Comparison of flow cytometry to routine testicular biopsy in male infertility

We compared DNA flow cytometry to morphologic evaluation of routine testicular biopsies as methods of monitoring spermatogenesis. The study group consisted of 14 azoospermic men and 5 others who underwent testicular surgery unassociated with fertility problems. The findings for both studies were divided into three groups: normal, moderately abnormal, and markedly abnormal. Correlations between the findings from routine biopsy and flow cytometry were good. Of 9 patients having normal testicular morphology, 7 had normal ploidy classes by DNA flow cytometry while 2 had moderately abnormal histograms. Of 5 cases with moderately abnormal morphology, 1 had normal, 1 had moderately abnormal, and 3 had markedly abnormal ploidy distributions. In 5 cases described as Sertoli cell only, all DNA histograms were markedly abnormal, consisting almost exclusively of diploid cells. DNA flow cytometry of testicular biopsies and aspirates has been demonstrated to be a rapid, reproducible, and objective approach in evaluating the infertile male and is a promising method to investigate spermatogenesis in an outpatient clinic in lieu of formal testis biopsy.     

Deoxyribonucleic Acid Flow Cytometry in the Assessment of Spermatogenesis

Purpose

We compared deoxyribonucleic acid (DNA) flow cytometric analysis of testicular tissue to quantitative assessment of spermatogenesis.

Materials and Methods

We studied 35 infertile men with azoospermia or oligospermia. All patients underwent incisional testicular biopsies. DNA flow cytometric analysis was performed on each specimen to evaluate the ability of the method to quantify alterations in spermatogenesis. The results were compared to quantitative histological examination. At least 100 spermatic tubules were examined on each specimen and the number of spermatids per tubule was counted. All histological specimens were examined by the same pathologist.

Results

Of the 35 specimens analyzed with DNA flow cytometry 5 were normal, while the percentage of haploid cells (spermatids and spermatozoa) was decreased (hypospermatogenesis) in 14, complete maturation arrest was noted in 2 and almost complete absence of haploid cells was found in 14. Comparing the findings on histological examination with histograms, excellent correlation was noted in cases of the Sertoli-cell-only syndrome and complete maturation arrest, while 3 of 14 histograms with hypospermatogenesis demonstrated normal spermatogenesis on histological examination. Additionally 1 of 5 histograms with normal spermatogenesis demonstrated hypospermatogenesis on histological examination.

Conclusions

DNA flow cytometry of the testicular tissue seems to be an objective and quantified method that can be used to investigate spermatogenesis in infertile men. It is also less time-consuming than any histological examination, permits management decisions within 1.5 hours after biopsy and may replace testicular histopathological study. Flow cytometric diagnoses correlated well with histopathological findings.     

The value of quantitative DNA flow cytometry of testicular fine-needle aspirates in assessment of spermatogenesis: a study of 137 previously maldescended human testes

In order to assess the suitability of DNA flow cytometry of fine-needle aspirates for quantiftring spermatogenesis, the results from DNA flow cytometry were compared to histological evaluation of testicular biopsies taken concomitantly from 171 previously maldescended testes. In 137 of 171 cases, sufficient material for flow cytometric as well as histological evaluation was obtained.
Histological analysis of surgical biopsy specimens revealed spermatogenesis including the spermatid stage in 117 of the 137 gonads. In six of the 117 gonads no haploid cells were found using flow cytometry. On the other hand, surgical biopsies failed to reveal spermatogenesis in five cases in which the corresponding aspirates contained haploid cells. Both methods therefore seem equally sensitive in detection of spermatogenesis.
Other types of histological patterns also corresponded to distinct DNA histograms.
Thus, in 11 of 12 cases with Sertoli-cell-only pattern in all tubules, at least 95% of the cells had a diploid DNA content. Furthermore, predominance of tubules with maturation arrest at the primary spermatocyte level corresponded to an increased proportion of tetraploid cells.
When compared to surgical biopsy, DNA flow cytometry of testiclar fine-needle aspirates is a more objective, easy and rapid method, which is more convenient for the patient. This study has indicatedthat DNA flow cytometry is a suitable method of quantitative assessment of spermatogenesis. One of the first target groups might be men with azoospermia. In such men, DNA flow cytometric analysis of fine-needle aspirates and surgical biopsy are apparently of equal sensitivity in detecting gonads with spermatogenesis. We conclude that DNA flow cytometry may become an alternative method for the quantification of spermatogenesis.     

