Dicentric Y chromosome in an azoospermic male

We describe a 28 year old male with a pseudodicentric Y chromosome who suffered from azoospermia attributed to maturation arrest of the primary spermatocyte, as diagnosed by testicular biopsy. Chromosome analysis, using G, Q and C banding techniques, revealed an abnormal karyotype of 45,X[7]/46,X,psu dic (Y)(pter-->q11.2::q11.2-->pter)[33]. Polymerase chain reaction (PCR) DNA analysis did not detect the absence of DAZ and RBM1 which are candidates for azoospermic factor (AZF) genes. Therefore, it is suggested that the maturation arrest of the primary spermatocyte in this patient was caused either by a pairing dysfunction between the X and Y chromosomes during meiosis or by deletions in the autosomal or the Y chromosomal spermatogenesis controlling genes, excluding DAZ and RBM1.      

Y染色体微缺失35例患者临床表型分析

目的分析35例Y染色体微缺失患者临床表型。方法按照WHO标准进行检查和精液分析,证实为非梗阻性无精子症或严重少精子症（〈1×10^6／mL）367例,然后应用改良多重多聚酶链反应（multiplex—PCR）,对367例不育患者进行Y染色体微缺失分子学诊断。将微缺失患者分为两组：严重少精子组和无精子组。再按缺失类型将无精子组分为两个亚组：单纯AZFc缺失组和其它类型缺失组。采集以下临床资料进行分析：结婚年龄、不育史、精液分析、睾丸体积、附睾睾丸穿刺情况、染色体核型分析以及性激素检测。结果367例中发现AZF微缺失35例（9．54％）,其中AZFa、AZFb微缺失各1例（2．86％）,AZFc微缺失29例（82．86％）,AZFb＋c微缺失2倒（5．71％）,AZFa＋b＋c微缺失2例（5．71％）。严重少精子患者14例,缺失类型均为AZFc;无精子症患者21例,其中对12例无精子症患者行睾丸穿刺活检,2例AZFc微缺失患者发现精子。其中未发现输精管缺如患者。染色体核型分析2例AZFc微缺失患者发现异常,其余均为46,XY。严重少精子组与无精子组患者年龄、不育年限、黄体生成素、雄激素及出生时父亲年龄无统计学差异,卵泡刺激素有统计学差异。结论 临床表型、染色体核型正常的严重少弱精子或无精子症患者可存在Y染色体微缺失,其发生率约为10％,最常见的类型为AZFc微缺失。单纯AZFc缺失对精子生成的影响比其他类型缺失较小,无精子症AZFc缺失者行睾丸穿刺活检有可能发现可用精子,AZFa、AZFb或AZFa＋b＋c缺失者基本不可能有精子,临床上无睾丸活检的价值。     

Y chromosome deletions in azoospermic and severely oligozoospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction

