Expression and distribution of OCTN2 in mouse epididymis and its association with obstructive azoospermia in juvenile visceral steatosis mice

AIM: L-carnitine, an essential cofactor for mitochondrial, beta-oxidation of long-chain fatty acids, is known to play important roles in sperm maturation and metabolism when spermatozoa pass and acquire motility in the epididymis. We reported that obstructive azoospermia occurred in the epididymis in the juvenile visceral steatosis (JVS) mice, which are OCTN2 dysfunction mice caused by mutations in the gene encoding OCTN2, have been used for animal models of primary systemic carnitine deficiency. The aim of present study is to investigate the expression of OCTN2 protein in the mouse epididymis and its relation between the localization of OCTN2 and obstructive azoospermia in JVS mice as animal models for human male infertility. METHODS: Animals used in this study were wild-type (C57BL/6 J) mice (n = 4) and JVS mice (n = 4). We made a specific polyclonal antibody against OCTN2 and examined immunohistochemically the localization of OCTN2 in the mouse epididymis. RESULTS: OCTN2 was localized on the apical membrane of the principal cells of distal corpus and cauda epididymides. Immunocytochemistry demonstrated that OCTN2 was localized on the surface of microvillus upon the principal cells. In JVS mice, immunoreactivity started in a region immediately distal to where the sperm obstruction occurred. CONCLUSIONS: Our results suggest that OCTN2 functions as a carnitine transporter between the epithelium and the lumen in distal corpus and cauda epididymides and provides a clue as to why obstructive azoospermia is induced in distal parts of epididymis.     

附睾输精管吻合术治疗梗阻性无精子症

目的探讨附睾输精管吻合术在梗阻性无精子症治疗中的作用。方法选择23例确诊为梗阻性无精子症并初步怀疑为附睾水平梗阻的患者进行阴囊探查，观察睾丸、附睾及输精管情况，对其中19例确定为附睾水平梗阻并在附睾液中找到活精子的患者用8-0尼龙线施行双侧或单侧附睾输精管端侧吻合术，术后随访其疗效。结果19例获随访8～34个月，9例(47％)于术后3～9个月从精液中检出活精子，其中5例配偶受孕成功。结论阴囊探查简单、易行，有助于梗阻性无精子症的诊断和治疗，附睾输精管吻合术治疗梗阻性无精子症取得初步效果，值得进一步探讨。     

146例炎症梗阻性无精子症的临床评估和ICSI治疗结局分析

目的分析炎症梗阻性无精子症的临床评估和单精子卵胞浆内注射(ICSI)的治疗结局。方法前瞻性研究近5年间接受ICSI治疗的炎症性梗阻性无精子症的临床特征、精液和超声特点,经皮附睾穿刺精子抽吸术(PESA)或经皮睾丸穿刺取精术(TEFNA)结合ICSI治疗后观察受精、临床妊娠等结果。结果146例患者体检附睾均有增粗变硬或伴头尾部结节。82例患者曾有生育史、附睾炎症史或输精管附睾吻合手术史,其中72例PESA找到附睾精子;53例无上述病史者49例PESA找到附睾精子:另有精道远端梗阻11例。ICSI治疗146例167周期炎症性梗阻性无精子症的受精率、每周期临床妊娠率分别为81.1%和42.1%。结论炎症梗阻性无精子症具备典型的临床和超声特征,PESA附睾精子获取率高,ICSI治疗获得较高受精率和临床妊娠率。     

Young's syndrome: report of two Japanese cases

Young's syndrome is characterized by azoospermia due to bilateral epididymal obstruction associated with chronic sinobronchial disease. We report two Japanese cases of Young's syndrome who were treated with microsurgical epididymovasostomy. The histopathology of the caput epididymis showed an obstruction at the most distal region of the ductuli efferentes. The incidence of Young's syndrome in patients with obstructive azoospermia was much lower for mongoloids than for Caucasians.     

