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.     

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.     

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.     

Flow Cytometric Analysis of the Maldescended Testis

Flow cytometric measurements of the cellular DNA contents of 16 testicular biopsies of 15 patients with maldescended testes were performed. 2 patients were children. 11 patients of pubescent or adult age had no haploid germ cells in the tissue of their maldescended testis. One patient with a testis mobilis had about a normal percentage of haploid germ cells. In one patient a malignant tumor was detected unexpectedly by an aneuploid stem line at 2.2 c. Flow cytometric analysis of human testicular biopsy material is an easy and a fast way to quantify the function of spermatogenesis and detect malignancies.     

Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy

Artificial unilateral cryptorchidism was performed in golden hamsters which were then held for different periods of time. The non-operated side was used as a control. At various times from 4 to 15 days, hamsters were killed, testes were removed and weighed, single cell suspensions were prepared for flow cytometry analysis and seminiferous tubules were fixed for confocal microscopy. Using DNA staining by propidium iodide or acridine orange followed by flow cytometry analysis, a marked decrease in the haploid condensed cell fraction was detected at the beginning stages of experimental cryptorchidism. In correlation with flow cytometry results, spermiogenic arrest at stages IX and X of seminiferous epithelium was detected in these animals by confocal microscopy and there were no mature forms of haploid cells in the cryptorchid testis. In the testis with more severe damage, there were almost no haploid cells in the seminiferous tubules of cryptorchid animals. In addition, a significant decrease in tetraploid cell fraction and an increase in S-phase fraction was obtained in severe cases. This may be explained by cell arrest before entrance into meiosis. Destruction of tubule structure and cell arrangement were also observed by confocal microscopy in such cases. In conclusion, flow cytometry, combined with confocal analysis, added useful information about spermatogenesis disturbances in cryptorchid testis and it may be used as diagnostic tools in other cases of spermatogenic disorders.     

Secondary changes in the scrotal testis in experimental unilateral cryptorchidism

Unilateral cryptorchidism was induced in under 2-day-old Wistar rat pups. A control group of rats underwent sham operation at the same age. The animals were killed at intervals from 5 to 120 days, both testes were excised, weighted, and processed for histological examination, morphometric measurement of the seminiferous tubules, and DNA flow cytometry. There was no difference in weight, Johnsen score, and tubular size between the scrotal testis of cryptorchid animals and control testes at any age. Significant decreases in all of these parameters occurred in the undescended testis from 30 days when compared with the scrotal testis in cryptorchid animals and controls. Using flow cytometry to measure changes in the DNA ploidy of the cells of the seminiferous epithelium during spermatogenesis, a significant decrease in the haploid population of cells occurred in the scrotal testis of cryptorchid animals at 40 days. This difference continued into adult life (P less than .005). Flow cytometry demonstrates a secondary decrease in spermatogenesis in the scrotal testis in experimental unilateral cryptorchidism.     

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).     

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.     

Calculation of spermatogenic transformations based on dual-flow cytometric analysis of testicular tissue in seasonal breeders

Summary.  The percentages of somatic and spermatogenic cells were quantified at different time intervals in testes of three seasonal breeders: roe deer, brown hare and mink. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry. Somatic and spermatogenic cells were distinguished by dual-parameter flow cytometry after DNA staining and selective labelling of the somatic cells with an antivimentin antibody. The portions of spermatogenic diploid and tetraploid cells were used to calculate total germ cell transformation (haploid:diploid cell ratio) as well as meiotic transformation (haploid:tetraploid cell ratio). During the breeding season the means of both ratios were 10.27 and 38.42 in roe deer, 7.55 and 15.81 in brown hare, and 11.25 and 53.47 in mink. The equivalent quotients calculated on the base of total diploid and tetraploid cells were considerably lower: 4.41 and 7.79, 5.13 and 7.86, 5.68 and 13.75, respectively. Portions as well as proportions of germinative and somatic cells changed during the annual cycle. The results demonstrate that the distinction of somatic and germinative cells in flow cytometric studies of spermatogenesis is a prerequisite for exact calculations of mitotic and meiotic processes and their alterations under different conditions.     