Y chromosome deletions encompassing the AZFc region have been reported in 13% of azoospermic men and 7% of severely oligozoospermic men. We examined the impact of these Y deletions on the severity of testicular defects in 51 azoospermic men undergoing intracytoplasmic sperm injection (ICSI) after testicular sperm extraction (TESE) and 30 men with severe oligozoospermia undergoing ICSI after ejaculation of spermatozoa. In addition, five azoospermic patients shown previously to have Y chromosome deletions underwent histological evaluation of their previously obtained testis biopsy specimens. A further 27 azoospermic men underwent TESE-ICSI, but not Y chromosome DNA testing. Ten of 51 azoospermic men (20%) who underwent TESE-ICSI and Y-DNA testing were found to be deleted for portions of the Y chromosome AZFc region. Of these 10, five had spermatozoa retrievable from the testis, and in two cases the wives became pregnant. Of the 41 azoospermic men with no Y chromosome deletion, 22 (54%) had spermatozoa retrievable from the testis, and in 12 cases (29%) the wives became pregnant. Four of 30 (13%) severely oligozoospermic patients were found to be deleted for AZFc and in three (75%) of these pregnancy was achieved. The other 26 severely oligozoospermic couples who had no AZFc deletions underwent ICSI, and 12 (46%) have an ongoing or delivered pregnancy. The embryo implantation rate was not significantly different for azoospermic (22%), oligozoospermic (16%), Y-deleted (14%) or Y-intact (18%) men. Of the total of 19 infertile men who had Y chromosome deletions, 14 had deletions within Y chromosome intervals 6D-6F, in the AZFc region. Twelve of those 14 had some spermatozoa (however few in number) in the ejaculate or testis. Five of the Y-deleted men had deletions that extended more proximally on the Y chromosome, and in none of these could any spermatozoa be observed in either ejaculate or testis. These results support the concept that, in azoospermic or oligozoospermic men with Y chromosome deletions limited to intervals 6D-6F (AZFc), there are generally very small numbers of testicular or ejaculated spermatozoa. Larger Y deletions, including and extending beyond the AZFc region and encompassing more Y genes, tend to be associated with a total absence of testicular spermatozoa. In those cases where spermatozoa were retrieved, the presence of Y deletions had no obvious impact on fertilization or pregnancy rate.      

Induction of spermatogenesis in azoospermic men after varicocele repair

BACKGROUND: The purpose of this study was to assess the treatment outcome after varicocele repair in azoospermic men and to correlate this outcome with the testicular histology patterns. METHODS: Medical records of 15 azoospermic men who underwent testis biopsy and microsurgical repair of clinical varicocele between July 1999 and November 2000 were reviewed. All patients had at least two semen analyses showing azoospermia taken before the surgery and two semen analyses post-operatively. Hypospermatogenesis was identified in four, maturation arrest in six, and germ cell aplasia in five men. RESULTS: Induction of spermatogenesis was achieved in seven men (47%). Of these, four had germ cell aplasia and three had maturation arrest. The improvement in sperm concentration and motility in patients with germ cell aplasia ranged from 1.8 to 7.9 x 10(6)/ml, and from 32 to 76% respectively. Of these seven patients with improvement in semen quality, five relapsed into azoospermia 6 months after the recovery of spermatogenesis (four germ cell aplasia and one maturation arrest). One patient with maturation arrest established a pregnancy. CONCLUSIONS: Azoospermic patients may have an improvement in semen quality following varicocelectomy. Semen samples should be cryopreserved after an initial improvement following varicocelectomy, as relapse to a state of azoospermia may occur.     

Y chromosome microdeletions, in azoospermic or near-azoospermic subjects, are located in the AZFc (DAZ) subregion

Submicroscopic deletions of the Y chromosome and polymorphisms of the androgen receptor (AR) gene in the X chromosome have been observed in men with defective spermatogenesis. To further define the subregions/genes in the Y chromosome causing male infertility and its relationship to polymorphisms of the AR polyglutamine tract, we screened the genomic DNA of 202 subfertile males and 101 healthy fertile controls of predominantly Chinese ethnic origin. Y microdeletions were examined with 16 sequence-tagged site (STS) probes, including the RBM and DAZ genes, spanning the AZFb and AZFc subregions of Yq11, and related to the size of trinucleotide repeat encoding the AR polyglutamine tract. Y microdeletions were detected and confirmed in three out of 44 (6.8%) of azoospermic and three out of 86 (3.5%) severely oligozoospermic patients. No deletions were detected in any of the patients with sperm counts of >0.5 x 10(6)/ml, nor in any of the 101 fertile controls. All six affected patients had almost contiguous Y microdeletions spanning the entire AZFc region including the DAZ gene. The AZFb region, containing the RBM1 gene, was intact in five of the six subjects. Y deletions were not found in those with long AR polyglutamine tracts. Our study, the first in a Chinese population, suggest a cause and effect relationship between Y microdeletions in the AZFc region (possibly DAZ), and azoospermia or near-azoospermia. Y microdeletions and long AR polyglutamine tracts appear to be independent contributors to male infertility.      

