A to G transitions at 260, 386 and 437 in DAZL gene are not associated with spermatogenic failure in Indian population

The autosomal DAZL (Deleted-in-Azoospermic-Like) gene, mapped to the short arm of the human chromosome 3, is the precursor for the Y-chromosomal DAZ cluster, which encodes for putative RNA-binding proteins. Mutations in the DAZL have been reported to be associated with spermatogenic failure in Taiwanese population but not in Caucasians. As there was no study on Indian populations, we have analysed the entire coding sequences of exons 2 and 3 of DAZL in a total of 1010 men from Indian subcontinent, including 660 infertile men with 598 non-obstructive azoospermia, 62 severe oligozoospermia and 350 normozoospermic fertile control men, to investigate whether mutation(s) in the DAZL is associated with male infertility. Interestingly, none of our samples (1010) showed A386G (T54A) mutation, which was found to be associated with spermatogenic failure in Taiwanese population. In contrast, A260G (T12A) mutation was observed in both infertile and normozoospermic fertile control men, without any significant association with infertile groups (chi2= 0.342; p = 0.556). Similarly, we have found a novel A437G (I71V) mutation, which is also present in both infertile and normozoospermic fertile control men without any significant difference (chi2 = 0.476; p = 0.490). Our study clearly demonstrates the complete absence of the A386G (T54A) mutation in Indian subcontinent and the other two mutations --A260G (T12A) and A437G (I71V)--observed are polymorpic. Therefore, we conclude that these mutations in the DAZL gene are not associated with male infertility in Indian subcontinent.     

Association study of single-nucleotide polymorphisms in FASLG, JMJDIA, LOC203413, TEX15, BRDT, OR2W3, INSR, and TAS2R38 genes with male infertility

Infertility is a major health problem today, affecting about 15% of couples trying to conceive a child. Impaired fertility of the male factor is causative in 20% of infertile couples and contributory in up to another 30%-40%. Based on association studies, an increasing number of gene polymorphisms have been proposed to modulate the efficiency of spermatogenesis. Here, we have investigated the possible association of 9 single-nucleotide polymorphisms (SNP) in 8 different genes-FASLG, JMJDIA, LOC203413, TEX15, BRDT, OR2W3, INSR, and TAS2R38--with male infertility. We analyzed a total of 136 men with idiopathic infertility (60 azoospermic and 76 oligozoospermic) and 161 fertile controls. Our study group included individuals of different ethnic origin: 93 of the infertile men were Macedonians, 32 were Albanians, and 11 were of other origin. The control group was composed of 125 Macedonian and 36 Albanian men. The methodology included multiplex polymerase chain reaction/SNaPshot analyses, followed by capillary electrophoresis on an ABI3130 Genetic Analyzer. Of the 9 SNPs evaluated, 3 are significantly associated (P < .05) with male infertility: SNPs rs5911500 in LOC203413, rs3088232 in BRDT, and rs11204546 in OR2W3. SNP rs5911500 showed the strongest association with infertility among Albanians (P = .0001), whereas rs3088232 was most significantly associated with azoospermia among Macedonians (P = .0082). Moreover, the frequency of co-occurrence of LOC203413 minor T allele with either homozygosity or heterozygosity for the BRDT minor G allele was significantly higher among both azoospermic (6 of 60 [10%]; P = .0057; odds ratio [95% confidence interval], 8.83 [1.73-45.08]) and oligozoospermic (10 of 76 [13.2%]; P = .0002; odds ratio [95% confidence interval], 12.04 [2.57-56.47]) men in comparison with fertile controls (2 of 161 [1.2%]).     

CAG-repeat variant in the polymerase γ gene and male infertility in the Chinese population： a meta-analysis