Procarbazine effects on spermatogenesis in golden hamster: a flow cytometric evaluation

The response of hamster testis to the administration of 450mg/kg procarbazine (PCB) over a period of 4 weeks was evaluated. Flow cytometry was used to investigate changes in cell populations in testicular single cell suspensions and to correlate these changes with those observed in histological sections. PCB caused significant decrease in testicular and epididymal weight and a drastic reduction in haploid cells and spermatogenic arrest, demonstrating variation among the test animals. The results obtained confirm previous observations concerning detrimental effects of PCB upon spermatogenesis in species such as the rat and mouse, though its effect on hamster testis is milder and does not include the germinal stem cells. The histological evaluation of the testis showed a good correlation with flow cytometric evaluation, emphasizing the usefulness of this method in providing quantitative and rapid results.     

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.     

Fine-needle aspiration biopsy cytology of the breast. A review of the Groote Schuur Hospital experience

The results of the first 1,500 cytological diagnoses of fine-needle breast aspirates performed at Groote Schuur Hospital Breast Clinic are compared with the histological diagnoses of Tru-cut and excision biopsies of the same lesions. A cytological diagnosis of malignant disease was made in 358 cases, 349 of which were confirmed histologically. The 10 false-positive and 1 false-negative diagnoses are discussed. The reliability of diagnosis in the first 500 cases is compared with that in the second and third 500 cases. There were no false-positive diagnoses in the last 500 cases, and only 3 in the second 500 cases compared with 7 in the first 500 cases. Similarly, the 1 false-negative diagnosis was made in the first 500 cases. The results confirm the reliability of fine-needle aspiration biopsy cytology as a diagnostic technique. The number of incorrect diagnoses decreases as experience with the technique increases.     

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.     

Cell kinetics by bromodeoxyuridine labeling and deoxyribonucleic acid ploidy in prostatic carcinoma needle biopsies

We studied 63 prostate carcinomas in needle biopsies after labeling in vitro by incubating the entire biopsy specimen with tritiated thymidine or bromodeoxyuridine before fixation. Biopsies from 176 patients were labeled, of which 98 were benign and 78 were carcinomas (15 carcinomas were excluded because of scant tissue). The procedure did not interfere with histological diagnosis. Median labeling index was 0.87% (range 0.1 to 29.3%). A labeling index exceeding 3% was unusual but it may indicate the potential for rapid clinical progression. Deoxyribonucleic acid (DNA) flow cytometry was performed on the last 44 carcinomas using cells shed by the prostatic needle biopsy tissue samples during transportation to the pathology laboratory. A sufficient number of cells were obtained for analysis in 37 cases, of which 6 were DNA aneuploid. Labeling index correlated with Gleason histological grade and score, and data from the 6 DNA aneuploid carcinomas suggest association between DNA aneuploidy and a high labeling index. Estimates of percentage of S-phase cells by flow cytometry did not correlate with variables other than DNA index, and appear to have been affected by a high noise-to-signal ratio (few proliferative cells relative to cellular debris) and inability to discriminate between benign and carcinomatous cells.     

Value of DNA analysis in addition to cytological testing in the diagnosis of malignant pleural effusions.

BACKGROUND--Aneuploidy appears to be a highly specific marker for cancer, and measurement of cellular DNA content by flow cytometry is rapid and reliable. This study was undertaken to determine if the addition of DNA analysis improved the sensitivity of cytological diagnosis of malignancy in pleural fluid. METHODS--Pleural effusions from 92 patients were studied by cytological examination and flow cytometry. RESULTS--In 41 patients the final diagnosis was malignancy, there were 40 cases of benign effusions including 22 with pleural tuberculosis, and in the remaining 11 patients with biopsy proven cancer the presence of malignant cells was not found by cytological and histological means in the pleural fluid. Aneuploidy and cytological malignancy were found in 14 samples. There were seven cases with abnormal flow cytometry and negative cytological results. In 12 patients the cytological test results were positive but DNA analysis was normal. Thirty six samples of fluid were both diploid and cytologically negative. Of the 22 tuberculous effusions seven contained aneuploid cells. The sensitivity of DNA and cytological analysis was 51.2% and 63.4%, respectively. The specificity of DNA analysis was 74.5%. CONCLUSIONS--DNA analysis of cells in malignant pleural effusions is both less sensitive and specific than the cytological diagnosis. Flow cytometric analysis is not recommended for routine use in the diagnosis of pleural effusions.     