先天性双侧输精管缺如不育患者睾丸生精功能的研究

目的 通过对先天性双侧输精管缺如(CBAVD)不育患者睾丸细针穿刺吸液(FNA)细胞学检查,了解睾丸生精功能。方法 对78例CBAVD不育患者进行FNA细胞学检查。结果 78例中56例涂片可见较多生精细胞、精子细胞及精子,提示睾丸生精功能正常,占71.8%(56/78);生精功能异常22例(28.3%),其中6例(6/78,7.7%)仅见支持细胞,8例(8/78,10.3%)见各级生精细胞及大量精子细胞,未见精子,8例(8/78,10.3%)涂片见较少精子(<2个/HP)。结论 部分CBAVD不育患者的睾丸生精功能有不同程度的损伤。     

Flow cytometric DNA analysis demonstrates contralateral testicular deterioration in experimental unilateral testicular torsion of prepubertal rats

Summary. It has been postulated that unilateral testicular torsion causes damage to the contralateral testis and reduces fertility. However, in animal studies such an effect has not been fully proven by histopathologic examination or other conventional assays of spermatogenesis. We investigated the effect of unilateral testicular torsion on contralateral spermatogenesis in prepubertal rats using quantitative flow cytometric DNA analysis. Male rats were divided into three groups which underwent sham-operation, simple hemiorchiectomy or unilateral testicular torsion. Five weeks after these operations, fertility and spermatogenesis by flow cytometry were evaluated. No significant differences were observed in body weight, contralateral testicular weight or serum testosterone concentration among the three experimental groups. In the torsion group, mean seminiferous tubular diameter, number of foetuses, fertility rate and percentage of haploid cells were all significantly decreased compared to the other two groups. These results suggest that unilateral testicular torsion causes damage to the contralateral testis and consequently can reduce the future fertility of prepubertal rats.     

Clinical evaluation of DNA flow cytometry of fine needle aspirates from testes of infertile men

DNA flow cytometry was performed on fine needle aspirates from the testes of 40 oligozoospermic or azoospermic men under investigation for infertility. The DNA distributions from men with increased FSH serum levels were all abnormal. The values were below the level of detection (or very low) with respect to both haploid (1c) and tetraploid (4c) cells, indicating reduced proportions of spermatids and primary spermatocytes. This confirms that increased FSH serum levels are indicative of severely damaged spermatogone-sis. The findings of both normal and abnormal testicular DNA distributions in the large group of oligozoospermic men indicate that the presented method may be of importance for evaluating prognosis, and for selection of men for further investigation and therapy. Many azoospermic men showed normal testicular DNA distribution patterns, suggesting the value of DNA flow cytometry for selection of such cases for surgical treatment (epididymovasos-tomia).     

Comparison of histologic and flow cytometric evaluation of cyclophosphamide-induced testicular damage

This study evaluates the ability of DNA histograms obtained by flow cytometry to detect and quantify reversible alterations in spermatogenesis induced by cyclophosphamide, a known inhibitor of spermatogenesis. Evaluation of per cent of cells in each of the haploid (lc), diploid (2c), and tetraploid peaks (4c) as determined by flow cytometry in treated and control Balb/C mice over a six-week period, and comparison with routine histologic evaluation have led us to conclude that DNA histogram evaluation is a rapid and accurate means of identifying testicular damage and recovery. This technique may be useful in sequential monitoring of the effects of malignancy and/or treatments applied on spermatogenesis in young men.     

Ultrastructure of the seminiferous tubules in oligoasthenoteratozoospermic men associated with varicocele

Varicocele is associated with venous reflux that may cause increased heat and interstitial pressure within the testes, with variable pathological effects on spermatogenesis. This study aimed to study the ultrastructural testicular changes in the seminiferous tubules of 20 infertile severe oligoasthenoteratozoospermia (OAT) men associated with varicocele and five patients with obstructive azoospermia without varicocele as controls. They were subjected to testicular biopsy which was evaluated by transmission electron microscopy. Ultrastructurally, the seminiferous epithelium in the testicular biopsies of infertile severe OAT men associated with varicocele was variably affected in the form of thickening of the peritubular connective tissue, vacuolation of Sertoli cell and germ cell cytoplasm, presence of degenerated and apoptotic cells among the germinal epithelium, altered spermatids and abnormal spermatozoa. It is concluded that varicocele in severe OAT men is associated with ultrastructural changes in the seminiferous tubule.     