High prevalence of testicular cancer in azoospermic men without spermatogenesis

BACKGROUND: An increased risk of testicular cancer in men with infertility and poor semen quality has been reported. Our aim was to investigate the prevalence of testicular nodules and cancer in azoospermic subjects with different spermatogenetic patterns. METHODS: A total of 1443 consecutive infertile men were investigated, out of which 145 (10.0%) were found to be azoospermic. By using clinical examination and testicular ultrasound, 11 out of the 145 patients showed testicular nodules (2.8-26 mm). To obtain spermatozoa for assisted reproduction, 97 subjects required testicular sperm extraction (TESE) and biopsy, including the 11 patients with nodules. They were divided into two groups according to biopsy results: Group A (n = 38) with complete Sertoli cell-only syndrome (SCOS) and Group B (n = 59) with varying spermatogenetic patterns. Ten nodules were found in Group A and one in Group B. RESULTS: In azoospermic men, the overall prevalence of nodules was 7.5%. In complete SCOS, the prevalence of nodules and cancer was 10/38 (26.3%) and 4/38 (10.5%), respectively. Amongst the cancers, one embryonal carcinoma, one seminoma and two in-situ carcinomas were found. CONCLUSION: The prevalence of testicular nodules and cancer in azoospermic men with complete SCOS is very high. In these subjects, the role of clinical evaluation, ultrasound and biopsy should be emphasized.     

Testicular sperm extraction in azoospermic men submitted to bilateral orchidopexy

BACKGROUND: This study was carried out to evaluate whether bilateral orchidopexy represents a poor or good prognostic factor in azoospermic men undergoing testicular sperm extraction (TESE). METHODS: One hundred and seven presumed non-obstructive azoospermia (NOA) patients, according to conventional clinical parameters (volume of testis, FSH, clinical history) were submitted to testicular biopsy with TESE. Thirty men (28%) had a history of bilateral orchidopexy for cryptorchidism. RESULTS: Normal spermatogenesis or mild hypospermatogenesis was diagnosed in 12/30 ex-cryptorchid patients and in 7/77 presumed NOA patients (P = 0.0004). Conversely, pure Sertoli cell-only syndrome or complete maturation arrest was found in 10/30 ex-cryptorchid patients and in 48/77 presumed NOA patients (P = 0.0094). In 53/107 patients (49.5%), TESE allowed a positive sperm retrieval. At least one spermatozoon was observed in 22/30 ( approximately 73%) ex-cryptorchid patients and in 31/77 ( approximately 40%) presumed NOA patients (P = 0.0026). A large number of spermatozoa (equivalent to an obstructive pathology) were retrieved in 13/30 ex-cryptorchid and in 10/77 presumed NOA patients (P = 0.001). A history of bilateral orchidopexy in presumed NOA patients correlates positively for the chance of retrieving testicular spermatozoa (odds ratio 3.8; 95% confidence interval 1.41-10.21; P = 0.008). CONCLUSIONS: Although bilateral cryptorchidism is usually considered a testicular secretive dysfunction, TESE permits retrieval of a large number of spermatozoa in almost 40% of cases. Our data suggest the existence of congenital or acquired obstructive anomalies of the seminal ducts in azoospermic orchidopexed men.     

Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men

The involvement of Sertoli cells in different spermatogenic impairments has been studied by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells. CK-18 is known to be expressed in Sertoli cells during prenatal and prepubertal differentiation and is normally lost at puberty. Forty-nine azoospermic men were included in the current study. Quantitative measurements on testicular biopsies revealed the highest CK-18 expression in the mixed atrophy biopsies (22 men), a lower expression in the Sertoli cell-only (SCO) biopsies (12 men), and minimal residual staining in the group considered as representing normal spermatogenesis (six obstructive azoospermia patients). The cytokeratin immunopositive-stained tubules were associated either with arrest in spermatogenesis or with SCO. Examination of sections from nine men with microdeletions in the AZF region of the Y chromosome revealed that these men were either negative for CK-18 expression or showed only weak residual staining. This may suggest that the spermatogenic defect in the AZF-deleted men originates in the germ cell and has no impact on Sertoli cell maturation. The cause that determined the spermatogenic defect in the other cases of male infertility with high CK-18 expression may have damaged both the Sertoli and the germ cells.     