睾丸细针穿刺吸液细胞学检查诊断阻塞性无精子症

目的 :观察睾丸细针穿刺吸液 ( FNA)细胞学检查的效果 ,为诊断阻塞性无精子症提供新的诊断方法。方法 :2 86例无精子症患者采用睾丸 FNA细胞学检查结合精浆生化指标测定及输精管造影对睾丸生精功能及阻塞部位进行诊断 ;以 42例精子密度在正常范围 ( 2 5～ 86× 1 0 6 / ml)的成年男性作为对照组。 2 4例做钳穿活检进行自身对照。结果 :( 1 )双侧输精管未触及者 58例 ,睾丸 FNA细胞学检查生精功能正常 2 6例 (可见较多生精细胞、精子细胞及精子 )、生精功能低下 2 4例、无生精功能 8例 ,精浆果糖在正常值范围 ,而肉毒碱及α-糖苷酶明显低于正常值范围 ;( 2 ) 3 2例睾丸 FNA细胞学检查见较多精子 ,精液沉渣涂片未见生殖细胞 ,其中 6例精浆果糖、肉毒碱及 α-糖苷酶明显低于正常值范围 ,结合输精管造影确诊为射精管阻塞 ,其余 2 6例精浆果糖在正常值范围 ,而肉毒碱及α-糖苷酶明显低于正常值范围 ,确诊为附睾尾部阻塞性无精子症 ;( 3 )睾丸生精功能极度低下或无生精功能 1 96例 ,其中 1 60例仅见各级生精细胞、精子细胞和支持细胞 (睾丸生精功能阻滞 ) ,3 6例仅见支持细胞 (唯支持细胞综合征 ) ,精浆果糖、肉毒碱及 α-糖苷酶均在正常值范围 ,为非阻塞性无精子症。结论 :睾丸 FNA细胞学检查可作为阻塞性无?     

Increased plasma malondialdehyde in patients with small intestinal strangulation obstruction

Plasma malondialdehyde (MDA) was evaluated as an index of lipid peroxidation in 20 patients with small intestinal obstruction, with or without strangulation. The gut proved to be strangulated in nine cases--irreversibly in five and reversibly in four--while 11 patients had simple obstruction. The MDA levels in these cases were compared with values obtained from 29 healthy control subjects. The mean MDA level in the control group was 2.3 +/- 0.7 (range 1.5-4.0) mumol/l. In the patients with strangulation obstruction the MDA concentration was 6.7 +/- 1.6 mumol/l, and in those with simple obstruction it was 2.9 +/- 0.5 mumol/l. The difference between the MDA level in strangulation and in the other investigated groups was statistically significant. Values above 4 mumol/l were found in all the patients with intestinal strangulation, whereas those with simple obstruction had lower values. The heightened MDA level in patients with intestinal strangulation may be used for diagnostic purposes.     

Glycerylphosphorylcholine in Selected Forms of Male Infertility

GPC was studied in the seminal plasma of 35 normozoospermic men, 34 cases of azoospermia due to bilateral deferent obstruction, 34 cases of azoospermia due to bilateral ejaculatory duct obstruction, 10 vasectomized patients, 6 vasectomized patients after vasovasostomy and 118 cases of spermatogenetic arrest without obstruction of the seminal ducts. Values of GPC in azoospermia due to deferent or ejaculatory ducts obstruction and to vasectomy are significantly lower than in normozoospermic subjects (p less than 0.001). Levels of GPC increased (p less than 0.05) following vasovasostomy. Although GPC values in secretory azoospermia were higher than those in cases of duct obstruction, they were still lower than in normozoospermic (p less than 0.001). The most likely source of GPC is the epididymis. These results support the assumption that GPC originates mainly in the epididymis. The absence of germinal cells in the epididymis could explain the decreased levels of GPC in azoospermia due to arrest of spermatogenesis.     