Value of flow cytometry in the evaluation of head and neck fine-needle lymphoid aspirates: a 3-year retrospective review of a community-based practice.

OBJECTIVE: To study the clinical efficacy of flow cytometry in patients suspected of having a lymphoma with head and neck pathology. STUDY DESIGN: Retrospective review of 49 patients with lymphoid aspirates having concurrent flow cytometry studies. SETTING: Private practice office-based study. CONCLUSION: Fine-needle aspiration with concurrent flow cytometry is a useful, convenient adjunct to the diagnosis of head and neck non-Hodgkins lymphomas. SIGNIFICANCE: Flow cytometry is a significant improvement over fine-needle aspiration derived cytologic diagnosis alone. Tissue open biopsy may be avoided altogether in some patients with non-Hodgkins lymphoma.     

The Diagnostic Impact of Aspiration Cytodiagnosis of Breast Masses in Association with Pregnancy and Lactation with an Emphasis on Clinical Decision Making

Abstract: Recently, in a review of fine-needle aspiration cytology of the breast that was done from January 1983 to June 1996, of the total of 18,695 cases, 331 aspirates had been submitted from women with breast mass(es) associated with pregnancy and lactation. As a result of this review it was noted that benign breast lesions were diagnosed in aspirates with minimal difficulty. In 10 (3.05%) of women in which the cytodiagnosis of carcinoma was made, the findings corresponded with subsequent cell blocks from the aspirate and tissue examination. All aspirates that were suboptimal (scanty, acellular) for cytodiagnosis were repeated as many as two to three times to minimize the chance of missing the abnormality. In cases with a persistent mass, the aspiration was repeated. With this protocol of follow-up, the effect on clinical decision making was significant in that the need of customary surgical biopsy was reduced to a bare minimum.     

Bilateral prepubertal testicular biopsies predict significance of cryptorchidism-associated mixed testicular atrophy, and allow assessment of fertility

INTRODUCTION: Mixed atrophy of the testis (MAT), a frequent finding in biopsies of formerly cryptorchid and/or infertile patients, is defined as the synchronous occurrence of both seminiferous tubules containing germ cells and Sertoli cell only-tubules in variable proportions. In tubules containing germ cells, different types of abnormalities in spermatogenesis may be seen. The presence of adult spermatids in the biopsy, even in small numbers, correlates with successful spermatozoa retrieval for "in vitro" fertilization techniques. Currently, it is unknown whether precursor lesions of MAT can be identified in cryptorchid patients during childhood. MATERIAL AND METHODS: Eighteen formerly cryptorchid adults who had undergone testicular biopsies in childhood had a repeat testicular biopsy to evaluate infertility. In prepubertal biopsies, abnormalities of the testicular parenchyma were classified into types I (slight alterations), II (marked germinal hypoplasia), and III (severe germinal hypoplasia). In postpubertal biopsies, the percentage of tubules containing germ cells and Sertoli cell only-tubules were estimated, as well as the presence of complete spermatogenesis. Abnormalities in spermatogenesis were classified into lesions of the adluminal or basal compartments of seminiferous tubules. RESULTS: Comparison between prepubertal and postpubertal biopsies revealed that most specimens developing from type III lesions presented with incomplete spermatogenesis (P<0.0001) and more severe lesions of the germinal epithelium (P=0.049). DISCUSSION: Type III lesions correlated with MAT characteristics that confer a worse prognosis for in vitro fertilization. Thus, MAT characteristics may be predicted in prepubertal cryptorchid patients, allowing a fertility prognosis. The pathogenesis of these lesions, and their possible inclusion into the spectrum of the testicular dysgenesis syndrome, are discussed.     