Fertilization and pregnancy outcome with intracytoplasmic sperm injection for azoospermic men

The evident ability of the intracytoplasmic sperm injection (ICSI) procedure to achieve high fertilization and pregnancy rates regardless of semen characteristics has induced its application with spermatozoa surgically retrieved from azoospermic men. Here, ICSI outcome was analysed in 308 cases according to the cause of azoospermia; four additional cycles were with cases of necrozoospermia. All couples were genetically counselled and appropriately screened. Spermatozoa were retrieved by microsurgical epididymal aspiration or from testicular biopsies. Epididymal obstructions were considered congenital (n = 138) or acquired (n = 103), based on the aetiology. Testicular sperm cases were assessed according to the presence (n = 14) or absence (n = 53) of reproductive tract obstruction. The fertilization rate using fresh or cryopreserved epididymal spermatozoa was 72.4% of 911 eggs for acquired obstructions, and 73.1% of 1524 eggs for congenital cases; with clinical pregnancy rates of 48.5% (50/103) and 61.6% (85/138) respectively. Spermatozoa from testicular biopsies fertilized 57.0% of 533 eggs in non-obstructive cases compared to 80.5% of 118 eggs (P = 0.0001) in obstructive azoospermia. The clinical pregnancy rate was 49.1% (26/53) for non-obstructive cases and 57.1% (8/14) for testicular spermatozoa obtained in obstructive azoospermia, including three established with frozen-thawed testicular spermatozoa. In cases of obstructive azoospermia, fertilization and pregnancy rates with epididymal spermatozoa were higher than those achieved using spermatozoa obtained from the testes of men with non-obstructive azoospermia.     

Diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men.

Various procedures for sperm recovery in azoospermic men have been described, from open testicular biopsy to simple needle aspiration from the epididymis and the testis. Fifty-one obstructive and 86 non-obstructive azoospermic men were treated to compare the recovery of spermatozoa obtained by percutaneous aspiration from the epididymis (PESA) and aspiration/extraction from the testis (TESA, TESE) with histopathology. If TESA failed, the work up proceeded with TESE. All patients were karyotyped. Spermatozoa were recovered by PESA or TESA in all obstructive men (51/51 patients). In 22 out of 86 patients with non-obstructive azoospermia, testicular spermatozoa could be successfully recovered by TESA. In five additional patients TESE was successful in recovering spermatozoa where TESA had failed. In 43 patients, neither TESA nor TESE was successful. Sixteen patients chose not to proceed with TESE. Seven out of 86 patients had an abnormal karyotype in the non-obstructive group (8%), none in the obstructive group. In the non-obstructive patient group testicular histopathology showed hypospermatogenesis, incomplete maturation arrest and germ cell aplasia with focal spermatogenesis in cases where spermatozoa were recovered and complete germ cell aplasia, complete maturation arrest and fibrosis in cases where no spermatozoa were found. Spermatozoa were recovered by PESA or TESA from all patients with obstructive azoospermia and from approximately 40% of patients with non-obstructive azoospermia by TESA or TESE. Retrieval of viable spermatozoa in the infertility work-up was highly predictable for sperm recovery in subsequent ICSI cycles. TESA performed under local anaesthesia seems almost as effective as more invasive procedures in recovering testicular spermatozoa, both in obstructive and non-obstructive azoospermic men.     