Several studies have reported a relationship between the length of the CAG-repeat in the polymerase γ (POLG) gene and male infertility. However, other studies have not reproduced this result. In our study, the POLG-CAG-repeat length was analyzed in 535 healthy individuals from six Chinese Han populations living in different provinces. The frequencies of 10-CAG alleles and genotypes were high (97.38 and 94.13%, respectively), with no significant difference among the six Chinese Han populations. Furthermore, we determined the distribution of the POLG-CAG-repeat in 150 infertile men and 126 fertile men. Our study suggested that the distributions of POLG-CAG-repeat alleles and genotypes were not significantly different between infertile (95.67 and 92.67%, respectively) and fertile men (97.22 and 94.44%, respectively). In a subsequent meta-analysis, combining our data with data from previous studies, a comparison of the CAG-repeat alleles in fertile versus infertile men showed no obvious risk for male infertility associated with any particular allele (pooled odds ratio (OR)=0.94; 95% confidence interval (CI): 0.60-1.48). The significance level was not attained with any of the following genetic models: homozygote comparison (not 10/not 10 versus 10/10: OR=1.34; 95% CI: 0.66-2.72), heterozygote comparison (10/not 10 versus 10/10: OR=1.04; 95% CI: 0.78-1.38), dominant model comparison (not 10/not 10+10/not 10 versus 10/10: OR=1.08; 95% CI: 0.79-1.47) and recessive genetic comparison (not 10/not 10 versus 10/not 10+10/10: OR=1.31; 95% CI: 0.68-2.55). In conclusion, there is no significant difference of the frequencies of POLG-CAG-repeat variants among six Chinese Han populations, and this polymorphism may not be associated with Chinese male infertility. On the basis of a meta-analysis, there is no obvious association between CAG-repeat variants of the POLG gene and male infertility.     

Kisspeptin and attributes of infertile males and females: A cross‐sectional study in a subset of Pakistani population

Kisspeptin, a peptide hormone, plays a pivotal role in fertility and neuroendocrine regulation of hypothalamo–pituitary–gonadal axis. Increased kisspeptin and reproductive hormones are responsible for fertility in male and females. This study aimed to explore the role of kisspeptin on hypothalamo–pituitary–gonadal axis by comparing the levels of kisspeptin in fertile and infertile subjects and identifying single nucleotide polymorphisms (SNPs) of KISS1 gene in exon 2 and exon 3 of infertile male and female cohorts. A cross‐sectional study was carried out on 80 males (44 infertile and 36 fertile) and 88 females (44 in each group). Significantly high levels of kisspeptin (KP), follicle‐stimulating hormone (FSH), luteinizing hormone and testosterone were observed in fertile male and female subjects except low FSH levels in comparison with infertile female subjects. One polymorphism in exon 2 (E1225K [G/A 3673]) and three in exon 3 (P1945A [C/G 5833]; Insertion of T at 6075; G2026G [C/G 6078]) in infertile group were detected, with low KP and hormonal levels. Male subjects had abnormal sperm parameters and unsuccessful attempt of intracytoplasmic sperm injection in females. Expression of SNP in exon 2 and exon 3 of KISS1 could be responsible for alteration in release of reproductive hormones and gonadal functions, hence causing infertility.     

Role of glutathione S‐transferase Mu‐1 (GSTM1) polymorphism in oligospermic infertile males

The aim of this study was to examine whether an association exists between glutathione S‐transferase Mu‐1 (GSTM1) gene polymorphism and idiopathic male infertility. Forty‐two men with infertility and 43 fertile men were recruited for this study. GSTM1 gene was analysed using PCR technique. The frequency of GSTM1 null (?) genotype was observed to be 45.2% in infertile men as against 20.09% in fertile men. Subjects with the GSTM1 null genotype had lower sperm concentrations and motility when compared with the subjects with GSTM1‐positive genotype in both the groups. This study shows that the frequency of GSTM1 null (?) genotype is significantly high in infertile males when compared with the frequency in fertile males (OR = 0.32, P = 0.017, 95% CI = 0.124–0.831).     

Cytogenetic and molecular analysis of the Y chromosome: absence of a significant relationship between CAG repeat length in exon 1 of the androgen receptor gene and infertility in Indian men

