Testicular testosterone concentration, interstitial cell density and spermatogenesis in infertile men

The concentration of testosterone was estimated in testicular biopsies taken from infertile men under general anaesthesia and was found to vary between 0.77 and 9.87 nmol/g wet tissue (median 3.41 nmol/g). Spermatogenesis was assessed by the Johnsen scoring technique but there was no evidence of a deficiency of intratesticular testosterone being associated with dysfunction of the seminiferous tubules. Similarly, there was no direct relationship with testicular size, clinical diagnosis, or the concentration of testosterone found in peripheral plasma taken at the same time as the testicular biopsy. These negative findings were not explained by variation in the volume densities of the Leydig cells assessed in 10 biopsies selected to be representative of the range of testosterone concentrations found in the entire series. A retrospective comparison between the concentration of testosterone in peripheral plasma at the time of surgery and the available preoperative levels found in 10 patients provided further evidence for the previously reported lowering of testosterone levels by general anaesthesia. It is suggested that future studies of testicular androgens should be conducted on tissue obtained under local anaesthesia in order to eliminate this effect.     

Seminal level of clusterin in infertile men as a significant biomarker reflecting spermatogenesis

The objective of this study was to assess the impact of seminal clusterin level on spermatogenesis in infertile men. This study included 89 men who visited our clinic due to infertility, consisting of 28, 33, and 28 diagnosed with normospermia, oligozoospermia and nonobstructive azoospermia (NOA) respectively. The seminal clusterin concentrations measured by enzyme‐linked immunosorbent assay were 47.9, 28.2 and 18.4 ng ml^?1 in men with normospermia, oligozoospermia and NOA, respectively, with significant differences among these three groups (P < 0.01). Microdissection testicular sperm extraction (MD‐TESE) was performed in the 28 men with NOA, and spermatozoon was successfully retrieved from 9. There was a significant correlation between seminal clusterin level and testicular clusterin protein expression evaluated by immunohistochemical staining in these men with NOA (P = 0.026). Of several parameters available before MD‐TESE, the univariate analysis identified serum follicle‐stimulating hormone (FSH) level <10 IU ml^?1 and seminal clusterin level ≥18 ng ml^?1 as significant predictors of sperm retrieval, and of these, only serum FSH level <10 IU ml^?1 was shown to be independently associated with sperm retrieval in the multivariate analysis. Accordingly, it might be worthy to further evaluate the significance of seminal clusterin level as a biomarker for the assessment of spermatogenic status in infertile men.     

无精子症和严重少精子症患者Y染色体微缺失和基因缺失研究

目的研究中国特发性无精子症和少精子症患者Y染色体无精子症因子(AZF)区缺失和其中RBMY1A1、DAZ基因缺失。方法选取AZFa、b和c区6个序列标签位点(STS)对56例少精子症和33例无精子症患者进行外周血Y染色体微缺失分析,对缺失样本进行RBMY1A1和DAZ基因缺失分析。结果共确认6例患者发生Y染色体微缺失和基因缺失、占7%(6/89);其中5例AZFc/DAZ基因缺失,1例AZFb+c/RBMY1A1和DAZ基因缺失。结论AZF部分区域缺失的患者同时伴有与精子生成具有重要作用的基因缺失,并可能由此导致精子生成障碍。     

Sensitivity and specificity of serum FSH and testis size in predicting the existence of spermatogenesis in azoospermic infertile men

The objective of research was to determine the sensitivity, specificity and positive predictive value of serum follicle stimulating hormone and testis size in predicting spermatogenesis in infertile men with azoospermia. In a prospective study, azoospermic men were studied. Serum follicle stimulating hormone measurement and scrotal sac ultrasonography were performed. Bilateral testis biopsy was performed for all of these patients. The sensitivity and specificity of follicle stimulating hormone and testis size were determined to predict the existence of different cellular steps of spermatogenesis. Of eighty infertile men who recruited into the study, 53 patients did not represent any different cellular steps of spermatogenesis, while 27 of them had various steps of such differentiation. Among the 53 patients without cellular steps of spermatogenesis in the biopsy, 41 were predicted to be azoospermic based on their serum follicle stimulating hormone levels (77.3% sensitivity), and of 27 patients with various cellular steps of spermatogenesis in the biopsy, 23 were predicted to have spermatozoa according to the follicle stimulating hormone level (85.2%) specificity. It is suggested that combination of these two indicators can substitute the invasive testis biopsy for predicting the existence of spermatozoa in infertile men with azoospermia.     