Fertility with testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermic men

In non-obstructive azoospermia spermatozoa can usually onlybe isolated from the testicles, and thus the most promisingtreatment model is testicular sperm extraction (TESE). Hormoneconcentrations, testicular volume determinations and testicularbiopsy results are not uniform enough to select potential candidatesfor successful TESE and intracytoplasmic sperm injection (ICSI)approaches in advance. The aim of this study was to assess theefficacy of using ICSI with testicular spermatozoa in casesof non-obstructive azoospermia and to compare the inclusioncriteria and sperm existence in the testicles in sperm obtainableand non-obtainable groups. All men showed either complete orincomplete (n = 14) maturation arrest in spermatogenesis, severehypospermatogenesis (n = 10) or Sertoli cell-only syndrome (n= 5) in their testicular biopsies. Only 14 out of a total of29 men provided enough spermatozoa for the ICSI procedure, whileno spermatozoa were found in the testicular samples of the remaining15 men. Out of 123 oocytes obtained from 14 females, 101 wereinjected with the husbands' testicular sperm cells. Total fertilizationfailure was observed in three cases. Of 39 oocytes fertilized,38 cleaved. The fertilization and cleavage rates were 38.6 and97.4% respectively. The pregnancy rate was 20.7% per initiatedcycle. In the group from whom spermatozoa were obtainable, thepregnancy rate was 42.9% per initiated cycle and 54.5% per embryotransfer. A total of six pregnancies were achieved, of whichtwo Were twins and four were singletons. One singleton pregnancyresulted in abortion in the first trimester. There was no statisticaldifference concerning the serum follicle stimulating hormoneconcentration, testicular volume and biopsy results in groupsin which spermatozoa were obtainable or not. In conclusion,although the association of TESE with ICSI obtained pregnanciesfor some patients with non-obstructive azoospermia, furtherstudies are needed to determine the inclusion criteria for successfulTESE.     

Testicular toxoplasmosis in two men with the acquired immunodeficiency syndrome (AIDS)

Testicular infection due to Toxoplasma gondii in two young men with the acquired immunodeficiency syndrome manifested as a multifocal necrotizing lesion of the testicular parenchyma. At the periphery of the necrotic area were inflammatory infiltrates consisting mainly of eosinophilic leukocytes. The Toxoplasma organisms were mainly found within the necrotic seminiferous tubules, where they were identified with periodic acid-Schiff or May-Grünwald-Giemsa stains and by electron microscopy. The histologic pattern of this orchitis is characteristic and should be suspected in patients with severe disorders of the immune response.     

Ultrastructural analysis of chromatin defects in testicular spermatids in azoospermic men submitted to TESE-ICSI.

BACKGROUND: Testicular sperm extraction (TESE) combined with intracytoplasmic sperm injection (ICSI) is offered to treat obstructive and non-obstructive azoospermia, but factors that influence the outcome of ICSI are not well defined. METHODS AND RESULTS: The percentage of elongated spermatids with normal chromatin condensation in azoospermic patients submitted for TESE-ICSI was determined. The quantitative analysis could be applied to nine of 19 biopsies classified as incomplete late maturation arrest (LMA) and compared with 10 biopsies with normal spermatogenesis. The percentage of elongated spermatids with normal chromatin was lower in LMA than in normal histology (mean 4.4%, range 0-20, and mean 52.9%, range 40-70 respectively; P = 0.0001). The percentage of elongated spermatids with normal chromatin was negatively correlated with the serum concentration of FSH (r = -0.86, P < 0.0001) and the number of degenerated germ cells per 100 Sertoli cells nuclei (r = -0.68; P < 0.0001), while it was positively correlated with the number of elongating spermatids per 100 Sertoli cell nuclei (r = 0.81; P < 0.0001). The percentage of elongated spermatids with normal chromatin was not correlated with the rate of oocyte fertilization, while the delivery rate/cycle was higher in cases with normal histology compared with cases of LMA. CONCLUSIONS: These preliminary data suggest that an altered chromatin condensation is a ubiquitous defect in spermatids of non-obstructed azoospermic men submitted for TESE-ICSI.     

