Identification of the second CFTR mutation in patients with congenital bilateral absence of vas deferens undergoing ART protocols

Congenital bilateral absence of vas deferens （CBAVD） is a manifestation of the mildest form of cystic fibrosis （CF） and is characterized by obstructive azoospermia in otherwise healthy patients. Owing to the availability of assisted reproductive technology, CBAVD patients can father children. These fathers are at risk of transmitting a mutated allele of the CF transmembrane conductance regulator （CFTR） gene, responsible for CF, to their offspring. The identification of mutations in both CFTR alleles in CBAVD patients is a crucial requirement for calculating the risk of producing a child with full-blown CF if the female partner is a healthy CF carrier. However, in the majority of CBAVD patients, conventional mutation screening is not able to detect mutations in both CFTR alleles, and this difficulty hampers the execution of correct genetic counselling. To obtain information about the most represented CFTR mutations in CBAVD patients, we analysed 23 CBAVD patients, 15 of whom had a single CFTR mutation after screening for 36 mutations and the 5T allele. The search for the second CFTR mutation in these cases was performed by using a triplex approach： （i） first, a reverse dot-blot analysis was performed to detect mutations with regional impact; （ii） next, multiple ligation-dependent probe amplification assays were conducted to search for large rearrangements; and （iii） finally, denaturing high-performance liquid chromatography was used to search for point mutations in the entire coding region. Using these approaches, the second CFTR mutation was detected in six patients, which increased the final detection rate to 60.8%.     

中国先天性双侧输精管缺如患者CFTR基因全部外显子突变检测

目的:探讨我国先天性双侧输精管缺如患者CFTR基因检测的必要性。方法:采用PCR技术结合DNA直接测序的方法检测9例先天性双侧输精管缺如患者CFTR基因全部外显子的突变情况,并在NCBI和Cystic Fibrosis Mutation Database在线比对。结果:除非编码区突变和已经报道的SNP位点之外,9例先天性双侧输精管缺如患者中4例新发现4种不同于西方人已知突变类型的外显子区突变,均为杂合子错义突变。结论:中国先天性双侧输精管缺如患者CFTR基因外显子区存在不同于西方人的突变,有必要对中国先天性双侧输精管缺如患者进行CFTR基因突变检测。     

Comorbidity of the congenital absence of the vas deferens

Congenital absence of the vas deferens (CAVD) is a relatively rare anomaly that may contribute to male infertility. The aim of this study was to evaluate the clinical features of patients with CAVD and to emphasise some pathological conditions that may be detected during the infertility work‐up or follow‐up of these patients. The charts of 150 males with the diagnosis of CAVD were evaluated retrospectively. The demographic characteristics, reasons for attendance, the way of diagnosis, interventions for infertility before and after attendance, physical examination findings, reproductive hormone levels, semen analysis results, genetical analysis results and resultant live birth events were all included in the study. There were 101 bilateral and 43 unilateral CAVD cases. Thirty‐two males (30.2%) had some renal abnormalities. Two cases, one with bilateral and one with unilateral agenesis, died because of colon cancer at a young age. One case with CUAVD had acute lymphoblastic leukaemia. Congenital absence of the vas deferens should not be seen only as a fertility problem because of the many genotypic or phenotypic disorders that may be present with it. These disorders can cause serious general health problems either presently or in future and can also be transmitted to future generations.     

Early microrecanalization of vas deferens following biodegradable graft implantation in bilaterally vasectomized rats

We evaluated a biodegradable graft for reconstruction of rat vasa deferentia with long obstructed or missing segments. A total of 47 Sprague-Dawley rats underwent bilateral vasectomy and were divided into groups according to length of the vas deferens affected （0.5, 1, 1.5 cm）. After 8 weeks, poly-（D,L-lactide） （PDLA） grafts were used to reconnect the vas deferens, Grafts and adjoining vasa deferentia were excised 8 and 12 weeks later and evaluated microscopically. At 8 weeks, microscopic changes included a robust inflammatory response around the grafts. All grafts were still intact but in the early stages of degradation. No microtubules, indicative of vas deferens recanalization, were identified. One specimen showed evidence of healing and neovascularization at the interface zone between the vas deferens and the graft. At 12 weeks, grafts were further degraded but still present. Microscopic evaluation showed decreased inflammation. Seven specimens showed neovascularization at the interface zone; two of these showed distinct epithelialized vas deferens microcanals at the graft edges. One specimen showed a microcanal spanning the entire 0.5-cm graft. A time period of 8 weeks is not ample enough for vas deferens regeneration in the setting of a biodegradable PDLA graft; however, early evidence of re-growth was seen at 12 weeks. A longer healing time should permit further biodegradation of the graft, as well as re-growth and possible eventual reconnection of the vas deferens, allowing passage of sperm. These findings suggest a potential role for biodegradable grafts in the reconstruction ofvas deferens with long obstructed segments.     

