Testicular fine needle aspiration cytology for the diagnosis of azoospermia and oligospermia

OBJECTIVE: To evaluate qualitative and quantitative cytologic features on testicular fine needle aspiration biopsy in the diagnosis of azoospermia and oligospermia and to correlate cytologic and histologic diagnoses. STUDY DESIGN: In this prospective study, 50 infertile males selected from the infertility clinic of Guru Tegh Bahadur Hospital were studied. Fine needle aspiration cytology (FNAC) smears from both testes of 27 azoospermic and 23 oligospermic patients (sperm count < 10 million per milliliter) were stained with May-Grünwald-Giemsa and Papanicolaou stain. Differential counting of 500 spermatogenic cells was done, and the number of Sertoli cells per 500 germ cells was determined for calculating the spermatic index and Sertoli cell index, respectively. FNAC and testicular biopsy were performed under local anesthesia as a minor surgical procedure. RESULTS: Six groups were identified on FNAC smears from azoospermic patients: I. normal spermatogenesis (8), II. hypospermatogenesis (2), III. maturation arrest (2), IV. Sertoli cells only (6), V. atrophic pattern (7), and VI. Leydig cell predominance (2). In oligospermic patients two groups were identified: I. those with normal spermatogenesis (4), and II. those with subnormal spermatogenesis (19). Correlation with histopathologic examination was seen in 81.5% azoospermic and 65.2% oligospermic patients. CONCLUSION: Qualitative and quantitative evaluation of testicular FNAC provides useful information on both azoospermic and oligospermic patients. FNAC performed under local anesthesia is an acceptable outpatient procedure that consistently yields sufficient diagnostic material in all patients.     

Role of fine needle aspirate mapping and touch imprint preparations in the evaluation of azoospermia

OBJECTIVE: To assess the utility of fine needle aspiration cytology (FNAC) and touch imprint cytology (TIC) in the evaluation of azoospermia. STUDY DESIGN: FNAC, TIC and open testicular biopsy (OTB) were used to evaluate 31 azoospermic men. RESULTS: OTB revealed normal spermatogenesis (10), spermatogenic arrest (12), Sertoli cell only syndrome (SCO) (7) and unsatisfactory cases (2). Cytologic examinations (TIC vs. FNAC) revealed normal spermatogenesis (11 vs. 9), spermatogenic arrest (13 vs. 7), SCO (2 vs. 1) and unsatisfactory cases (5 vs. 5). Sensitivity and specificity of TIC and FNAC were 98% vs. 83% and 100% vs. 93%, respectively. CONCLUSION: Testicular FNAC is a reliable and simple method for the evaluation of azoospermia.     

Expression of connexin 43 in human testis

In order to further characterize the Sertoli cell state of differentiation, we investigated the expression of connexin 43 (cx43) protein in the testis of adult men both with normal spermatogenesis and associated with spermatogenic impairment, since cx43 is first expressed during puberty. Cx43 protein was found as a single 43-kDa band on western blots of extracts of normal human testicular material. Cx43 immunoreactivity was generally present between Leydig cells. Within the normal seminiferous epithelium cx43 immunoreactivity was localized between adjacent Sertoli cells, except at stages II and III of the seminiferous epithelial cycle when primary spermatocytes cross from the basal to the adluminal compartment suggesting a stage-dependent Sertoli cell function. While testes with hypospermatogenesis and spermatogenic arrest at the level of round spermatids or spermatocytes revealed a staining pattern similar to that of normal adult testis, the seminiferous tubules showing spermatogenic arrest at the level of spermatogonia and Sertoli-cell-only syndrome were completely immunonegative. We therefore assume that severe spermatogenic impairment is associated with a population of Sertoli cells exhibiting a stage of differentiation deficiency. Accepted: 10 June 1999     

The cytologic identification and quantification of testicular cell subtypes. Reproducibility and relation to histologic findings in the diagnosis of male infertility

