Image Analysis Assessment of the Abnormal Testis Biopsy in Male Infertility

Purpose

We previously demonstrated that testis biopsy image analysis is an effective method for quantifying intratubular spermatogenic cells in the obstructed testis with normal spermatogenesis. As an extension of the initial report, we describe using the quantitative ploidy and morphological characteristics of cells counted with image analysis in abnormal testis biopsies obtained for a male infertility evaluation.

Materials and Methods

Image analysis using a specifically designed filter was performed on Feulgen stained 5 micro m. sections of paraffin embedded testicular tissue. Archival testicular tissue had been obtained using standard biopsy techniques from patients with azoospermia or severe oligospermia. Qualitative classification was based on standard evaluation of hematoxylin and eosin processed tissue.

Results

There were 62 biopsies performed in 58 men. Significant differences in the intratubular content of haploid (spermatozoa and spermatids), diploid and tetraploid cells were found among the 5 categories of abnormalities: the Sertoli-cell-only syndrome, spermatocyte arrest, spermatid arrest, hypospermatogenesis and normal spermatogenesis. Moderate variability was found in the proportion of cell types in spermatid arrest and hypospermatogenesis.

Conclusions

Testis biopsy image analysis provides a quantitative method for categorizing abnormalities of intratubular cell content present in male infertility states by using deoxyribonucleic acid content and morphology characteristics. The limitations of the present qualitative analysis system are emphasized by the moderate variability evident within the current categories of spermatid arrest and hypo-spermatogenesis states.     

Flow-cytometric analysis of testes in infertile men: a comparison of the ploidy to routine histopathologic study.

DNA flow-cytometric analysis was performed on the testicular needle biopsies of 25 infertile men with azoospermia or oligozoospermia to evaluate the ability of DNA histograms in order to detect and quantify alterations in spermatogenesis. Concomitant histopathologic study was performed on the tissues from needle biopsy. In contrast to difficulty in quantifying spermatogenesis in histopathologic examination, flow-cytometric analysis revealed characteristic ploidy patterns in the relative proportions of haploid (1 n), diploid (2 n) and tetraploid (4 n) cells corresponding to the histopathologic appearances of normal spermatogenesis, hypospermatogenesis, maturation arrest and aspermatogenesis. Findings evaluated with flow cytometry were well correlated with those from routine histopathologic study. In 21 of these patients (84%) there was concordance between histopathologic and flow-cytometric diagnoses. However, in 4 patients (16%) there was discordance between two diagnostic modalities. In conclusion, DNA flow cytometry of testicular biopsies was a reproducible, objective and quantitative approach in evaluating infertile men, and it is a promising method to investigate spermatogenesis in an outpatient clinic in lieu of formal testicular histopathologic study.     

DNA flow cytometric evaluation of spermatogenesis. Part 2: Studies on male infertility

Flow cytometric DNA analysis was performed on the testicular biopsy tissue obtained from 17 oligospermic men, 25 azoospermic men and 5 normal men. DNA histograms were made after viewing a small piece of biopsy tissue for a short time. The DNA histograms were classified by eye into four types (Type A: Aspermatogenesis without haploid cell, Type B: Maturation arrest at primary spermatocyte, Type C: Hypospermatogenesis, Type D: Normal spermatogenesis). Analysis of the DNA histograms accurately revealed the proportion of haploid, diploid and tetraploid cells. The DNA distributions for 5 normal men were 58.9 +/- 3.6% haploid cells, 24.3 +/- 3.8% diploid cells and 16.8 +/- 0.8% tetraploid cells. Significant correlation was found between the proportion of haploid cells (%haploid) and the testicular volume. The results of the investigation of the correlation between the DNA distributions and histological evaluations show that testicular degeneration increase proportionally to the decrease in haploid cells. Therefore, the %haploid appears to be an effective index for the quantitative evaluation of spermatogenesis.     

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.     

IMAGE ANALYSIS ASSESSMENT OF TESTICULAR TOUCH PREPARATION CYTOLOGIES EFFECTIVELY QUANTIFIES HUMAN SPERMATOGENESIS

Purposes

We have recently demonstrated that computer assisted image analysis of paraffin embedded testicular tissue based on deoxyribonucleic acid content and morphology characteristics is an effective method for the quantitative assessment of spermatogenesis. We assess the use of testicular touch preparation image analysis as a technique for quantification of spermatogenesis.