The CFTR gene mild variants poly‐T,TG repeats and M470V detection in Indian men with congenital bilateral absence of vas deferens

The aim of the study was to detect the frequency of the CFTR gene variants poly‐T, TG repeats and c.1408A>G p.Met470Val (M470V) in Indian men with congenital bilateral absence of the vas deferens (CBAVD). Men diagnosed with CBAVD (n = 76), their female partners (n = 76) and healthy men from general population (n = 50) were recruited. Genomic DNA was isolated and the polymorphic regions of IVS9‐ c.1210‐12T [5] and M470V were amplified using specific primers followed by Sanger's DNA sequencing. A statistically significant increase in the frequency of heterozygous IVS9‐ c.1210‐12T [5] (39.4%) was observed in CBAVD men as compared to controls (14%). The allelic distribution of c.1210‐12T [5], c.1210‐12T [7] and c.1210‐12T [9] in CBAVD men was 21%, 64.4% and 13% and that in healthy controls was 7%, 73% and 20% respectively. Longest TG repeat c.1210‐34TG [13] was found in association with c.1210‐12T [5] with an allelic frequency of 5.9% in CBAVD men. We found a significant association of c.1210‐34TG [12]/c.1210‐34TG [13] ‐ c.1210‐12[5] –V470 allele in CBAVD men. Twelve female partners harboured a heterozygous c.1210‐12T [5] allele. The study emphasises the need to screen both partners for the polymorphisms M470V, poly‐T, TG tract repeats in addition to population‐specific known CFTR gene mutations.     

Cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation associated with a congenital bilateral absence of vas deferens

Abstract:   Cystic fibrosis transmembrane conductance regulator ( CFTR ) gene mutations associated with cystic fibrosis have been reported to be rare in Japanese patients with congenital bilateral absence of vas deferens (CBAVD).
A 28-year-old Japanese male was referred for infertility. Vas deferens and epididymis were not palpable bilaterally. Semen analyses showed azoospermia with volumes below 2.0 ml. Serum follicle-stimulating hormone value was slightly elevated. Seminal fructose concentration was also very low. Scrotal ultrasonography showed absence of the bodies and tails of the right and left epididymides. Imaging studies showed cystic dysplasia of the right seminal vesicle and agenesis of the left seminal vesicle. A CFTR gene mutation of I556V was found.
Recent studies show that prevalence of CFTR gene mutation in Japanese CBAVD patients may be approximately equal to that of the Caucasian population. Genetic counselling may be recommended for any couple attempting assisted reproduction technology when the man has CBAVD.     

74例先天性双侧输精管缺如者精浆生化测定

目的 对74例先天性双侧输精管缺如(congenital bilateral absence of the vas deferens,CBA VD)者精浆生化测定和分析,评价精浆生化对输精管道通畅功能的指标意义.方法 从2 775例男性不育患者中筛选出74例CBAVD,测定精浆生化指标,同时选取健康成年男性作对照进行统计分析.结果 CBAVD患者精浆生化指标中果糖,α-糖苷酶,pH值与对照组比较,差异有统计学意义(P<0.05),酸性磷酸酶、锌、白细胞染色计数差异无统计学意义(P>0.05):CBAVD患者与正常人群精液中α-糖苷酶值有部分交叉.结论 精浆生化指标对评价输精管道功能有重要临床意义;在以α-糖苷酶作为精路通畅指标时应考虑附睾和精囊腺以外的附属性腺的分泌部分,同时结合精浆果糖浓度、总量和pH值更有价值.     