Alpha-glucosidase in the human epididymis: topographic distribution and clinical application

Alpha-glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of alpha-glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of alpha-glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean +/- SEM): normozoospermia (control): 20.2 +/- 1.4 mU ml(-1); azoospermia: normal value: 17.6 +/- 2.2 mU ml(-1), low value: 7.4 +/- 1.8 mU ml(-1); oligozoospermia: normal value: 22.3 +/- 2.5 mU ml(-1), low value: 7.3 +/- 0.7 mU ml(-1); leucocytospermia: increase value: 38.9 +/- 3.7 mU ml(-1), low value: 11.1 +/-1.3 mU ml(-1). This study suggests that determination of alpha-glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.     

不明原因梗阻性无精子症的诊断与治疗

在51例梗阻性无精子症中发现6例不明原因的梗阻,经造影及探查证实梗阻在附睾头1例,附睾尾4例,附睾输精管连接部位1例。对其中5例进行了附睾输精管吻合,术后3例精液中发现精子,但无一例妇方怀孕。结合文献对其诊断条件及治疗进行了讨论。     

Epididymal microsurgery: current techniques and new horizons

Surgical treatment for obstructive azoospermia was introduced about 30 years ago with the development of Bayle's vasoepididymal fistula technique (Bayle: Enc Med Chir 41:435, 1966). More recently this has been replaced by single tubule end-to-end microvasoepididymal bypass procedures with improved success rates. We describe the use of animal models in the development and application of a modified end-to-end microanastomosis technique in which the vas deferens is attached to a single surface convolution of the ductus epididymidis. In comparison with other microvasoepididymostomy (micro-VE) procedures, this technique results in less postoperative scarring and allows for easier access to the epididymis in those patients requiring subsequent epididymal surgery. With this procedure 60% of patients produced spermatozoa after operation, and 10% of 102 patients have so far achieved pregnancies. For patients, such as those with congenital absence of the vasa deferentia, whose infertility cannot be corrected by microvasoepididymal surgery, we describe a microaspiration procedure that can be used to collect spermatozoa from the epididymis for use in in vitro fertilization (IVF). This procedure has a low success rate at present, with an 18% fertilization and 3% pregnancy rate per cycle. Improvements in treatment procedures for aspirated sperm samples, such as the use of motility stimulators and in vitro maturation by coculture with epididymal tubule segments before IVF, may enhance the success for this technique. Microinjection of sperm collected by epididymal microaspiration into oocytes may be an alternative method of treatment for these patients in the future. Two procedures (microepididymoepididymostomy and the vas bridge bypass) that are currently being modelled in the rabbit may provide new directions for epididymal microsurgery and for examining epididymal function. Although the two methods are technically more difficult than standard micro-VE procedures, preliminary studies are encouraging and suggest a future role for these techniques in treating obstructive azoospermia. Such techniques make use of the epididymis distal to the obstruction site and may be particularly important in improving the success of surgery for obstructive azoospermic patients with high-level obstructions in whom sperm quality following micro-VE surgery is often poor.     

Why do we determine α-glucosidase activity in human semen during infertility work-up?

alpha-Glucosidase is a normal constituent of human semen, produced mainly in the epididymis. It is significantly correlated to sperm count. Its activity is low in cases of epididymal obstruction. We evaluated alpha-glucosidase activity in 653 semen samples of patients, who attended our department for marital infertility, with respect to associations to clinical and other seminal parameters. The normal range (mean +/- 2 SD) in samples with normal parameter values was 7.2-46.4 mU ml-1. The determination in patients with azoospermia revealed mean values of 7.7 +/- 9.5 mU ml-1 in obstructive azoospermia, and 15.8 +/- 11.5 mU ml-1 in nonobstructive azoospermia. The difference was not statistically significant in that the sensitivity of determination with respect to the presence of obstruction was only 0.66, and the specificity 0.83. A significant correlation (r = 0.34) of alpha-glucosidase activity with log sperm count was observed. The mean alpha-glucosidase activity was not significantly different in groups formed according to sperm motility, according to leucocyte count or according to semen volume. A difference between smokers and nonsmokers with comparable sperm count, as reported in the literature, did not occur. We conclude from our results that the determination of alpha-glucosidase activity does not give additional information of the fertility status exceeding that of other clinical investigations or parameters of semen analysis.     