The genetic basis of male infertility remains unclear in the majority of cases. Recent studies have indicated an association between microdeletions of the azoospermia factor a (AZFa)-AZFc regions of Yq and severe oligospermia or azoospermia. Increased (CAG)n repeat lengths in the androgen receptor (AR) gene have also been reported in infertile men. Therefore, in order to assess the prevalence of these genetic defects to male infertility, 183 men with non-obstructive azoospermia (n = 70), obstructive azoospermia (n = 33), severe oligospermia (n = 80) and 59 fertile men were examined cytogenetically and at molecular level for Yq deletions, microdeletions, and AR-CAG repeat lengths along with hormonal profiles [luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone (T)]. We used high resolution cytogenetics to detect chromosome deletions and multiplex polymerase chain reaction (PCR) involving 27 sequence-tagged site (STS) markers on Yq to determine the rate and extent of Yq microdeletions. PCR amplification with primers flanking exon 1 of AR gene was used to determine the AR-(CAG)n repeat lengths. Hormonal profiles (LH, FSH and T levels) were also analysed in infertile and fertile men. Testicular biopsies showed Sertoli cell only (SCO) morphology, maturation arrests (MA) and hypospermatogenesis. No chromosome aberrations were found in infertile men but there was a significant increase (p < 0.001) in the association of acrocentric chromosomes including the Y chromosome. Yq microdeletions were found in 16 non-obstructive azoospermic men (16 of 70; 22%) and seven severe oligospermic individuals (seven of 80; 8.7%) and most of them had deletions in the sY240 locus. No Yq microdeletions were detected in patients with obstructive azoospermia. No statistically significant difference in the mean length of CAG repeats in AR gene was observed between infertile and fertile men (22.2 +/- 1.5 and 21.5 +/- 1.4 respectively). No significant increase or decrease in levels of LH, FSH and T was observed in infertile and fertile men. In some infertile men, significantly elevated levels of FSH alone or in combination with LH were found to be indicative of failure of spermatogenesis and/or suggestive of testicular failure. Y-chromosome microdeletions contribute to infertility in some patients but no relationship could be established with the (CAG)n repeat lengths in exon 1 of the AR gene in infertile Indian men.     

Soluble Fibrinogen-like protein 2 plays a role in varicocele induced male infertility

Varicocele is the most common cause of male infertility. Several theories have been proposed to explain how varicocele induces infertility. The role of epididymis in male infertility is not fully well established. Fibrinogen-like protein 2 is one of serine proteases and is a potent coagulant in membranous form and immune-modulator in soluble form (sFGL-2) and expressed in the epididymis. There are no previous reports about its possible role in varicocele. This case-controlled study aimed to evaluate the seminal level of sFGL-2 in infertile men with varicocele and in men with idiopathic infertility. This study included 85 participants divided into three groups; 25 normal fertile men, 30 infertile men with varicocele and 30 infertile men of idiopathic cause. Clinical examination, Doppler ultrasound, semen analysis and measurement of seminal level of sFGL-2 were done to all participants. Seminal level of sFGL-2 was significantly elevated in infertile than normal fertile men. Seminal level of sFGL-2 showed negative correlations with sperm concentration, motility and normal morphology. Seminal level of sFGL-2 had a positive correlation with seminal liquefaction time. This study concluded that seminal level of sFGL-2 is increased in infertile men with idiopathic cause and with varicocele induced infertility and affects seminal liquefaction.     

The CAG repeat polymorphism of mitochondrial polymerase gamma (POLG) is associated with male infertility in Tunisia

Male fertility largely depends on sperm quality, which may be affected by environmental and genetic factors. Recent data emphasised the implication of the polymorphism of mitochondrial DNA polymerase gamma (POLG) CAG repeats in male infertility. In this report, we explored a possible role of the (POLG) gene polymorphism in male infertility in Tunisian men. The polymorphic CAG repeat in the nuclear POLG gene was studied in 339 male subjects (216 patients with infertility (69 azoospermic, 115 oligoasthenoteratospermic and 32 normospermic) and 123 fertile) after DNA amplification by PCR, followed by genotyping using an automatic sequencer. The heterozygous and the homozygous mutant genotypes (10/ ≠ 10 and ≠ 10/ ≠ 10) were significantly more frequent among infertile patients than among fertile controls (11.2% versus 1.6%, P = 1.3 × 10(-3) and 4.6% versus 0.8%, P = 4.2 × 10(-7) respectively). We also found a significant difference between the frequencies of 10/ ≠ 10 genotype in azoospermic (4.4%) and in oligoasthenoteratospermic (15.6%) infertile patients (P = 2.6 × 10(-2) ). However, the homozygous mutant genotype (≠ 10/ ≠ 10) was seen at similar frequencies in azoospermic, normospermic and oligoasthenospermic men (4.4%, 3.1% and 5.2% respectively). Under our conditions, the findings showed an association between POLG CAG repeat polymorphism and male infertility in Tunisian population.     