Investigation of serum vitamin D levels in Chinese infertile men

Recently, the question of whether vitamin D exerts an effect on the pathogenic process of infertility has become the centre of attention. There are some controversial conclusions on this issue. Based on previous studies, we sought to explore the difference of serum 25‐hydroxyvitamin D₃, 1,25‐dihydroxyvitamin D₃ levels between infertile patients and fertile men, and to find the influence on semen quality. The analysis of serum 25‐hydroxyvitamin D₃ level showed no significant difference between infertile patients and fertile men. However, the levels of serum 1,25‐dihydroxyvitamin D₃ in oligospermia (P < 0.05), asthenospermia (P < 0.01), oligoasthenospermia (P < 0.05) and azoospermia (P < 0.01) patients were significantly lower than those in fertile men. Moreover, serum 1,25‐dihydroxyvitamin D₃ level was positively correlated with progressive motility and total sperm number in infertile patients. In addition, a positive correlation between serum prolactin and 1,25‐dihydroxyvitamin D₃ was observed in fertile men. Our results indicated that lower vitamin D could be a risk factor for poor semen quality in infertile men. The 1,25‐dihydroxyvitamin D₃, as the biologically active form of vitamin D, may be more significant.     

Nitric oxide produced in the testis is involved in dilatation of the internal spermatic vein that compromises spermatogenesis in infertile men with varicocele

OBJECTIVE: To determine the concentration of nitric oxide (NO) and the location and change in the expression of NO synthase (NOS) isoforms in the testes of subfertile men with varicocele, and to compare the NO concentration or NOS expression with clinical variables, to determine the role of NO on the pathophysiology of varicocele. PATIENTS AND METHODS: In all, 27 men who had a left varicocelectomy and five with 'normal' spermatogenesis (controls) who had scrotal surgery for other reasons were enrolled. Intratesticular fluid was taken from the men and the NO concentration determined colorimetrically. The expression and location of NOS isoforms were examined by Western blotting and immunohistochemistry, using testicular biopsy specimens, and NADPH-diaphorase (NADPH-d) staining used to identify NO-producing cells. The relationship between the NO concentration and the expression of NOS isoforms or clinicopathological variables was investigated. RESULTS: In testes with grade 2 and 3 varicoceles there were significant increases in the concentration of NO or the expression of inducible NOS. There was no change in the expressions of endothelial NOS, which is located in vascular endothelial cells, while NADPH-d activity was mainly located in these cells. The concentration of NO was significantly correlated with the maximum and total vein diameter (both P<0.01). In patients aged>35 years, the concentration of NO significantly correlated with a deterioration in total motile sperm count (P<0.05). CONCLUSIONS: Increased production of NO in the testis is involved in the enlargement of varicocele and indirectly deteriorates spermatogenesis.     

Seminal leucocyte subpopulations and sperm function in fertile and infertile Chinese men

The level of seminal leucocytes and the prevalence of leucocytospermia was determined in a group of fertile and infertile southern Chinese men in Hong Kong. Sixteen normal fertile semen donors and 49 men with male factor infertility were studied prospectively. None had antisperm antibodies and past or present evidence of genital tract infection. Seminal leucocytes and their subsets were analysed using monoclonal antibodies and an immunocytochemical alkaline phosphatase-anti-alkaline phosphatase conjugate technique. Seminal leucocytes were detectable in 94% and 86% of the fertile and infertile men respectively, with the predominant subset being granulocytes. Leucocytospermia (> 1 × 10⁶ leucocytes/ml) was found in only one of the 49 (2%) infertile men without clinical evidence of genito-urinary infection. Inverse correlations were observed between (1) the percentage of spermatozoa with normal morphology and the number of T-helper/inducer cells, (2) the linearity of sperm movement and the number of T-lymphocytes. In conclusion, the level of seminal leucocytes and the prevalence of leucocytospermia is low in infertile Chinese subjects. The effect of seminal leucocytes on sperm function in these subjects needs further evaluation.     