Materials and Methods

Air dried, touch imprints of testicular tissue from obstructed azoospermic and severely oligozoospermic patients were obtained at the time of biopsy. Image analysis using a filter based on deoxyribonucleic acid content and cellular morphological characteristics was performed on Feulgen stained touch preparation imprints as well as paraffin embedded sections.

Results

Image analysis of 52 testicular touch preparations from 48 azoospermic or severely oligozoospermic men revealed significant differences (p <0.05) in the percentages of spermatid and spermatozoa, and 2N and 4N cells among seminiferous tubules exhibiting the 5 diagnostic categories of obstruction with normal spermatogenesis, maturation arrest at the spermatocyte stage, maturation arrest at spermatid stage, hypospermatogenesis and Sertoli cell only. Similar differences were observed in the image analysis data of the corresponding paraffin embedded testicular sections.

Conclusions

Computer assisted image analysis of testicular touch preparation is an effective quantitative method of spermatogenesis evaluation.     

Testis Biopsies Frequently Demonstrate Sperm in Men With Azoospermia and Significantly Elevated Follicle-Stimulating Hormone Levels

Purpose

Men with azoospermia, markedly elevated serum follicle-stimulating hormone levels and testicular atrophy were previously considered irreversibly infertile. Thus, testicular biopsy in this patient population was considered unnecessary. However, presently men with even the most severe infertility disorders are potentially able to initiate a pregnancy with intracytoplasmic sperm injection provided sperm can be recovered in even relatively few numbers directly from the testicular tissue. For these reasons we sought to reevaluate the findings from testicular biopsies in these men in the era of advanced micromanipulation techniques.

Materials and Methods

Chart review identified men with azoospermia, confirmed on a pelleted specimen, and a serum follicle-stimulating hormone level of 3 or more times normal. Mature sperm in the touch preparation cytology and testis biopsy specimen were confirmed.

Results

A total of 57 men, most with testicular atrophy, underwent a testicular biopsy and in 17 (30%) mature sperm were identified. The most common diagnosis in these men was severe hypospermatogenesis. Men without sperm most commonly had a pure Sertoli-cell-only pattern.

Conclusions

Men with azoospermia and testicular atrophy with significantly elevated follicle-stimulating hormone levels should undergo testicular biopsy if in vitro fertilization with intracytoplasmic sperm injection is an acceptable approach for the couple.     

睾丸精曲小管精子发生功能量化评价法分析无精子症患者睾丸生精功能

目的 :分析无精子症患者临床和病理资料 ,研究病理学量化评价睾丸精曲小管精子发生功能的方法的临床意义。　方法 :无精子症患者 112例 ,年龄 2 2～ 4 6 (2 9.0± 4 .4 )岁 ,婚龄 2～ 12 (4 .0± 2 .8)年、病程 2～ 6 (2 .70±1.0 2 )年 ,其中原发性无精子症 96例 ,继发性无精子症 16例 ;梗阻性无精子症 7例。不育症患者精液常规检查 3次确认无精子症 ,检测性激素水平 ,常规消毒下睾丸活检病理检查 ,在高倍镜下计数每个精曲小管中各类生精细胞数 ,测定小管直径、生精上皮高度和固有层厚度 ,按制定的精曲小管精子发生功能 10分 5级分度法加以评分 ,进行统计学分析。　结果 :精曲小管生精上皮 10分分度法评分结果 ,1分 5例 (4 .5 % ) ,2分 38例 (33.9% ) ,3分 2例(1.8% ) ,4分 6例 (5 .4 % ) ,5分 2例 (1.8% ) ,6分 17例 (15 .2 % ) ,7分 6例 (5 .4 % ) ,8分 19例 (17% ) ,9分 10例(8.9% ) ,10分 7例 (6 .3% )。精曲小管精子发生功能 5级分度法结果 ,1级 5例 (4 .5 % ) ,2级 38例 (33.9% ) ,3级 33例 (2 9.5 % ) ,4级 2 9例 (2 5 .9% ) ,5级 7例 (6 .3% )。多元回归分析结果 ,精曲小管精子发生功能分级与生精上皮高度、固有层厚度、精曲小管直径和血清卵泡刺激素 (FSH)具有极显著相关性 (P <0 .0 1)。组合     