Mutation analysis of the BOULE gene in men with non-obstructive azoospermia: identification of a novel polymorphic variant in the black population

The BOULE gene is a member of the 'deleted in azoospermia'DAZ family. As in flies and worms, disruption of the BOULE homologues leads to meiotic defects during the first meiotic division, we screened the coding region of the BOULE gene from 40 infertile men with non-obstructive azoospermia or severe oligoasthenoteratozoospermia by denaturing high-pressure liquid chromatography and direct sequencing in order to make progress in elucidating the aetiology of male infertility. Two infertile patients were heterozygous for a novel G to C transversion in exon 2 resulting in a Q2E amino acid substitution. As these two infertile men were from African origin, screening of fertile African subjects identified this novel variant in two fertile male subjects suggesting that this novel Q2E substitution had non-pathologic role. Taking into account the size of our sample, we conclude that BOULE coding sequence mutations are not an important factor in the aetiology of azoospermia.     

CA repeat and RsaI polymorphisms in ERbeta gene are not associated with infertility in Indian men

Oestrogen Receptor β (ERβ) gene plays an important role in the regulation of fertility in both males and females. Polymorphism in CA repeat located in the flanking region of ERβ has been shown to be associated with several diseases, but its association with male infertility has not been analysed so far. However, Rsa I polymorphism (rs1256049) in exon 5 of ERβ has been shown to be associated with male infertility in Caucasian patients. Hence, we have analysed 695 Indian men, including 443 infertile and 252 fertile men to evaluate the association of CA repeat length and Rsa I polymorphisms in male infertility. Our results revealed no significant difference in the distribution of CA repeat length between infertile (mean ± SD 23.24 ± 2.06, median 24) and fertile men (mean ± SD 23.16 ± 2.27, median 24). The analysis of dosage effect by classifying samples into SS (short/short), SL (short/long) and LL (long/long) groups also did not show any significant difference between infertile and fertile men. Similarly, Rsa I polymorphism also did not show any significant difference between infertile and fertile men. Furthermore, the combined analysis of CA repeat and Rsa I polymorphisms by haplotyping showed that the distribution of haplotypes was not significantly different between fertile and infertile men. Our results suggest that CA repeat length and Rsa I polymorphisms in ERβ are not associated with infertility in Indian men.     

睾丸微结石的超声诊断及与男性不育的关系

目的:探讨睾丸微结石(TM)的超声声像图表现及其与男性不育症的相关性。方法:应用高分辨率二维超声常规检查1198例阴囊疾病患者。结果:其中男性不育患者379例,TM检出21例(5.54%),阴囊内其他疾病患者819例,TM检出12例(1.47%)。TM与男性不育相关(r=0.115,P<0.01),TM在男性不育中发病率显著高于阴囊内其他疾病患者(P<0.05)。TM超声声像图表现为于睾丸实质内可见稀疏?散在或密集分布的强光点,直径在1mm左右,不伴有声影。结论:TM超声声像图特征明显,易于明确诊断;TM与男性不育存在一定相关性。     

Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men

The aim of the study was to examine the relationships between concentrations of zinc in blood and seminal plasma and sperm quality among infertile and fertile men. One hundred seven male (infertile group) partners of couples who were undergoing investigation for infertility with no known cause for the infertility and 103 men (fertile group) whose wives were pregnant at the time of the study were recruited. The subjects' blood and seminal plasma concentration of zinc were determined by atomic absorption spectroscopy. Except for semen volume, all the other semen parameters for the infertile men were significantly lower than those for the fertile group. The geometric means of the seminal plasma zinc concentration were significantly lower in the infertile group compared with those in the fertile group; 183.6 mg/L (range, 63-499) versus 274.6 mg/L (range, 55-420). There were no significant differences in the geometric means of the blood zinc concentration between the 2 groups. Seminal plasma zinc concentration was significantly correlated with sperm density (r = 0.341, P < .0001), motility (r = 0.253, P < .0001), and viability (r = 0.286, P < .0001). On the basis of the findings of this study and those of other reports, zinc may contribute to fertility through its positive effect on spermatogenesis.     