In testicular imprint smears from 100 infertile men (both testicles), stained using the Pappenheim and Papanicolaou methods, the cell forms (light and dark spermatogonia, primary and secondary spermatocytes, spermatids, spermatozoa and Sertoli cells) were identified and quantified by the counting of 500 consecutive cells. Identification of the cell types, which are described and illustrated, was consistent and reproducible; the advantages of the different stainings in their analysis is documented. The cell counts were tested for reproducibility and compared to the histologic diagnoses and sperm counts. Statistical analysis showed the highest reproducibility for cells frequently encountered in smears (0.99 for Sertoli cells and 0.98 for spermatozoa) and the lowest but still satisfactory reproducibility for rare or arbitrarily defined cell forms (0.71 for dark spermatogonia and 0.76 for secondary spermatocytes). The high reproducibility of the smear quantification permits the introduction of a number of indices, defining clinically useful relations between cell types that are indicative of various types of infertility. The data obtained by cytologic quantification showed reasonably good correlation with the histologic diagnoses of desquamation and focal fibrosis and excellent correlation with Sertoli cells only, arrested spermatogenesis and complete fibrosis. The cytologic quantification of testicular smears adds considerable information to the diagnosis of impaired fertility and should be instituted in properly equipped centers.     

Correlation of cell counts and indices in testicular FNAC with histology in male infertility.

OBJECTIVE: To evaluate the value of percentage cell counts and cell indices in testicular fine needle aspiration cytology (FNAC) in male infertility and their correlation with histologic categories as seen in open testicular biopsies. STUDY DESIGN: Differential cell counts were performed, and cell indices, including spermatic index, Sertoli cell index and sperm-Sertoli cell index, were calculated in testicular fine needle aspiration (FNA) smears in 30 azoospermic males whose open testicular biopsies were classified as normal spermatogenesis in 10 cases, maturation arrest in 5, hypospermatogenesis in 6, Sertoli cell only syndrome in 5 and tubular/peritubular sclerosis in 4. RESULTS: In normal spermatogenesis, FNA smears showed up to 40% Sertoli cells, and spermatozoa were the predominant spermatogenetic cell type. There was a progressive increase in Sertoli cell percentage and Sertoli cell index and reduction in spermatozon percentage, spermatic index and sperm-Sertoli cell index with increasing severity of reduction in spermatogenesis in different histologic categories. The differences between mean counts and indices in normal spermatogenesis and other histologic categories were statistically significant (P < .01). CONCLUSION: The percent cell counts and cell indices in testicular FNAC correlate with histologic categories and are useful in evaluating male infertility.     

Clinical value of cell counts and indices in testicular fine needle aspiration cytology in primary infertility: diagnostic performance compared with histology

OBJECTIVE: To compare the diagnostic value of testicular fine needle aspiration (FNA) cytology with that of open biopsy in primary infertility and nonobstructive azospermia or severe oligozoospermia, to evaluate the reliability of percentage cell counts and cell indices. STUDY DESIGN: Thirty patients (21 azospermic and 9 severe oligozoospermic) who had samples for testicular FNA obtained from both testis (mean age = 28.7) and open biopsy were included in the prospective study. Primary infertility, history, complete physical examination, hormonal assay and testicular ultrasound data were evaluated. One case was excluded because of an unsatisfactory result in aspiration cytology. The percentage population of Sertoli cells and spermatogenetic cells, in addition to spermatic index, sertoli cell index and sperm-Sertoli cell indexes, was calculated. The statistical analysis was determined using the paired t test. RESULTS: Progressively increasing values of the Sertoli cell index and progressively decreasing values of the sperm--Sertoli cell index were seen in maturation arrest, hypospermatogenesis and Sertoli cell-only syndrome. The difference between mean counts and indices in normal spermatogenesis and other histologic categories was statistically significant (p < 0.05). CONCLUSION: Percentage cell counts and cell indices in testicular FNA significantly correlate with histological categories. In primary male infertility, testicular FNA can be performed instead of open biopsy.     

Fine needle aspiration cytology of clear cell sarcoma. Report of a case with immunocytochemical, immunohistochemical and ultrastructural studies.