先天性输精管缺如的临床特点与诊疗策略

目的:探讨先天性输精管缺如(CAVD)的临床特点,提高其诊断和治疗水平。方法:分析81例CAVD患者的临床诊治资料,总结其临床特点、诊断和治疗方法。结果:79例因不育就诊,从有生育要求到确诊平均经过4.8年;2例术中探查发现。先天性双侧输精管缺如(CBAVD)40例,先天性单侧输精管缺如(CUAVD)25例,节段性输精管缺如16例。74例患者的配偶接受辅助生殖治疗,其中12例接受卵细胞胞质内单精子注射(ICSI)治疗,4例成功妊娠。结论:CAVD患者多以不育就诊。CBAVD均表现为梗阻性无精子症;CUAVD和节段性缺如者可表现为少精子症、弱精子症或梗阻性无精子症。本病诊断多无困难,但常因体检不仔细而漏诊。CAVD治疗的原则主要是恢复患者的生育能力,可借助辅助生殖技术(ART)进行治疗。     

Micro-recanalization in a biodegradable graft for reconstruction of the vas deferens is enhanced by sildenafil citrate

This study investigated the effect of sildenafil citrate on micro-recanalization and neovascularization, which were previously demonstrated in a rat model using biodegradable grafts （BGs） for vas deferens reconstruction. A total of 24 male rats underwent bilateral vasectomy with removal of a 0.5-cm vasal segment and were randomly assigned to four groups. Groups 1 and 2 underwent immediate vasovasostomy. Groups 3 and 4 underwent interposition of a 0.5-cm BG in the vasal gap. Groups 1 and 3 were given 5 mg kg-1 day-1 oral sildenafil. Other groups were given placebo. Rats were housed with females 12 weeks postoperatively. Reconstructed vasal segments were harvested 16 weeks postoperatively and analyzed histologically. Fluid from the distal vasal stump was analyzed for motile sperm. Urine samples obtained 16 weeks postoperatively were analyzed for cGMP levels. cGMP levels in rats treated with sildenafil were signifcantly higher than in control rats. No pregnancies were sired by grafted groups. In all, 5/6 rats in group 1 and 3/6 rats in group 2 sired litters. No motile sperm were noted in the vasal fluid of the grafted groups. Motile sperm were noted in all rats in group 1 and in 5/6 rats in group 2. In addition, 29 and 4 microcanals were detected in the sildenafil and placebo groups, respectively （P = 0.023）. No microcanal exceeded 3 mm in length. An average of 12 and 28 blood vessels per graft were noted in the placebo and sildenafil groups, respectively （P 〈 0.0001）. In conclusion, sildenafil enhances micro-recanalization and neovascularization in BG used for vas deferens reconstruction, but does not increase the microcanal length after 16 weeks.     

Significance of CFTR gene mutations in patients with congenital aplasia of vas deferens with special regard to renal aplasia

Between 1994 and 2010, a total of 123 patients with obstructive azoospermia due to aplasia of vas deferens (CAVD) were surgically treated. In 110 patients, the condition was bilateral (CBAVD), 13 men had unilateral aplasia (CUAVD), and 10 patients additionally had aplasia of one kidney. All patients underwent CFTR genetic testing, which detected two mutations (homozygous or compound heterozygous condition) in 38%, one mutation in 34% and no mutation in 28% of the patients with CBAVD. Neither the azoospermic patients with congenital unilateral aplasia of vas deferens nor those with CBAVD and renal aplasia were found to have CFTR mutations. The results militate against the assumption that there is an association between the CFTR gene and unilateral aplasia of vas deferens or bilateral aplasia of vas deferens with renal involvement.     

Microsurgical anastomosis of vas deferens by a telescopic technique

Summary A telescopic microsurgical anastomosis of the vas deferens was performed on 24 rats. Sequential histological examination demonstrated mucosal healing in 7 days. Healing was complete in 21 days, and tubal patency was confirmed by histology in 100% of the cases.     

The CFTR polymorphisms poly-T,TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.     