Reproductive capacity of round spermatids compared with mature spermatozoa in a population of azoospermic men

The present study aims to evaluate the injection of testicular round spermatids from patients with complete failure of spermiogenesis compared with that of mature epididymal and testicular spermatozoa. Over a period of 8 months, 188 azoospermic patients were evaluated with a view to their inclusion in our intracytoplasmic sperm injection (ICSI) programme. All patients had had a previous testicular biopsy; 38 had pure obstructive azoospermia, while 150 had non-obstructive azoospermia. Mature spermatozoa were found in 93 patients, whereas spermatozoa were entirely absent, with a predominance of round spermatids in 87. In eight patients, spermatids could not be found and therefore their cycles were cancelled. There was an early appearance of the two pronuclei stage in the round spermatid group compared with the mature spermatozoa group of patients (10.2 and 16 h respectively). The fertilization rate was also significantly lower (P = 0.00001) in the round spermatid group. The numbers of embryos developed and of embryo transfers in the round spermatid injection group were significantly lower compared with the mature spermatozoa injection group (P = 0.05 and 0.0001 respectively). No pregnancies resulted from round spermatid injection, while 18 pregnancies were achieved from the injection of mature spermatozoa. In conclusion, injection of round spermatids from patients with complete failure of spermiogenesis resulted in a significantly lower fertilization rate and a higher developmental arrest compared with injection of mature spermatozoa. With no pregnancies achieved, one may question the unusual variability of reported success rates and stress the need for further research in order to improve the outcome of this novel technique.     

The results of 154 ICSI cycles using surgically retrieved sperm from azoospermic men

BACKGROUND: The effects of source of sperm, aetiology and sperm cryopreservation on ICSI cycles in azoospermic men were evaluated. The effect of aetiology of azoospermia on embryo development was also assessed. METHODS: This study was a retrospective analysis of 154 cycles (91 couples) using surgically retrieved sperm. Outcome measures were fertilization rate (FR), implantation rate (IR), and clinical pregnancy rate (CPR) and livebirth rate (LBR) per transfer. RESULTS: Our data demonstrated similar outcome between the use of epididymal or testicular sperm in men with obstructive azoospermic (OA). FR and IR were significantly lower (P < 0.05) using sperm from men with non-obstructive azoospermic (NOA), but although pregnancy outcome appeared lower, this did not reach statistical significance (P = 0.08). Cryopreservation of epididiymal sperm did not alter outcome, but the use of frozen-thawed testicular sperm did demonstrate a lower FR, with no statistical difference in IR or pregnancy outcome. Embryos derived from NOA sperm had impaired development beyond day 2 post-oocyte retrieval (OA, 44% <5 cell; NOA, 71% <5 cell; P = 0.002). CONCLUSIONS: The use of sperm from men with NOA significantly affects fertilization and implantation in ICSI cycles. The use of frozen-thawed testicular sperm affects fertilization rate without significantly altering pregnancy outcome. The use of such data on which to base clinical decisions needs to be supported by the meta-analyses of previous reports.     

Towards the molecular localisation of the AZF locus: mapping of microdeletions in azoospermic men within 14 subintervals of interval 6 of the human Y chromosome.

We have used a series of 30 DNA probes previously mapped to the long arm of the human Y chromosome, to screen a panel of 21 patients with structural abnormalities in Yq, by genomic blot hybridisation. The results have allowed us to construct a detailed map of interval 6 of the Y chromosome, in which 28 of the probes could be assigned to 14 sub-intervals within interval 6. Some probes detect two or more loci within this region, each of which has been localised. The same set of probes has been used to screen a panel of 19 chromosomally normal azoospermic men, two of whom have been found to carry microdeletions within this region. With the completion of this map we have been able accurately to localise these microdeletions within interval 6 and show that they do not overlap. We believe these microdeletions may disrupt the azoospermia factor (AZF) involved in spermatogenesis, and which is known to lie in this region. These results are an important step towards the localisation of the AZF locus.