Spermatogenesis in the contralateral testis of patients with testicular germ cell cancer: histological evaluation of testicular biopsies and a comparison with healthy males

OBJECTIVE: To assess histologically signs of testicular dysgenesis (TD) in the contralateral testes of patients with testicular germ cell tumours (GCTs) and to compare these findings with the spermatogenetic quality in healthy men, as the contralateral testis is considered to be involved with dysgenetic features such as poor sperm production, and accordingly, GCTs are hypothesized to be part of the 'TD syndrome' (TDS). One testicular biopsy is thought to represent spermatogenesis in the entire testis. We evaluated this view by using testicular two-site biopsies. PATIENTS AND METHODS: 2318 patients with testicular GCT had a contralateral testicular two-site biopsy. Testicular biopsies taken on forensic autopsy from 1388 presumably healthy men served as controls. Spermatogenesis was rated histologically according to a modified Johnsen score. Clinical factors were recorded to explore associations with reduced spermatogenesis. Differences in spermatogenesis scoring results among two-site biopsies were noted. Statistical analysis involved Wilcoxon-Mann-Whitney and Jonckheere-Terpstra tests for comparing patients and controls, and for studying associations with clinical factors. Classification and regression-tree analysis was used to explore multivariate associations. RESULTS: Histologically, patients had significantly poorer spermatogenesis than healthy men. Clinically, hypospermatogenesis was significantly associated with testicular atrophy, undescended testes, male infertility, and advanced clinical stage; 5.4% of cases (95% confidence interval 4.43-6.27) had discordant findings of >2 points on double biopsy and 9.8% had differences of 1 point. Discordance was significantly associated with poor spermatogenesis and testicular atrophy. CONCLUSIONS: We confirmed histologically that there is markedly reduced spermatogenesis in the contralateral testes of patients with GCT. This result lends credence to the view that GCT is part of the so-called TDS. But as hypospermatogenesis is associated with advanced clinical stage, impairment of sperm production might at least partly be acquired secondary to the endocrine activity of GCT. There were clinically relevant discordant results on double biopsy in 5.4%, predominantly in infertile patients and in atrophic testes. Thus the histological evaluation of male infertility is best done by multiple biopsies.     

Sonographic testicular microlithiasis as an indicator of premalignant conditions in normal and infertile men.

Sonographic detection of multiple, small hyperechogenic lesions in the testis (testicular microlithiasis; TM) can indicate germ cell tumors. However, it has not been well established whether this finding signifies a risk factor for development of testicular neoplasm in all cases or whether it indicates premalignant changes only in those men with additional risk factors for germ cell cancer, such as infertility, a history of testicular maldescent, or the presence of an atrophic testis. In a retrospective analysis of 1701 consecutively performed scrotal sonographies of patients with (n = 1399) and without (n = 219) infertility or with contralateral testicular tumors (n = 83), the prevalence of TM was compared with that in 198 healthy men who volunteered for different clinical trials. TM was equally frequent in all groups (2.3% [32/1399] of infertile patients, 2.3% [5/219] of other patients without infertility, and 1.5% [3/198] of healthy men). Results of testicular biopsies were available for a subgroup of infertile men. Carcinoma in situ (CIS) was present only in cases with TM (2/11). In addition, sonographic follow-up examinations were performed in another 14 men with TM. Testicular tumors had developed in 2 patients, one whom was infertile and one in the control group. None of these patients had a history of testicular maldescent but all testes affected either by CIS or tumors were reduced in volume. We conclude that diagnosis of TM, especially if it is present in an atrophic testis, demands a diagnostic biopsy or at least sonographic follow-up examinations.     

Yield and efficacy of biopty gun testis needle biopsy

OBJECTIVES: To assess whether a preliminary skin incision enhances diagnostic yield of percutaneous testis biopsy and to further evaluate the clinical efficacy of this procedure. METHODS: A total of 45 men (67 testes) underwent testicular biopsy with two passes of a Biopty gun spring-loaded needle. Twenty-seven biopsies were performed without a preliminary skin incision (group 1), and 40 were performed after a small scrotal incision (group 2). In 56 testes, needle biopsy histopathologic diagnosis was compared with that of open biopsy or orchiectomy specimens from the same patient. Needle and surgical specimens were fixed in Bouin's solution and sent separately for independent, blinded, histologic interpretation. RESULTS: Complications of the procedure were negligible. In all 67 needle biopsies, specimen quality was adequate for histopathologic interpretation. The mean number of seminiferous tubules obtained from needle biopsy was 28% higher among patients having a preliminary skin incision (25.9) compared with those without (18.7, P = 0.023). Correlation between needle and open histopathologic diagnosis was excellent (55 of 56, 98%). CONCLUSIONS: A preliminary skin incision made before needle biopsy increases the diagnostic yield of percutaneous testis biopsy. Percutaneous testis biopsy using the Biopty gun needle provides equal diagnostic information when compared with open testis biopsy or orchiectomy specimens. The concomitant reduction in morbidity and cost make this an attractive diagnostic procedure.     