先天性双侧输精管缺如不育患者附睾精子功能及妊娠率的研究

目的了解先天性双侧输精管缺如(CBAVD)不育患者附睾精子功能及经皮穿刺附睾吸精后,子宫腔内人工授精(IUI)和卵浆内单精子显微注射(ICSI)的效果。方法对78例CBAVD不育患者进行经皮穿刺附睾吸精子(PESA),对吸出的精子进行精子低渗膨胀实验(HOST)、IUI、ICSI。结果附睾头部PESA精子活率、畸形率、密度、总精子数及HOST膨胀率分别为(18．4±11．2)%、(56．1±21．3)%、(26．0±14．0)×10~6/ml、(18．3±11．1)×10~6及38%±18%,而附睾尾部PESA精子各项指标分别为(8．3±7．2)%、(78．2±32．0)%、(9．0±12．0)×10~6/ml、(10．2±10．8)×10~6及(16．0±14．0)%,PESA附睾尾部的精子活率、密度、总精子数及HOST膨胀率都明显低于附睾头部的精子(P＜0．01),畸形精子比率明显高于附睾头部的精子(P＜0．01)。PESA-IUI及PESA-ICSI-IVF-ET分别有2例和6例妊娠,周期妊娠率分别为3．8%和33．3%。结论(1)CBAVD患者附睾尾部精子质量明显低于附睾头部精子；(2)PESA-IUI及PESA-ICSI-IVF-ET是CBAVD不育患者治疗较为有效的方法。     

Duplicated vas deferens: A case report and comprehensive review of the literature

Duplication of vas deferens is a very rare anomaly which two vasa deferentia are found in the spermatic cord. It can be recognised during autopsy or cadaveric dissection and also several surgical procedures which require spermatic cord dissection including inguinal hernia repair, orchiopexy, vasectomy, varicocelectomy, vasectomy reversal and radical prostatectomy. Recognition of the duplicated vas deferens is important to avoid surgical complications such as an unsuccessful vasectomy or transection of the vas. It was reported in only three cadavers and 31 patients since 1959. In this study, we describe a new case of duplicated vas deferens found incidentally during routine inguinal hernia repair in a 66-year-old patient. We also review all previously reported cases in the literature to draw attention to this rare but important anomaly.     

Evaluation of the azoospermic male

When presented with an azoospermic patient, a thorough history and careful, considered physical examination often leads to a definite or presumptive diagnosis. An algorithmic, logical thought process is important to have in mind when embarking on the evaluation. Adjunctive laboratory tests, such as hormonal assays or genetic studies, are often complementary and/or additive and allow a very precise determination to be made as to the etiologies, either genetic or acquired. It is only with this information that a therapeutic plan can be made for the patient. As will be discussed, a targeted approach to testing is far more satisfying and cost-effective than a blind, shotgun approach.     

Histological study of vas deferens following intravasal laser irradiation

Aim: To study the histologic changes of the vas deferens following Nd: YAG laser irradiation. Methods:Intravasal laser irradiation was given to (i) 52 segments of rabbit (laser dosage: 2 seconds at 40W～50W) and 16 segments of human (3 seconds at 45W～55W) vas deferens in vitro, (ii) 25 rabbit vasa (2 seconds～2.5 seconds at 40W～45W) in vivo and (iii) 2 human vasa (3 seconds at 55W) in vivo. Segments of vasa were removed from the in vivo irradiated vasa deferentia 15 days ～ 180 days (rabbit) or 15 days (man) after the exposure. All vas segments were embedded in methacrylate resin. Serial sections (thickness 25μm～30μm) were obtained and observed under a light microscope. Results: (i) Laser-induced damage reached the muscularis layer in 27% and 94% of the rabbit and human vas segments in vitro, respectively. (ii) Fourteen of the 25 in vivo rabbit vasa were completely occluded by fibrous tissue and the longer the time interval after treatment, the more likely was the vas occluded. Those unoccluded vasa had either a normal histology or a mucosal damage. (iii) One in vivo human vas was almost completely occluded by the fibrous tissue but the other had a relatively large lumen packed with sperm granulomatous tissue and partial destruction of the smooth muscle layer. Conclusion: Laser irradiation can induce long-term vas occlusion; for rapid occlusion, laser doses just completely destroying the mucosal layer will be advisable.     

Genetic, andrological and clinical characteristics of patients with congenital bilateral absence of the vas deferens