男性不育症患者DAZ基因缺失研究

为探讨ＤＡＺ基因在男性生精过程中的作用及ＤＡＺ基因与男性不育症的关系。本文应用多重聚合酶链反应对男性不育症患者ＤＡＺ基因进行检测。并结合临床表型进行分析。结果在正常有生育力的男性、精子数正常不育男性、已知原因无精子少精子症患者中皆无ＤＡＺ基因缺失;在特发性无精子症患者中检测出ＤＡＺ缺失,缺失比例高达１５％（５／３４）,其中无精子症１７％（３／１８）,少精子症１３％（２／１６）。提示ＤＡＺ基因在生精过程中起重要作用,ＤＡＺ基因缺失是导致特发性无精子少精子症的原因之一。     

泛素特异蛋白酶26基因多态性与特发性男性不育症的相关性研究

目的:研究泛素特异蛋白酶26(Usp26)基因多态性与特发性男性不育的关系及其在精子发生过程中的作用机制。方法:按照WHO标准(第4版)从150例不育患者中筛选出41例特发性不育患者,同时选取50例正常生育男性作为对照。采用PCR-SSCP法,从特发性不育患者中筛选突变样本,通过基因测序以确定突变方式和位点。结果:筛选出的41例特发性男性不育患者主要表现为精子浓度低、活动率差。基因测序分析结果显示:41例不育患者中9例(22.0%,P=0.01)存在Usp26基因的改变。其中,从8例(19.5%,P=0.01)患者中检测出复合突变:364位插入ACA和460位A置换了G;从1例(2.4%,P>0.05)患者中检测出1 044位A置换了T。以上3种变化均导致编码氨基酸的改变。50例正常生育男性均未发现该基因的突变。结论:Usp26基因的多态性可能与特发性男性不育症密切相关,且影响睾丸功能。     

A single-nucleotide polymorphism in TP53 may be a genetic risk factor for Iranian patients with idiopathic male infertility

Male infertility is a heterogeneous disorder that contributes to the impairment of spermatogenesis. The purpose of this study was to assess whether tumour suppressor p53 gene (Tp53) polymorphism is associated with idiopathic male infertility in the Iranian population. The prevalence of G → C substitution at codon 72 in exon 4 was determined in 110 idiopathic infertile azoo-/oligospermic patients and 180 fertile healthy control men. PCR-restriction fragment polymorphism analysis was employed to determine the genotypes. PCR amplicons were subjected to restriction digestion with Bstu1 and separated by gel electrophoresis. The Arg/Arg genotype was found more frequently among men with idiopathic infertility (32.7%) than among controls (17.7%). No significant difference was observed between Pro/Pro genotype and Arg/Arg + Arg/Pro genotypes among men with idiopathic infertility and controls (P = 0.11; OR: 0.36; 95% CI: 0.36-1.10). In contrast, a significant difference was observed in the comparison of the Arg/Arg genotype and Arg/Pro + Pro/Pro genotypes among patients and controls (P = 0.004; OR: 2.25; 95% CI: 1.29-3.90). Allele frequency evaluation suggested a significantly higher incidence of the Arg allele among infertile men compared with controls (56% versus 44%; OR: 1.6; 95% CI: 0.92-2.80). In conclusion, arginine allele appears to be at greater risk of developing idiopathic infertility in Iranian men.     

Assessment of seminal granulysin in infertile men with varicocele

Varicocele has a common association with male infertility, but its exact role is still debated. Apoptosis has been suggested as one of the mechanisms of varicocele‐associated infertility. Granulysin is a molecule that plays a role in apoptosis with no previous study about its role in male infertility. This case‐controlled study aimed to assess seminal plasma granulysin level in infertile patients with varicocele. This study involved 90 men that were allocated into fertile normozoospermic men (n = 20), infertile men without varicocele (n = 30) and infertile men with varicocele (n = 40). These men were subjected to history taking, clinical examination, semen analysis and estimation of seminal granulysin. In general, seminal granulysin level was significantly elevated in infertile men compared with fertile men. Infertile men with varicocele showed significantly higher seminal granulysin compared with infertile men without varicocele, in bilateral varicocele cases and in grade III varicocele. Seminal granulysin level was negatively correlated with sperm concentration, sperm motility, sperm normal forms percentage and testicular volumes. It is concluded that increased seminal granulysin has a negative impact on spermatogenesis in infertile men in general and in infertile men associated with varicocele in particular.     