Testicular seminoma after the complete remission of extragonadal yolk sac tumor : a case report

Background

Between 2% and 5% of malignant germ-cell tumors in men arise at extragonadal sites. Of extragonadal germ cell tumors, testicular carcinoma in situ (CIS) are present in 31–42% of cases, and CIS are reported to have low sensitivity to chemotherapy in spite of the various morphology and to have a high likelihood of developing into testicular tumors. A testicular biopsy may thus be highly advisable when evaluating an extragonadal germ cell tumor.

Case presentation

A 36-year-old man was diagnosed as having an extragonadal non-seminomatous germ cell tumor, that was treated by cisplatin-based chemotherapy, leading to a complete remission. In the meantime, testicular tumors were not detected by means of ultrasonography. About 4 years later, a right testicular tumor was found, and orchiectomy was carried out. Microscopically, the tumor was composed of seminoma.

Conclusions

We herein report a case of metachronous occurrence of an extragonadal and gonadal germ cell tumor. In the evaluation of an extragonadal germ cell tumor, a histological examination should be included since ultrasonography is not sufficient to detect CIS or minute lesions of the testis.

     

Quantification of seminal germ cells in azoospermia: correlations with testicular histology and TESE outcome

In the treatment of male infertility by intra-cytoplasmic injection of spermatozoa (ICSI) extracted from testicular tissue (TESE), the high incidence of negative TESE outcome calls for non-invasive prognostic methods. Literature suggests that seminal haploid germ cell detection could be one. For this purpose, a multi-parametric stringent flow cytometric method was applied to 50 TESE patients for the quantification of ejaculated germ cells. Cells from 50 ejaculates were identified and quantified as spermatozoa (HC, highly condensed), round spermatids (1N), primary spermatocytes (SPC) (4N) or diploid cells (2N, including somatic and non-testicular cells) by their DNA and mitochondria staining and laser scatter characteristics, and compared with testicular biopsy histology and TESE outcome. Whereas 96% of patients displayed a diploid peak in the distribution histograms, the HC, 1N and 4N peaks were absent from the majority of samples. In 13 ejaculates, either a HC or 1N or 4N peak, or a combination of these, was discernible. Although seminal germ cell numbers bore no overall association with elongated spermatids (ES) in histology or spermatozoa retrieval in TESE outcome, 4N cells per ejaculate were correlated with the percentage of tubule sections showing SPC as the most advanced germ cells. The incidence of HC peaks was higher in patients showing some ES in histology or sperm retrieval than in the sperm-negative groups. In groups with suspected obstruction showing nearly full spermatogenesis and maximal sperm retrieval, there was no incidence of a HC peak. Germ cell peaks were associated with germ cell degeneration noted in testicular histology. In conclusion, seminal germ cells cannot provide good prognosis for TESE, although their presence could indicate the spermatogenic activity in the testis.     

Potential role of reactive oxygen species on testicular pathology associated with infertility

Aim: To investigate the level of malondialdehyde (MDA), a direct indicator of lipid peroxidation-induced injury by reactive oxygen species (ROS), in testicular biopsy specimens from infertile patients. Methods: Levels of MDA were measured in testicular biopsy specimens from 29 consequent-randomized infertile men, aged 29.58±4.76 (21-45) years. All patients were evaluated by a complete medical and reproductive history, physical examination, semen analysis (at least two), serum follicle-stimulating hormone and free testosterone levels, testicular biopsy and contact imprint. Scrotal colour Doppler ultrasonography was used to confirm suspected varicocele. The testicular MDA level was measured using the thiobarbituric acid test and the results were expressed per unit tissue weight. Results: As a causal factor in infertility, varicocele was identified in 17 (58.6 %) patients, and idiopathic infertility, testicular failure and obstruction in 4 (13.8 %) patients each. The testicular MDA level was 13.56 (6.01),     

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.     

