant increased active caspases-3 and -9. CONCLUSIONS: Results suggest that increased active caspase-3 might be involved in Sertoli-cell-only syndrome etiology. In cases of hypospermatogenesis, intrinsic lesions at the meiotic stage seem to be related to the pathology. In secondary obstruction apoptosis is suggested to be initiated due to extrinsic and intrinsic lesions, whereas in primary obstruction only the intrinsic apoptotic pathway seems to be present. Finally, in oligozoospermic patients spermatogonia death by mitochondrial damage additionally to meiosis malfunctioning, might be on the origin of the decreased sperm output.

The RNase III enzyme DROSHA is essential for microRNA production and spermatogenesis.

DROSHA is a nuclear RNase III enzyme responsible for cleaving primary microRNAs (miRNAs) into precursor miRNAs and thus is essential for the biogenesis of canonical miRNAs. DICER is a cytoplasmic RNase III enzyme that not only cleaves precursor miRNAs to produce mature miRNAs but also dissects naturally formed/synthetic double-stranded RNAs to generate small interfering RNAs (siRNAs). To investigate the role of canonical miRNA and/or endogenous siRNA production in spermatogenesis, we generated Drosha or Dicer conditional knock-out (cKO) mouse lines by inactivating Drosha or Dicer exclusively in spermatogenic cells in postnatal testes using the Cre-loxp strategy. Both Drosha and Dicer cKO males were infertile due to disrupted spermatogenesis characterized by depletion of spermatocytes and spermatids leading to oligoteratozoospermia or azoospermia. The developmental course of spermatogenic disruptions was similar at morphological levels between Drosha and Dicer cKO males, but Drosha cKO testes appeared to be more severe in spermatogenic disruptions than Dicer cKO testes. Microarray analyses revealed transcriptomic differences between Drosha- and Dicer-null pachytene spermatocytes or round spermatids. Although levels of sex-linked mRNAs were mildly elevated, meiotic sex chromosome inactivation appeared to have occurred normally. Our data demonstrate that unlike DICER, which is required for the biogenesis of several small RNA species, DROSHA is essential mainly for the canonical miRNA production, and DROSHA-mediated miRNA production is essential for normal spermatogenesis and male fertility.

Possible role for glutathione-S-transferase in the oligozoospermia elicited by acute zearalenone administration in Swiss albino mice.

Zearalenone (ZEA) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium, commonly found in the soil in temperate and warm countries and is a frequent contaminant of cereal crops worldwide. Accordingly, it has been implicated in several mycotoxicosis in farm animals and in humans, but the underlying mechanisms remain largely unknown. Therefore, the current study was aimed to investigate the effect of an acute dose of ZEA (40 mg/kg, p.o.) on reproductive and hematological parameters, as well as on markers of oxidative stress in liver, kidney and testes in mice. Adult Swiss albino male mice were exposed to a single oral administration of ZEA, and 48 h thereafter behavioral and biochemical tests were performed. No differences in locomotor or exploratory activity were observed in the open-field test. On the other hand, ZEA increased the number of leukocytes, segmented neutrophils, sticks, eosinophils, monocytes and decreased platelets and lymphocytes number. Moreover, ZEA drastically reduced the number and motility of live spermatozoa. Additionally, while levels of thiobarbituric acid reactive substances (TBARS), non-protein thiols (NPSH) and ascorbic acid in liver, kidney and testes were not altered by ZEA administration, superoxide dismutase activity increased in all tissues evaluated, catalase activity increased in the kidney, and glutathione-S-transferase activity decreased in kidney and testes. In summary, we showed that ZEA have acute toxic effects mainly in reproductive system of adult male Swiss albino mice and its effect probably is related to a reduced activity of GST and increased in SOD activity in testes.

Glutathione peroxidase activity in seminal plasma and its relationship to classical sperm parameters and in vitro fertilization-intracytoplasmic sperm injection outcome.

OBJECTIVE: To relate the glutathione peroxidase (GPX) activity level in human seminal plasma with standard semen parameters and spermatozoa fertilization potential in terms of fertilization and pregnancy rates in an IVF program. DESIGN: Prospective study. SETTING: Human Reproduction Unit at Cruces Hospital (Vizcaya, Spain). PATIENT(S): Three hundred consecutive males from infertile couples participating in the IVF program. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Analysis of GPX activity in seminal plasma by spectrophotometry. RESULT(S): GPX activity in seminal plasma was significantly lower in patients with abnormal sperm as assessed by 1999 and 2010 World Health Organization (WHO) criteria, compared with normozoospermic individuals. There was a more significant decrease in those samples with severe sperm pathologies. GPX values were significantly lower in samples with severe asthenozoospermia, oligozoospermia, and teratozoospermia compared with normal samples. However, there was no correlation between GPX activity in seminal plasma in IVF patients and fertilization rates or pregnancy outcome. CONCLUSION(S): Although seminal plasma GPX activity was related to semen quality according to WHO parameters, such an association was not found with IVF-intracytoplasmic sperm injection (ICSI) outcome, presumably because of the well-known ability of IVF-ICSI procedures to overcome sperm deficiencies in the fertilization process.

Sperm caspase-9 in oligoasthenoteratozoospermic men with and without varicocele.