Expression of hyperacetylated histone H4 during normal and impaired human spermatogenesis

Histone-to-protamine exchange in haploid spermatids is preceded by hyperacetylation of core histones resulting in decreased DNA-histone interaction. During normal spermatogenesis, immunohistochemistry with a polyclonal antihyperacetylated histone H4 antibody displayed a strong signal in nuclei of elongating spermatids and, in addition, spermatogonia. Quantitative analysis revealed 98.2 +/- 1.1% of immunopositive spermatids. The percentage of positive spermatids was significantly reduced in infertile men exhibiting at least qualitatively normal spermatogenesis (scores 10-8, 93.1 +/- 6.6%) and impaired spermatogenesis (scores 7-1, 74.9 +/- 23.4%). In seminiferous tubules showing spermatogenic arrest at the level of round spermatids, only 59.5 +/- 16.5% of spermatids were immunopositive for hyperacetylated histone H4. These data demonstrate that the decrease of histone acetylation in spermatids associated with impaired spermatogenesis corresponds with the well known reduction of protamine expression in these cells and confirms the essential role of histone hyperacetylation for correct histone-to-protamine exchange. In seminiferous tubules exhibiting round spermatid maturation arrest, there was an additional signal in nuclei of spermatocytes, suggesting that premature hyperacetylation of histone H4 may result in precocious histone-to-protamine exchange followed by infertility. This is in accordance with data from transgenic mice, where it has been demonstrated that premature expression of protamine-1 results in precocious chromatin condensation followed by sterility.     

中国男性不育患者染色体核型及Y染色体微缺失分析

目的 探讨男性不育症与染色体畸变及Y染色体微缺失之间的关系.方法临床诊断男性不育患者1975例,采集外周血淋巴细胞常规培养,Giemsa染色,镜下观察并分析染色体核型;选取Y染色体特异性序列标签点(STS),应用PCR技术对无精子症及少精子症患者进行Y染色体微缺失检测.结果 1975例患者中,染色体核型异常305例(15.44%),其中常染色体异常101例(5.11%),患者主要表现为少精子症、畸形精子症;性染色体异常204例(10.33%),主要表现以克氏征(5.62%)为主.728例无精子症或少精子症患者中,Y染色体微缺失109例(14.97%),其中AZFa区缺失3例(2.75%),均表现为无精子症;AZFb区缺失5例(4.59%),表现为无精子症2例、严重少精子症2例,精液正常1例;AZFc区缺失者68例(62.39%),患者主要表现为无精子症和严重少精子症;AZFa区和AZFc区均缺失者5例(4.59%),均表现为无精子症;AZFb区和AZFc区均缺失者15例(13.76%),患者以无精子症表现为主;AZFa区、AZFb区和AZFc区均缺失者6例(5.50%),均表现为无精子症.结论染色体异常及Y染色体微缺失均为男性不育的重要病因.

Abstract：

Objective To study the relationship between chromosomal abnormality and Y chromosome microdeletions and male infertility. Methods Lymphocytes were cultured from peripheral blood of 1975 male infertility patients and stained with Giemsa. The chromosomes were analyzed under microscope. Y chromosome specific sequence tags (STS) were selected, then the Y chromosome microdeletions in AZF regions were screened by polymerase chain reaction (PCR) in azoospermia and oligozoospermia patients. Results There were 305 cases of detected chromosomal abnormalities (15.44%) in the 1975 cases. There were 101 cases (5.11 %) with autosome abnormalities which clinically manifested as oligozoospermia and teratospermia. There were 204 cases (10. 33%) of sexual chromosome abnormalities and the patients were mainly characterized with Klinefelter's syndrome. Y chromosome microdeletions were detected in 109 (14.97 %) of the 728 cases of azoospermia or oligozoospermia. The most common microdeletion of Y chromosome was AZFc (62.39%) and these patients were characterized with azoospermia and oligozoospermia. Five patients (4. 59%) who suffered Y chromosome microdeletion in AZFa region and AZFb region were characterized with azoospermia. Fifteen cases (13.76%) with microdeletion in AZFb region and AZFc region were mainly characterized with azoospermia. There were 6 cases (5. 50 % ) of microdeletion in AZFa, AZFb and AZFc regions,these patients were all characterized with azoospermia. Conclusions Both Chromosome abnormalities and Y chromosome microdeletions are important causes for male infertility.     