精浆游离L-肉毒碱水平及其与精子密度、活动率及活力的相关性研究

目的:研究正常生育及不育男性精浆中游离L-肉毒碱水平差异及其与精子密度、活动率(a+b+c级精子百分率)及活力(a+b级精子百分率)之间的相关性,探讨精浆中游离L-肉毒碱水平对男性生育力的影响及其在不育症检查和治疗中的作用。方法:分别采用高效液相色谱法和计算机辅助精液分析系统,测定了230例不育症患者(精子密度正常117例,少精子症81例,无精子症32例)和30例正常生育男性精浆中游离L-肉毒碱水平及精子密度、活动率、活力等参数。根据检查结果对不育症患者分组后,以SPSS12.0软件包进行统计学分析,比较各组间游离L-肉毒碱水平的差异以及游离L-肉毒碱水平与精子密度、活动率、活力之间的相关性。结果:正常生育组精浆游离L-肉毒碱水平明显高于不育组(P<0.01)。精液中精子密度越低、活力越弱,这种差异性越显著。相关性分析结果显示,精浆游离L-肉毒碱水平与精子密度呈显著正相关关系(r=0.521,P<0.01),与精子活动率和活力之间也具有正相关关系(r=0.319,P<0.01;r=0.251,P<0.01)。结论:精浆L-肉毒碱水平与精子密度、活动率和活力之间密切相关,其含量测定作为一项有用的生化指标,可为男性不育症检查及临床诊治和进行有关男性生殖功能机制研究提供参考。     

Screening of ΔF508 mutation and IVS8-poly T polymorphism in CFTR gene in Tunisian infertile men without CBAVD

It is well established that cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations are involved in congenital bilateral absence of the vas deferens (CBAVD), causing obstructive azoospermia and male infertility. Also, several studies reported a relatively high prevalence of CFTR gene mutations in healthy men presenting reduced sperm quality. In this study, we investigate ΔF508 mutation and IVS8-polyT polymorphism in CFTR gene in Tunisian infertile men without CBAVD. Genetic analyses were performed in 148 infertile patients and 126 fertile individuals. The polymorphic IVS8-polyT tract in CFTR gene was analysed in only 129 infertile patients and 54 individuals of control group. As well, we screened for Y chromosome microdeletions in all infertile patients. No ΔF508 mutation was diagnosed either in infertile patients or in control group. 5T allele of IVS8-polyT tract was found in both infertile men (4.26%) and fertile individuals (8.33%). 5T/5T genotype was observed only in two azoospermic patients without Y microdeletions. The most frequent genotype of IVS8-polyT tract in infertile men and controls was 7T/7T (69.75% and 59.25% respectively). There was no association between IVS8-polyT polymorphism and reduced semen quality. Neither ΔF508 mutation nor 5T allele is involved in pathogenesis of male infertility in Tunisian infertile patients without CBAVD.     

Molecular evaluation of CFTR sequence variants in male infertility of testicular origin

Although the involvement of the CFTR gene has been well established in congenital agenesia of vas deferens, its role in non-obstructive (NOb) infertility is still a matter of debate. In order to definitively define the involvement of the CFTR gene in spermatogenic impairment and a potential synergistic contribution to known genetic and clinical factors, genetic variants in the entire coding sequence and the immediately flanking regions of the CFTR gene, along with a thorough clinical evaluation, were analysed in 83 NOb infertile patients and 87 clinically well-defined fertile individuals as controls. The results of our study showed no statistical difference between CFTR carrier frequency in the infertile and fertile population. Specifically, the IVS8-6(5T) allele carrier frequency was similar in NOb infertile patients when compared with fertile men, but it is noteworthy that, when fertile men were classified into having optimal and suboptimal fertility, no 5T allele was found among the 35 men with optimal fertility parameters. In conclusion, extensive CFTR analysis in infertile individuals and fertile population as adequate control definitively excludes the involvement of the CFTR gene variants in sperm production and stresses the importance of carefully identifying those individuals with obstructive defects, in whom CFTR screening will be beneficial.