A Comparative Study of the Effects of Non-Union of Testis and Epididymis and Cryptorchidism on Testicular Development and Spermatogenesis in the Rat

Malunion of testis and epididymis is a congenital anomaly of human undescended testicles which may be of considerable clinical importance. In an experimental model in rat the effects of early disconnection of testis and epididymis (16 days) on testicular development have been testis and puberty. The results are compared with corresponding effects of cryptorchidism and sham-operation. The testes were weighed, and the relative proportions of haploid, diploid and tetraploid cells were quantified by DNA flow cytometry. The histology was evaluated by light and electron microscopy. The non-union operated testes showed a close to normal weight development until about 40 days of age, whereafter a decline occurred. The progression of the maturation division was only slightly reduced at 30 and 37 days of age, but later the spermatogenesis was significantly inhibited. At 58 days of age the maturation division had practically ceased in non-union operated testes. In cryptorchid animals the onset and progression of maturation divisions were almost totally depressed, and only traces of haploid cells were seen in some specimens. The transient increase in tetraploid cells, reflecting primary spermatocytes seen at about 30 days during normal testicular development, was neither depressed in non-union operated nor in cryptorchid animals. This indicates that the major block in spermatogenesis in both situations is at the stage of the primary spermatocytes. At 58 days of age the testicular histology was similar in non-union operated and cryptorchid testicles with many tubular sections showing only Sertoli cells. Scattered and partly degenerated germinal cells were seen in some sections. No histological signs of increased intratubular fluid pressure were detected, suggesting that pressure atrophy is not decisive for the reduced spermatogenesis in non-union operated gonads.     

False-negative biopsies for the diagnosis of testicular intraepithelial neoplasia (TIN)--an update

PURPOSE: Testicular intraepithelial neoplasia (TIN; or carcinoma in situ of the testis) is the precursor of testicular germ-cell tumours (GCT). It is detected by conventional surgical biopsy of the testis. To date, only little information is available in regard to the accuracy of the biopsy. False-negative biopsies have been reported only sporadically. PATIENTS AND METHODS: Twenty-one patients who developed a testicular GCT despite a testicular biopsy negative for TIN were analysed clinically and histologically. The median age of the patients is 34 years. The median interval from biopsy to the clinical appearance of GCT is 39 months. Four of the 21 patients had their biopsy done within a previously reported multicentric study (n=1859 cases with negative biopsy including five cases with false-negative biopsy hitherto known). All of the biopsy specimens were re-examined immunohistologically. In 15 cases, the orchiectomy specimens were re-examined for the presence of TIN in the tumour-surrounding tissue. RESULTS: In five cases, TIN was found in the biopsy specimen upon re-examination. In all of the 15 orchiectomy specimens there was evidence of TIN in the tissue adjacent to the tumour. In three biopsy specimens there were microcalcifications in the seminiferous tubules. Severe impairment of the spermatogenesis was observed histologically in only 3 of the 21 patients. The relative proportion of false-negative biopsies is 0.5% (95% confidence intervals (CI): 0.22%; 0.92%). The sensitivity of the biopsy to detect TIN is 0.914 (95% CI: 0.842; 0.959) and the overall accuracy is 0.995 (95% CI: 0.991; 0.9979). A total of 44 false-negative biopsies are reported to date. CONCLUSIONS: False-negative biopsies for TIN do occur but the proportion is only 0.5%. There is no clear-cut clinical nor histological feature associated with false-negative biopsies. However, young age (i.e. <18 years) and intratubular microcalcifications should increase the clinician's and pathologist's vigilance. The majority of false-negative biopsies are caused by the non-random distribution of TIN in the testis while some few cases are caused by technical problems. Two-site biopsies would probably increase the accuracy of the biopsy in high risk cases.