The contractile wall of the caput epididymidis in men affected by congenital or postinflammatory obstructive azoospermia

The transport and storage of spermatozoa in the epididymis depend on the contractile activity of its tubular wall. It is not known what differences exist in the contractile wall of the human epididymis in cases of obstructive azoospermia. The contractile wall in the tubules of the caput epididymidis was analyzed by light microscopy and transmission electron microscopy in 10 azoospermic men, 5 with a bilateral congenital absence of vas deferens (CBAVD) and 5 with a bilateral postinflammatory congestive obstruction of the epididymis. Five specimens from the same region of the caput epididymidis, obtained from fertile men who had undergone an orchidectomy because of testicular cancer, served as controls. No differences were observed between congenital and congestive obstructions. The contractile wall in caput tubules proximal to the obstructed level was strongly thickened when compared with controls (62.98 +/- 5.84 micro; 80.82 +/- 7.72 micro vs 19.59 +/- 2.23 micro, respectively, for congestive and congenital obstructions vs controls; P <.0001 vs controls), and the spindle-shaped myoid cells, which formed the contractile wall in normal cases, were replaced by large smooth muscle cells (SMCs) that showed features of coexisting contractile and secretory functions. The former included crowded cytoplasmic bundles of thin myofilaments (5-6 nm in diameter) converging to a large number of dense bodies, numerous micropinocytotic vesicles of the plasma membrane, and a continuous cell basement membrane. The presence of a developed rough endoplasmic reticulum and a Golgi complex, associated with the accumulation of thick layers of pericellular basement membrane-like material and ground substance, was indicative of a secretory phenotype of SMCs. The increased mechanical forces on the epididymal wall upstream from the obstruction might eventually activate the differentiation of myoid cells into SMCs, leading to an altered physiology of the contractile wall that could have possible clinical relevance in the case of microsurgical epididymovasostomy.     

Free and total plasma theophylline concentrations in chronic airflow obstruction

It has been suggested that the variability in clinical response between individuals at any given total plasma theophylline concentration may be related to interpatient variability in theophylline plasma protein binding. We therefore measured the plasma protein binding of theophylline in plasma from 39 outpatients with chronic airflow obstruction who were receiving long term oral theophylline treatment. The protein binding was measured at 37 degrees C and pH 7.4 by equilibrium dialysis. Total plasma theophylline concentration was measured by high performance liquid chromatography and the free concentration calculated by multiplying total concentration by the free (unbound) fraction. The total plasma theophylline concentration varied from 0.7 to 22.0 micrograms/ml, mean 10.6 (SD 5.3) micrograms/ml (3.9-121 mumol/l, mean 58.3 (29.2) mumol/l). The free fraction of theophylline varied only from 0.58 to 0.69 (mean 0.626 (0.024] microgram/ml and was not related to the total concentration (r = -0.236, n = 39, p greater than 0.05). There was a very close relationship between free and total plasma concentrations (r = 0.996, n = 39, p less than 0.001). It is concluded that there is little variability in plasma protein binding of theophylline in patients with chronic airflow obstruction. Other factors appear to be responsible for variability between individuals in the response to a given total plasma theophylline concentration.     

先天性输精管缺如患者生育问题的研究

自1988年至1991年共收治25例先天性输精管缺如患者,在20例行手术诊治术中15例用自体睾丸精索鞘膜制成人工精池囊,5例又在囊内放置用微涤纶制成的异质管。术中抽吸10例附睾内精子进行快速活化,其中5例活化成功,行人工授精,结果2例怀孕,1例已生育一女孩。认为该症是有生育可能的,但需要解决3个问题:(1)采用显微外科技术把附睾内部活着的精子取出一定数量;(2)选配好快速活化剂使精子快速活化成功;(3)用患者的精浆培养自己的精子。建议对梗阻性无精子症在用手术疏通精路的同时可以采用快速活化精子技术行人工授精,以提高生育的效果。     