Shape and position of the haploid peak in flow cytometric sperm histograms for the assessment of andrological diseases

Ejaculates from 45 patients with various andrological diseases and from healthy men were analyzed by flow cytometric DNA measurements. The sperm histograms obtained by this method were subjected to mathematical analysis. The height, width and position of the haploid (1 C) peak proved to be a useful criterion for the discrimination of spermatozoa from patients with heterogeneous testicular disorders (SHTD, "mixed atrophy") and homogeneous testicular disorders (SETD) with delivery of immature spermiogenetic cells as compared to healthy persons.     

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.     

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.     

Microfluorometric assessment of sperm maturation in testicular biopsies from men with histologically normal or reduced spermatogenesis

Spermatogenesis has been studied in testicular biopsies by means of microfluorometric assessment of the DNA-DNP complex using ethidium bromide in 10 men with histologically normal spermatogenesis and in 10 men with histologically reduced spermatogenesis. The expected haploid DNA value was found in the round spermatids, whereas only 70% of the haploid fluorescence value was found in the elongated spermatids and around 60% in the testicular spermatozoa. No difference was found in the mean fluorescence values between the pathological group and the controls. It is suggested that structural changes or an increase in basic nuclear proteins gradually exclude the ethidium bromide from the binding to the DNA molecule and that this phenomenon occurs concomitantly with a decrease in double-stranded RNA. The difference in fluorescence intensity before and after RNAase treatment was regarded as an approximate estimate of the RNA content. There was a significant decrease of these values during spermatogenesis.     

Laparoscopic repair of pediatric inguinal hernia--is vascularity of the testis at risk? A study of 125 testes

Aim

The aim of this study was to study the effects of laparoscopic inguinal hernia repair on testicular perfusion and size.

Materials and Methods

A prospective study concerning laparoscopic inguinal hernia repair was performed for an 18-month period to evaluate testicular perfusion and size in the preoperative, early postoperative (within 48 hours of surgery), and late postoperative periods (6 months after surgery) using Doppler ultrasound (DUS) (both duplex and power Doppler mode). Laparoscopic closure of the deep inguinal ring was accomplished with a purse string suture (Nylon 3-0) using standard 3-port technique. The testis units were divided in 2 groups: group 1 comprising testis units in which a resistive index (RI) could be calculated and group 2 with instances in which an RI could not be calculated but showed blood flow consistently on DUS.

Results

A total of 112 boys underwent laparoscopic inguinal hernia repair with 100 available for complete follow-up and data analysis. One hundred twenty-five inguinal (25 bilateral) hernia repairs were performed. Group 1 had 80 testis units. There was no significant difference in values of RI between preoperative, early postoperative, and late postoperative periods. Group 2 had 45 testis units. Resistive index could not be calculated. Seventy-five percent showed only systolic blood flow on spectral analysis; hence, RI, 1; and the rest showed the presence of blood flow on power Doppler scan. All testis units consistently showed blood flow in the early and late postoperative period.No testicular atrophy was found at 6-month follow-up examination on DUS.

Conclusion

Laparoscopic repair of inguinal hernia in children does not affect testicular perfusion or growth.     

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.     

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.     

Are hormone measurements and ultrasounds really predictors of sperm retrieval in testicular sperm extraction? A case report and literature review

Azoospermia can be diagnosed in about 10%–15% of the infertile male population. To overcome the problem of failure to produce spermatozoa in the ejaculate in patients with nonobstructive azoospermia (NOA), testicular sperm extraction (TESE) may be performed to find the focal area of spermatogenesis. A 47‐year‐old man with NOA presented for treatment of secondary couple infertility. The patient underwent a first TESE 7 years earlier with cryopreservation, and an intracytoplasmic sperm injection–embryo transfer ended in a term pregnancy. He reported a history of repeated testicular traumas. At the present time, a complete medical workup was carried out, including clinical history, general and genital physical examination, scrotal and transrectal ultrasounds. Hormone measurements showed follicle‐stimulating hormone level of 42.7 IU/L, luteinising hormone of 11.4 IU/L, total testosterone of 2.6 ng/ml and right and left testicular volume, respectively, of 4 and 3.9 ml. He underwent a second TESE, with successful sperm retrieval and cryopreservation. The histological pattern was hypospermatogenesis. In cases of extreme testicular impairment, although in the presence of very high follicle‐stimulating hormone value and small testicular volume, estimating poor sperm recovery potential, the integration of clinical and anamnestic data, could help the surgeon to practise the more appropriate method of treatment.