OBJECTIVE: To assess sperm caspase-9 activity in infertile oligoasthenoteratozoospermic (OAT) men with and without varicocele. DESIGN: Prospective. SETTING: Academic setting. PATIENT(S): Eighty men: healthy fertile control subjects (n = 20), OAT (n = 25), and OAT associated with left-side varicocele (n = 35). INTERVENTION(S): History taking, clinical examination, semen analysis,assessment of seminal caspase-9. MAIN OUTCOME MEASURE(S): Semen analysis, sperm caspase-9. RESULT(S): Sperm caspase-9 was significantly increased in infertile OAT men associated with varicocele compared with OAT men without varicocele and healthy fertile control subjects. Sperm casapse-9 activity demonstrated significant negative correlation with sperm count, sperm motility, sperm velocity, sperm linear velocity, sperm linearity index, and sperm normal morphology. CONCLUSION(S): Sperm caspase-9 is exaggerated in infertile OAT cases with varicocele compared with infertile OAT cases without varicocele or healthy fertile men. Sperm caspase-9 demonstrated significant negative correlation with semen variables.

Study of GT-repeat expansion in Heme oxygenase-1 gene promoter as genetic cause of male infertility.

PURPOSE: The length of GT-repeats polymorphic region in the promoter of human Heme oxygenase-1 gene (HO-1) alters the level of its transcriptional activity in response to oxidative stresses. Decreased level of HO-1 protein in the seminal plasma has been reported to be associated with oligospermia and azoospermia in male infertility. This is the first study to investigate the association between GT-repeats expansion in the promoter of the HO-1 gene and male infertility. METHODS: The frequencies of different GT-repeats alleles in the promoter of HO-1 gene were determined in 100 cases and 100 normal controls using PCR-PAGE, ABI fragment analysis genotyping and sequencing analysis. RESULTS: All alleles were classified into S and L alleles. S alleles were specified as number 0 to 3 with <27 GT-repeats and L alleles were specified as number 4 to 6 with >27 repeats. The L allele frequency was significantly higher among case group (54.5%) than that was obtained in the normal control group (37.5%). Statistical analysis provided a significant relationship between L allele and male infertility (P < 0.001). CONCLUSIONS: This study shows for the first time that GT-repeats expansion in promoter of the HO-1 gene is associated with oligospermia and azoospermia among Iranian infertile cases.

Reduced expression of DNMT3B in the germ cells of patients with bilateral spermatogenic arrest does not lead to changes in the global methylation status.

DNA methylation events during spermatogenesis have important implications for gamete integrity and transmission of epigenetic information to the next generation. However, the role of DNA methyltransferases in the disorders of human spermatogenesis has not been elucidated. The aim of the present study was to evaluate the expression of DNMT3B, crucial for full germ cell methylation, in testicular germ cells of patients with spermatogenic arrest and to determine whether or not there is an association with the global methylation status. In order to determine the DNMTs expression status at various stages of spermatogenesis, immunohistochemical localization was performed on 16 fertile controls having normal spermatogenesis and 11 patients with bilateral spermatogenic arrest. DNMT3B was expressed in most of the germ cell types in both controls and patients with bilateral spermatogenic arrest. The number of DNMT3B positive preleptotene/zygotene cells and pachytene spermatocytes was significantly lower in patients with bilateral arrest. However, evaluation of 5-methylcytosine, a global methylation marker, in the few matured germ cells of these patients did not reveal altered methylation. In conclusion, the global methylation status of germ cells is not affected by spermatogenic defects in spite of aberrant DNMT3B expression indicating the necessity of proper methylation for full spermatogenesis.

The association of aromatase (CYP19) gene variants with sperm concentration and motility.

The irreversible transformation of androgens into oestrogens is catalysed by cytochrome P450 aromatase. In the present study, we explored the contribution of the (TTTA)(n) polymorphism in the aromatase gene (CYP19) to sperm concentration and motility. Ninety normozoospermic and 60 oligospermic men were examined during infertility examinations. DNA was extracted from spermatozoa, and the CYP19 (TTTA)(n) polymorphism was genotyped by PCR. Genotype analysis revealed six CYP19 (TTTA)(n) alleles with 7-12 repeats. The allelic distribution of the CYP19 (TTTA)(n) polymorphism differed between normozoospermic and oligospermic men (P<0.01). Oligospermic men less frequently had long CYP19 alleles than did normozoospermic men (25 and 37.8%, respectively; P<0.02). The higher frequency of short CYP19 alleles in oligospermic men compared to normozoospermic men (43.3 and 28.3%, respectively; P<0.01) was primarily due to the distribution of the CYP19 (TTTA)(7) allele. The CYP19 (TTTA)(7) allele was associated with lower sperm concentration in normozoospermic men (P<0.01) and in the total study population (P<0.01); it was also associated with lower sperm motility in normozoospermic men (P<0.05) and in the total study population (P<0.01). In conclusion, the CYP19 (TTTA)(7) allele probably impairs aromatase activity, which in turn alters aromatase and oestrogen levels in the testis, leading to decreased sperm concentration and motility. These findings support the significance of cytochrome P450 aromatase in human spermatogenesis and consequently in semen quality.