Cytologic findings of clear cell sarcoma obtained by fine needle aspiration (FNA) of a tumor are described. The tumor probably originated in the retroperitoneal tissue, and the diagnosis was confirmed histologically by open biopsy. Percutaneous needle aspirates of the intraabdominal tumor and touch preparations obtained from the open biopsy specimen revealed numerous atypical cells with an extremely hyperchromatic nucleus, prominent nucleoli and clear cytoplasm. The cytoplasm was rich in glycogen. The immunocytochemical technique demonstrated S-100 protein and neuron-specific enolase in the cytoplasm, both of which were exhibited also in the histologic specimen. Clear cell sarcoma is a rare tumor of soft tissue, and to our knowledge, detailed cytologic appearances of this tumor obtained by FNA have not been reported. In addition, the present tumor was unique in location. It is possible to diagnose clear cell sarcoma accurately on an FNA cytologic specimen if the periodic acid-Schiff stain and immunocytochemical technique are utilized in addition to the routine Papanicolaou method.     

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.     

Imprint cytology in the diagnosis of Helicobacter pylori. Does imprinting damage the biopsy specimen?

OBJECTIVE: To investigate the efficacy of imprint cytology in the diagnosis of Helicobacter pylori infection and whether it damages the biopsy specimen for subsequent histologic examination. STUDY DESIGN: Two antral biopsies were taken from 76 patients with dyspeptic symptoms undergoing upper gastrointestinal endoscopy. Imprint cytology was made from the first specimen. This specimen was fixed in 10% formalin and sent for histopathologic examination. The second specimen was directly fixed in 10% formalin for routine histopathologic examination without being used for an imprint. The imprint smears were examined by cytopathologists. The biopsy specimens were examined by pathologists who did not know which specimens were used for the imprints. RESULTS: H pylori was seen in smears from 55 (72%) patients and in both biopsy specimens from the same patients. The pathologists could not recognize the biopsy specimens from which the imprints were made. Concordance between imprint cytology and histopathology was 100%. CONCLUSION: Imprint cytology is a suitable test for H pylori diagnosis, and imprints do not adversely affect the quality of the biopsy specimen.     

Patterns of testicular cytology in men with primary infertility: any change since the Gulf War?

OBJECTIVE: To evaluate the patterns of testicular cytology in men with primary infertility, to compare the morphologic patterns between the periods 1990-1995, immediately after Gulf War II, and 1997-2001 and to determine whether there is a correlation between hormonal profile, testicular volume and morphologic pattern. STUDY DESIGN: Retrospective study of men with primary infertility. History, complete physical examination, hormonal assay and testicular ultrasound were evaluated. A total of 545 patients had samples for testicular cytology obtained from both testes. The patient's consent was obtained in all cases. Smears were interpreted under light microscopy after treatment with Diff-Quik. A total of 104 healthy, fertile subjects were used for comparison of the hormonal profile and testicular volume. RESULTS: The mean (+/- SD) age was 28.66 +/- 4.36 years and duration of marriage 4.4 +/- 4.36 years. There were 11.2% patients with normal cytology, 55.8% with hypospermatogenesis, 28.4% with testicular atrophy, 2.9% with Sertoli cells only and 1.7% with maturation arrest. A significant increase in hypospermatogenesis and decrease in the Sertoli cell-only pattern were noted in 1997-2001 when compared with 1990-1995. The mean left testicular volume was 10.53 +/- 5.51 mL3 in the infertile group vs. 15.2 +/- 4.97 in the fertile group (p < 0.003); right testicular volume was 10.84 +/- 4.77 vs. 15.15 +/- 5.31 (p < 0.003). The hormonal profile revealed higher luteinizing hormone and follicle-stimulating hormone levels in the infertile group vs. control group (8.53 +/- 9.03 and 16.44 +/- 19.243 vs. 6.98 +/- 4.53 and 7.37 +/- 6.63, respectively [p < 0.001]). Free testosterone was higher in the fertile group (39.69 +/- 12.76 vs. 20.28 +/- 8.5 [p < 0.000]). CONCLUSION: The majority of infertile males in our cohort had hypospermatogenesis; testicular atrophy was the next most common disorder. There was no major change in overall absolute numbers since the Gulf War. Testicular cytology by fine needle aspiration is a safe and well-tolerated complementary investigation for unexplained male infertility.     