Intracytoplasmic sperm injection using surgically retrieved epididymal and testicular spermatozoa in cases of obstructive and non-obstructive azoospermia

This was a retrospective study of 115 patients who underwent 124 cycles of ICSI using surgically retrieved spermatozoa. The objective was to compare the results of ICSI in patients with obstructive azoospermia using epididymal spermatozoa (36 cycles) or testicular spermatozoa (58 cycles) with ICSI in patients with non-obstructive azoospermia using testicular spermatozoa (30 cycles). When epididymal spermatozoa were used for ICSI, the fertilization rate per injected metaphase-II oocyte and the clinical pregnancy rate per ICSI cycle were 60.4 and 25%, respectively. When testicular spermatozoa were used in obstructive cases, the fertilization rate and pregnancy rate were 57.9 and 34.5%. In non-obstructive cases the fertilization and pregnancy rates were 41.2 and 16.6%. When patients with obstructive azoospermia were regrouped according to the cause of obstruction, the fertilization and pregnancy rates were 59.1 and 35.1% in acquired obstruction and 58.7 and 24.3% in congenital obstruction. The fertilization and pregnancy rates were not statistically different ( p  > 0.05) when testicular or epididymal spermatozoa were used in obstructive cases; neither was statistically different ( p  > 0.05) when compared in patients with congenital and acquired obstruction. On the other hand, the fertilization and pregnancy rates in cases with non-obstructive azoospermia were significantly lower ( p  < 0.05) than in obstructive cases.     

显微外科技术下梗阻性无精子症术前诊断及治疗方式探讨

目的:探讨梗阻性无精子症(OA)在显微外科技术下术前诊断及其治疗策略。方法:57例不育症患者确诊为OA并初步怀疑为附睾梗阻,行阴囊探查术观察附睾及输精管梗阻情况;术中对确定为附睾梗阻并在附睾液中找到活精子的患者施行附睾输精管端侧吻合术,同时对探查至附睾头部才发现精子或术中发现双侧附睾以远输精管梗阻、缺如的患者留取精子冷冻以备卵细胞胞质内单精子注射(ICSI);术后随访其疗效。结果:53例(92.9%,53/57)行阴囊探查术确诊为附睾水平OA,47例(82.5%,47/57)完成显微手术,10例(17.5%,10/57)术中留取精子冷冻。22例(46.8%,22/47)于显微手术后1～18个月从精液中检出活动精子;5例(10.6%,5/47)配偶自然受孕成功,6例(18.5%,6/32)留取精子行ICSI后配偶怀孕。结论:在显微外科技术日益成熟下,OA的术前诊断应尽量采取无创的手段,在手术探查中进行梗阻部位的确诊及决定治疗方式。     

The influence of kidney transplantation on carnitine metabolism

In this study, we examined all three plasma carnitine fractions, free carnitine (FC), short-chain acyl- (SCC), and long-chain acylcarnitine (LCC), as well as the urinary excretion of FC and SCC in 62 patients with functioning renal allografts 1-70 months following the kidney transplantation (KT). Patients were classified into three groups according to their transplant function, as characterized by the serum creatinine concentration (CR). A comparison with normal subjects (n = 20) and with patients on chronic hemodialysis (HD, n = 46) revealed a complete normalization of all carnitine plasma fractions for group I patients (Cr less than 120 mumol/l). In contrast, significant elevations of plasma free and esterified carnitine were found in group II (Cr greater than 120, less than 200 mumol/l; with FC + 27%, SCC + 82%, and LCC + 49% and group III patients (Cr greater than 200 mumol/l; with FC + 39%, SCC + 122%, and LCC + 106%), as compared to healthy subjects (p less than 0.001). The elevations in concentrations of SCC were more pronounced than those of FC; consequently, we found a higher ratio of acylcarnitine (AC) to FC in group III than in group I patients (+ 41%, p less than 0.001). Yet even in the former group, this ratio was found to be markedly reduced when compared to HD patients (-41%, p less than 0.001). We found no significant differences in the urinary excretion of FC and SCC between the 3 groups of KT patients. It is thus to conclude that in patients with a well-functioning transplant, the pattern of carnitine fractions in plasma is fully normalized. The decrease in the ratio of AC to FC following a successful KT might suggest a better availability of FC and thereby be associated with an enhanced fatty acid oxidation.     