Aneuploidy in spermatozoa of infertile men with teratozoospermia

Recent studies have shown that aneuploidy in spermatozoa of infertile men with poor semen quality is increased. The purpose of this study was to determine whether poor sperm morphology is associated with the incidence of spermatozoa with numerical chromosome abnormalities. Semen samples from 20 infertile teratozoospermic men were studied using multicolour fluorescence in situ hybridization (FISH). Men were divided into four groups according to the proportion of normal sperm morphology: infertile men with <10% (group A, n=7), 10-19% (group B, n=6), and 20-29% (group C, n=7) of morphologically normal spermatozoa, and controls (group D, n=5) with > or =30% normal forms. Two hybridizations were performed. All the samples were analysed using probes for chromosomes 1 and 7 and, in addition, in group A and in controls with normal semen parameters probes for chromosomes X, Y and 18 were also used. Ten thousand spermatozoa were scored per hybridization. Severely teratozoospermic men (<10% normal forms) had significantly higher frequency of disomy 7, 18, YY, XY and diploidy in their spermatozoa when compared with controls. The results suggest that poor sperm morphology is associated with numerical chromosome abnormalities of spermatozoa. Severely teratozoospermic men may be at an increased risk of producing aneuploid offspring.     

十一酸睾酮对正常男性精子发生的影响(4例报告)

为了解肌注十一酸睾酮对男性精子发生的影响以及所用剂量对人体是否有毒副作用。作者对４例健康男性每月肌注５００ｍｇ十一酸睾酮,直到严重少精症或无精症。每月随访了解有无不适反应、精子变化和血清生殖激素的水平。结果２例用药５月时达严重少精症,３例用药３～６月时达无精症,停药后无精症可持续３～５个月。在注药期开始后６～９个月精子密度恢复到正常,未发现近期毒副反应。随访期间血清ＦＳＨ、ＬＨ、精子密度、精子活力     

Apoptosis-related proteins in the testes of infertile men with varicocele

OBJECTIVES: To assess immunohistochemically the expression of apoptosis-related proteins in the testes of infertile men, to determine which of these proteins were related to hypospermatogenesis, as a previous report suggested that apoptosis was suppressed in infertile men with varicocele. MATERIALS AND METHODS: The expression of Bcl-2, Bax, caspase-1 (ICE) and caspase-3 (CPP32) were examined in bilateral testicular specimens from 26 infertile men with varicocele and six normal testicular specimens, using the avidin-biotin-peroxidase complex method. Clinical variables were also assessed. RESULTS: Bax, ICE, and CPP32 were expressed in germ cells, while Bcl-2 was not. Differences in staining in left or right testes were not significant. In both testes of infertile patients with varicocele, significantly fewer germ cells stained for CPP32 than in controls (P < 0.001). For Bax and ICE, total germ cell staining was similar between these groups. Staining was less frequent in infertile patients for both CPP32 and ICE when the analysis was restricted to spermatogonia. Serum luteinizing hormone levels correlated positively with CPP32 staining (P = 0.0457). CONCLUSIONS: The reduced expression of CPP32 participates in regulating apoptosis in the testes of infertile men with varicocele.     

Morphology of spermatozoa in infertile men with and without varicocele

This study was carried out to evaluate the morphology of spermatozoa in infertile men with and without varicocele. A series of 285 ejaculates were fully evaluated for seminal volume, sperm count, motility, and morphology, and classified into fertile (165 subjects), infertile without varicocele (93 subjects) and infertile with varicocele (27 subjects). Sperm morphology was classified by multiple entry criteria and recorded as normal, abnormal with head, midpiece, or tail single anomaly or abnormal with simultaneous multiple abnormalities. Semen volume was almost identical in the three groups. Among the infertile men, sperm count was lower in those having a varicocele, but conversely those with varicocele had a higher percentage of motile spermatozoa, higher percentage of spermatozoa with forward movement and higher sperm velocity. There were higher proportions of spermatozoa with abnormal morphology, total number of anomalies, and multiple anomalies in infertile men, both with and without varicocele, than in fertile men. The percentage of abnormal spermatozoa was higher in infertile men with varicocele than in those without varicocele. The pattern of sperm morphology differed between the infertile and the fertile group, and with the presence or absence of varicocele. In the presence of varicocele, only the incidence of elongated (tapered) forms was significantly increased.     