The possibility of retrieving spermatozoa from the epididymis allows patients with congenital bilateral absence of the vas deferens (CBAVD) to father a child by means of assisted reproduction techniques. This has, however, increased the chance of transmitting a mutated allele of the cystic fibrosis transmembrane conductance regulator (CFTR) gene which increases the risk of generating offspring with cystic fibrosis (CF). Because of the increased heterogeneity of the CFTR locus, the study of a discrete number of mutations, as usually carried out in a diagnostic work-up, is unable to ascertain the presence of a mutation in a relatively high proportion of the patients screened. In an attempt to increase the chance of detecting the presence of CFTR gene abnormalities, 37 patients with CBAVD and one patient with congenital unilateral agenesis of the vas deferens (CUAVD) underwent an enlarged diagnostic protocol, which included screening for the most expected mutations of the CFTR gene in our population, evaluation of the five thymidine (5T) allelic variant, sweat test, respiratory function tests, evaluation of steatocrit, and an accurate evaluation of the history of the patient to search for symptoms commonly found in patients with CF. A single CFTR gene mutation was found in 18 patients (48.6%) with CBAVD and in the patient with CUAVD. The most frequent mutation observed was the Delta F508. Eleven patients (45.8%) had the 5T variant and in five of them it was not associated with any detectable mutation of the CFTR gene. Two female partners were found to be carriers of a mutation, whereas 5 (18.5%) had the 5T variant. As many as 71% of CBVAD patients had the simultaneous presence of at least two signs and/or symptoms suggestive of CF, albeit they were of mild intensity and the patients felt fit and healthy. In conclusion, these results suggested that some patients with CBAVD without CFTR gene mutation or 5T variant, even when their sweat test is negative, may show clinical suspicion of carrying a CFTR gene mutation and therefore are at risk of generating children affected by CF if the partner carries a mutation as well. The screening for mutations and a careful clinical examination may contribute to better identification of patients with CFTR-related CBAVD.     

Abnormalities of the vas deferens and epididymis in cryptorchid boys with congenital rubella.

Cryptorchidism was present in 12% of 316 boys with congenital rubella (CR) followed by The Roosevelt Hospital Rubella Project. Eight of these patients, age 4 through 16 yr, had a recent orchiopexy, 4 on the left, 2 on the right, and 2 bilaterally. The vasoepididymal system was absent or apparently obstructed in 60% of the 10 sides. The epididymis was abnormal in 6 instances and the vas deferens in 5. Sixty-one boys of the entire series had an intravenous pyelogram (IVP) that was significantly abnormal in 18%. The 8 patients described all had a normal IVP except for 2 who had a malrotated kidney on the involved side. In 5 of the 8, a known maternal rubella infection has occurred during the first 8 wk of gestation. As the rubella virus is known to interfere with cellular growth and tissue differentiation in early pregnancy, it apparently altered the developing testis and mesonephric duct system. Similar vasoepididymal abnormalities have been described previously in patients with uncomplicated cryptorchidism, inguinal hernia, kidney defects, cystic fibrosis and male sterility. Their presence should alert the clinician to perform an IVP and also consider a diagnosis of congenital rubella.     

Three cases of Klinefelter's syndrome with unilateral absence of vas deferens

Genital abnormalities such as congenital uni/bilateral absence of the vas deferens are very rare in Klinefelter's syndrome. Here, we report three cases of Klinefelter's syndrome with unilateral absence of the vas deferens. All cases had small testicles, and unilateral vas deferentia were not palpable. Hormonal evaluations revealed hypergonadotropism. One case had elevated prolactin level, and pituitary adenoma was detected by magnetic resonance imaging. All cases were diagnosed as Klinefelter's syndrome (one of them had mosaicism) cytogenetically, and some CFTR gene mutations were detected. To our knowledge, this is the first case series of both conditions existing simultaneously.     

先天性双侧输精管缺如患者睾丸超微结构的改变

目的 了解先天性双侧输精管缺如（Congenital bilateral absence of vas deferens,CBAVD)患者睾丸超微结构的改变,以预测单精子卵胞浆内注射（ICSI）治疗效果。方法 对13例光镜下睾丸组织形态学正常的CBAVD患者睾丸组织进行电镜观察。结果 曲细精管界膜改变表现变睾丸曲细精管基膜增厚,分层,向管腔形成不同程度的指状突起,基膜外胶原纤维呈不同程度增多,增粗、排列紊乱,极性消失,生精上皮表现为精子细胞头部畸形,胞核染色质呈现明显颗粒状结构。出现核内空泡,精子细胞中段线粒体鞘部分或完全缺失,顶体内陷,顶体囊增大,内有电子密度不高的片层结构（5例）。支持细胞表现数目增多,胞浆内脂质颗粒及降解小体明显增多。有时可见支持细胞之间的紧密连接消失（2例）。结论 CBAVD患者睾丸组织超微结构异常主要表现为界膜与精子细胞的改变,CBAVD所致的无精子症除了梗阻因素外,同时合并了睾丸性因素。