A comparison of plasma and muscle carnitine levels in patients on peritoneal or hemodialysis for chronic renal failure

We studied plasma, dialysate, and muscle carnitine levels in patients with stable chronic renal failure on hemodialysis, and intermittent peritoneal, or continuous ambulatory peritoneal dialysis (CAPD). In patients on hemodialysis, plasma carnitine levels fell from 46.2 +/- 4.5 mumol/l (mean +/- SEM) to 18.8 +/- 2.7 mumol/l immediately after the procedure (p less than 0.001). Depletion of muscle carnitine was found after hemodialysis (1,518 +/- 273 nmol/g wet weight of tissue) compared to normal levels of 5,230.5 +/- 142.7 nmol/g tissue (p less than 0.01). However, the plasma and muscle carnitine levels remained in the normal range in patients on intermittent peritoneal dialysis and CAPD. We postulate that the rapid decline in plasma levels of carnitine caused by hemodialysis initiates unilateral transport of the compound from muscle to the plasma, thus depleting the skeletal muscle stores of carnitine.     

Animal models for epididymoepididymostomy. Development of an alternative microsurgical procedure for vasoepididymostomy.

The present success rate for high level microsurgical vasoepididymostomy in patients with obstructive azoospermia is generally poor in comparison with more distal vasoepididymal bypasses, suggesting that the development of a high level bypass method which preserves the distal maturation and storage functions of the epididymis might increase the fertility success rate. To achieve this we have developed a segmental bypass of the distal caput-proximal corpus regions of the epididymis (epididymoepididymostomy) using the rat and rabbit as animal models. In this procedure the epididymides of 11 adult male Sprague-Dawley rats were exposed through a scrotal incision and a convolution of the duct in the proximal corpus region was attached to an opened convolution in the mid-caput epididymides using a standard microsurgical technique. Each male was rested for at least 3 months after the microsurgical bypass operation to allow the preoperative sperm contents of the caudal storage region to be replaced by post-bypass spermatozoa. Six of the 11 rats were proven fertile after surgery by siring litters from mating trials up to 11 months later. The patency of anastomoses was confirmed in 3 animals by laparotomy and recovery of large numbers of sperm with normal motility from the cauda epididymidis distal to the anastomosis site and also from the vas. Similar unilateral surgery in 2 adult male rabbits resulted in normal post-operative semen profiles in 1 and an in vivo fertilisation rate of 100% from 1 mating trial 8 months after surgery. The successful development of a reliable animal model for epididymoepididymostomy provides an experimental tool for studying the function of the epididymis. This technique may also have clinical application as an alternative to high level vasoepididymostomy in selected patients with obstructive azoospermia.     

彩色多普勒超声在无精子症诊断中应用价值的评估

目的 评价超声在梗阻性无精子症诊断中的作用.方法 158例梗阻性无精子症患者,经过体格体检查、精液分析及血液激素水平测定后,均进行了阴囊超声及经直肠彩色多普勒超声检查.结果 在158例梗阻性无精子症患者的超声检查中,25例无阳性结果,133例超声检查结果异常,阳性率为84.2%.其病因是多方面的,包括远端梗阻和近端梗阻,共分为4种分类：（1）先天性发育异常,如：精囊缺失或精囊发育不良以及输精管单侧及双侧缺失（55例）;（2）射精管梗阻（EDO）（34例）;（3）附睾病变（25例）;（4）炎症性病变（19例）.结论 阴囊超声及经直肠超声检查是临床诊断梗阻性无精子症的重要手段.