USP26基因突变与精子发生研究进展

泛素特异蛋白酶26(USP26)基因位于Xq26.2,仅有单一外显子,编码由913个氨基酸组成的蛋白质,USP26属于去泛素化酶家族,特异表达于睾丸。USP26基因常见突变类型有插入突变和点突变。目前,该基因与精子发生的关系各研究报道还不一致。本文从USP26基因突变与精子发生障碍之间的关系,USP26基因突变的种族、地域分布差异和USP26基因进化方面,综述了USP26基因与精子发生障碍的关系和研究进展。     

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.     

Single nucleotide polymorphisms in succinate dehydrogenase subunits and citrate synthase genes: association results for impaired spermatogenesis

Evaluation of the possible implication of the SDHA, SDHB, SDHC, SDHD and CS genes in non-obstructive male infertility was performed on the basis that sperm concentration in the ejaculate has been previously correlated with nuclear-encoded mitochondrial enzyme activities (the four subunits of succinate dehydrogenase/complex II of the respiratory chain and citrate synthase). We performed an exhaustive analysis of the five genes for the presence of sequence variants that could be associated with impairment of sperm production. blastn searches in the genomic sequence NCBI database evidenced the presence of highly homologous sequences elsewhere on the genome that can interfere with polymerase chain reaction experiments. Therefore, a careful design of the analytical strategy to search for sequence variants was performed. In this report, we provide primer sequences that allowed selective amplification of coding and immediate flanking regions of the five genes. Fifty-five sequence variations in the five genes were identified in infertile and normozoospermic fertile individuals as controls and only one of them (SDHA c.456+32G>A) showed significant genotype association with impairment of sperm production. Moreover, new single nucleotide polymorphisms identified should be useful in future association studies for other human diseases related to nuclear-encoded genes, leading to mitochondrial respiratory chain activity impairment revealing the physiological role of these genes.     

Histological lesions in the testis of infertile men with varicocele.

Varicocele frequently causes male infertility and histological lesions at the contralateral testis. The most frequent lesions found in this study included maturation arrest in the spermatidic phase, cellular and acellular thickening of the tubular wall, and degeneration of the Leydig cells. These lesions were typical of varicocele and their simultaneous presence suggests that scrotal temperature and modified endocrine secretion of the interstitial testis play a role in the pathogenesis of this type of infertility.     

男性不育患者Y染色体微缺失液态芯片筛查方法的建立及应用

目的建立一套全新的基因诊断方法,检测Y染色体无精子因子(azoospermia factor,AZF)区域微缺失,并对Y染色体微缺失与男性不育相关性进行初步探讨。方法按照欧洲男科协会和欧洲分子遗传实验质控网检测指南推荐标准,采用多重PCR-液态芯片技术对648例精子发生障碍的患者和100例合格捐精者进行Y染色体微缺失筛查。结果648例精子发生障碍的患者中,发现62例患者存在Y染色体AZF区域微缺失,对应于5种缺失模式AZFa,AZFb,AZFc,AZFb c,AZFa b c。按区域统计,AZFc区域缺失的频率最高,其次是AZFb,AZFa的检出率最低。无精子症患者中微缺失的发生率为12．31％,严重少精子患者中微缺失发生率为5．43％。100例对照组没有发现任何缺失,两组比较,差异显著(P<0．001)。结论男性不育与Y染色体微缺失密切相关,本研究建立的多重PCR方法-液态芯片技术平台,用于男性不育患者的YqAZF区域筛查,结果可靠、快捷、重复性好、通量高。     

Association of PIWIL4 genetic variants with germ cell maturation arrest in infertile Spanish men

Dear Editor,
The PIWI proteins （originally P-element-induced wimpy testis in Drosophila） are predominantly present in the germ-line in diverse organisms and are involved in the processing of a class of small RNAs known as piRNAs （see Refs. for review）. The human PIWI protein family consists of four members： PIWIL1-4. Of these, PIWIL4 is known to have essential roles in the first phases ofspermatogenesis： its expression is restricted to gonocytes and it is required for transposon silencing. The lack of this gene in mice causes meiotic arrest in spermatogenesis.