Mast cells and fibrosis on testicular biopsies in male infertility

Testicular dysfunction correlates with increased testicular mast cells. Mast cells can activate fibroblasts and promote collagen synthesis. The aim of the study was to examine testicular mast cells containing tryptase, and the relationship between mast cells and different fibrosis stages of interstitium and peritubular region of testes. Testicular biopsies obtained from 33 infertile men were assigned to 2 groups: normal spermatogenesis (n = 10) and defective spermatogenesis (n = 23). Total, interstitial, and peritubular mast cells were examined immunohistochemically using antihuman tryptase. The fibrosis stage was evaluated using vimentin and alpha-smooth muscle actin. The ratio of tubules with sclerosis to total tubules was also calculated. In all cases, mast cells were mainly localized in the interstitium. The number of total mast cells was significantly higher in defective spermatogenesis than in normal spermatogenesis (p = .048). In both groups, interstitial mast cells were higher than peritubular mast cells. However, the increase in peritubular region was much higher than the increase in interstitium. Total, peritubular, and interstitial mast cell counts were not different from each other, according to the changing fibrosis stages. Total and interstitial mast cells were significantly higher in the cases with sclerosing seminiferous tubules than in the cases with no sclerosis (p = .04 and p = .024, respectively). The mast cells and the mast cell product tryptase could be involved in the etiology of defective spermatogenesis, especially whenever the last stage (tubular hyalinization and sclerosis) takes place.     

Clinical aspects of testicular carcinoma-in-situ

Carcinoma-in-situ germ cells were demonstrated in testicular biopsies from 9 of 826 patients (1.1%) from a selected group of Danish infertile men. A similar observation was noted in testicular biopsies from 9 Swiss patients (representing 0.55% of the total number of infertile patients biopsied in that study). Such changes were also seen in 8 testicular biopsies from the contralateral testis of 180 patients (4.4%) with carcinoma of the teitis. Moreover, carcinoma-in-situ has beer, found in maldecended testes and in gonads of patients with the testicular feminization syndrome although the incidence of carcinoma-in-situ in these two latter groups is unknown.
The malignant potential of carcinoma-in-situ of the testis in infertile men has been clearly demonstrated, whereas its clinical significance in other groups of patients remains to be determined.     

Canine model of infertility after spinal cord injury: time course of acute changes in semen quality and spermatogenesis

PURPOSE: We established a canine model of subfertility after spinal cord injury and examined the time course of acute changes in semen quality and spermatogenesis after spinal cord injury. MATERIALS AND METHODS: Seven dogs underwent surgical T7 spinal cord injury. Six dogs were used as controls. Electroejaculation and testicular fine needle aspiration were performed at baseline and twice weekly for 3 weeks after spinal cord injury. Semen quality change was examined by standard semen analysis. Spermatogenesis was assessed by flow cytometry of testicular fine needle aspiration in all dogs as well as by testicular histology at study conclusion in 4 controls and 4 spinal cord injured dogs. RESULTS: No significant changes in spinal cord injured dogs were noted before 3 weeks after injury. From baseline to 3 weeks after injury certain changes were evident in spinal cord injured dogs. Mean antegrade sperm motility decreased from 62.9% to 20.1% (p = 0.008), mean total sperm (antegrade plus retrograde total sperm) decreased from 423 to 294 x 106 which was not statistically significant, and the incidence of testicular haploid cells decreased from 75.6% to 48.3% (p = 0.028). No significant change in any parameter was present in control dogs. The mean number of mature spermatids per cross-sectional tubule on final testicular histology was significantly decreased in spinal cord injured dogs compared with controls (13.6 versus 43.9, p = 0.02). CONCLUSIONS: In the canine model tested the dogs readily survived spinal cord injury, electroejaculation was effective for obtaining ejaculate and fine needle aspiration allowed serial examination of spermatogenesis. Three weeks after spinal cord injury but not before 3 weeks sperm motility and spermatogenesis were significantly decreased. However, at the same point this decrease in spermatogenesis was not yet reflected in the total ejaculated sperm count.     

DNA flow-cytometric,histological and hormonal analysis of sertoli cell only syndrome (SECOS)

Sertoli cell only syndrome (SECOS) was identified on histology in 21 cases(16,28%) among 129 testicular biopsies performed in our department forazoospermia over the last 5 years. In these patients history, clinicalfeatures, hormonal levels, and histological findings were analyzed. Inaddition DNA flow-cytometric analysis was performed and showed an almostcomplete absence of haploid cells. All patients presented with elevatedserum FSH levels suggesting a Sertoli cell damage or reduced productionof inhibin due to the absence of sermatogenic cells. An good correlation wasfound between histological findings and DNA histograms.In conclusion SECOS is a syndrome of unknown aetiology presenting in menwith azoospermia. DNA flow-cytometric analysis is a reliable, rapid and easymethod in the diagnosis of SECOS, and can replace histological examination.     

Diagnostic findings from testis fine needle aspiration mapping in obstructed and nonobstructed azoospermic men

PURPOSE: Although helpful for defining extratesticular obstruction, the testis biopsy offers limited information on nonobstructive azoospermic testes. Guided by diagnostic biopsies, testis sperm extraction procedures fail in 25% to 50% of patients with nonobstructive azoospermia, largely because it is clinically difficult to know where sperm are located. To provide a more complete assessment of spermatogenesis in nonobstructive azoospermic patients and to simplify the confirmation of sperm production in men with obstruction, we use a systematic, fine needle aspiration "mapping" procedure. We summarize the diagnostic findings in a series of azoospermic men. MATERIALS AND METHODS: From 118 azoospermic infertile men (22 with obstructed and 96 with nonobstructed azoospermia) fine needle aspiration data were used to generate location specific, sperm frequency maps for obstructed and nonobstructive azoospermic testes to determine if "sperm rich" locations existed. RESULTS: Fine needle aspiration map analysis revealed that all aspiration locations from obstructed cases showed sperm. In men with nonobstructive azoospermia, sperm was identified in the right testis in 134 of 652 (20.5%) and in the left testis in 151 of 716 (21.1%) separate aspirations. Rates of sperm detection among various intratesticular sites were not statistically different. In 27.1% of cases the fine needle aspiration map found sperm in men with sperm negative biopsies. The likelihood of heterogeneity in fine needle aspiration sperm findings was 25% within individual nonobstructive azoospermic testes and 19.2% between testis sides. At post-procedure followup of 88 patients (74%), no clinical or surgical complications were observed. CONCLUSIONS: Testis fine needle aspiration mapping is a simple, reliable and informative diagnostic tool in the evaluation of azoospermic infertile men.     

The Significance of Testicular Reactive Oxygen Species on Testicular Histology in Infertile Patients

This study was designed to investigate the relationship between the effects of testicular reactive oxygen species (ROS) levels and testicular histology on infertile patients with the aid of xanthine oxidase system and testicular tissue malondialdehyde levels. Forty patients with idiopathic infertility constituted our study group. Bilateral testicular biopsies were performed and spermatogenesis was assessed histopathologically. Patients were divided into 4 groups according to spermatogenic pattern (normal spermatogenesis; hypospermatogenesis; maturation arrest; Sertoli cell only syndrome). Testicular tissue xanthine oxidase and malondialdehyde (MDA) concentrations were analyzed in each sample by spectrophotometric assay and thiobarbituric acid reaction assay, respectively. Testicular tissue MDA and xanthine oxidase concentrations were not statistically different in patients having normal spermatogenesis, with respect to Sertoli cell only syndrome, maturation arrest and hypospermatogenesis, respectively. As a result of our study we think that there are still some factors other than ROS which may be important contributors to spermatogenetic injury that need to be examined.     

Mast cells in testicular biopsies of infertile men with 'mixed atrophy' of seminiferous tubules

Mast cells in the bilateral testicular biopsies of 30 patients with a 'mixed atrophy' of seminiferous tubules were analysed. Seven biopsies from vasectomized patients served as controls. With regard to their characteristic location within testicular tissue, two groups of mast cells could be distinguished, in both control and infertile patients: 'interstitial' mast cells (located between Leydig and other interstitial cells as well as in the vicinity of blood vessels) and 'peritubular' mast cells (located in the close proximity of the tubular lamina propria or incorporated in the lamina propria itself). Morphometric data indicated a significant increase in the number and volume of mast cells in infertile patients when compared with controls. In the biopsies of infertile patients that were analysed both 'interstitial' and 'peritubular' mast cells showed a significant increase in their number and volume, although it appeared that 'peritubular' mast cells increased at a higher rate than 'interstitial' mast cells. A significant negative correlation was found between the following variables: volume and number of mast cells, testis volume and the status of spermatogenesis evaluated by Johnsen's scoring. It was concluded that the increased presence of mast cells is closely associated with an impairment of spermatogenesis.     

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?