Spermatogenesis in azoospermic,formerly cryptorchid men. Use of needle aspiration techniques

OBJECTIVE: To assess the use of testicular needle aspiration techniques to evaluate fertility potential in azoospermic, formerly cryptorchid men. STUDY DESIGN: Fifteen consecutive adult azoospermic, formerly cryptorchid patients (eight unilateral and seven bilateral) were examined by needle aspiration techniques, fine (FNA) and large needle (LNAB) testicular aspiration biopsy, for cytologic and histologic analysis. Five of the 15 subsequently underwent surgical biopsy for attempted assisted fertilization. RESULTS: Spermatozoa or spermatids were detected by FNA cytology or LNAB histology in one or both testicles in 87.5% of the unilateral and 28.6% of the bilaterally affected, formerly cryptorchid patients (P = .041, Fisher's exact test). The addition of LNAB to FNA identified spermatids in one patient with unilateral cryptorchidism and only Sertoli cells on FNA cytology. Furthermore, LNAB differentiated testicles with the cytologic finding of only Sertoli cells into those with or without diffuse fibrosis. In the five patients in whom assisted fertilization was attempted, the needle aspiration techniques predicted the presence or absence of spermatozoa in the subsequent surgical biopsy. CONCLUSION: The two needle aspiration techniques can be used to assess the fertility potential of azoospermic, formerly cryptorchid men and to select patients for assisted fertilization.     

Specific expression of VCY2 in human male germ cells and its involvement in the pathogenesis of male infertility

Abnormal spermatogenesis in men with Y-chromosome microdeletions suggests that genes important for spermatogenesis have been removed from these individuals. VCY2 is a testis-specific gene that locates in the most frequently deleted azoospermia factor c region in the Y chromosome. We have raised an antiserum to VCY2 and used it to characterize the localization of VCY2 in human testis. Using Western blot analysis, the affinity-purified polyclonal VCY2 antibody gave a single specific band of approximately 14 kDa in size, corresponding to the expected size of VCY2 in all the collected human testicular biopsy specimens with normal spermatogenesis. Immunohistochemical analyses showed that VCY2 localized to the nuclei of spermatogonia, spermatocytes, and round spermatids, except elongated spermatids. At the ultrastructural level, VCY2 expression was found in the nucleus of human ejaculated spermatozoa. To determine the possible relationship of VCY2 with the pathogenesis of male infertility, we examined a group of infertile men with and without Y-chromosome microdeletions and with known testicular pathology using VCY2 antibody. VCY2 was weakly expressed at the spermatogonia and immunonegative in spermatocytes and round spermatids in testicular biopsy specimens with maturation arrest or hypospermatogenesis. The specific localization of the protein in germ cell nuclei indicates that VCY2 is likely to function in male germ cell development. The impaired expression of VCY2 in infertile men suggests its involvement in the pathogenesis of male infertility.     

Testicular fine needle aspiration cytology in male infertility

OBJECTIVE: To compare the accuracy of testicular fine needle aspiration and biopsy for diagnostic and therapeutic purposes. STUDY DESIGN: A comparison of testicular fine needle aspiration and biopsy was performed on 34 infertile men over a 2-year period. RESULTS: Concordance of the 2 tests was evident in 88% of cases. In discordant cases fine needle aspiration yielded more than biopsy, considering the existence of secondary spermatocytes and spermatids. Charcot-Bottcher crystalloids were frequently seen in the cytoplasm of Sertoli cells in normal and abnormal spermatogenesis. CONCLUSION: Testicular fine needle aspiration is a simple and cost-effective method of evaluating testicular pathology in male factor infertility. It may yield more diagnostic and therapeutic material than does biopsy.     

Sulfhydryl oxidase immunoreactivity in seminiferous tubules of infertile men

Summary Sulfhydryl oxidase (SOx) immunoreactivity was investigated in the seminiferous epithelium of human biopsy material from the testes of 33 adult men with disturbed fertility. SOx immunoreactivity was expressed in normal seminiferous epithelium in type-A spermatogonia (27±4% of all spermatogonia) (n=4), in spermatocytes and round spermatids. Mature spermatozoa as well as Sertoli cells were unlabelled. within the interstitium, Leydig cells were immunopositive. In biopsies of oligozoospermic men showing hypospermatogenesis (n=24), an increase in labelled spermatogonia up to more than 90% was observed in biopsies, where seminiferous epithelia revealed only spermatogonia and Sertoli cells. Within the group of oligozoospermic patients there was a significant increase of labelled spermatogonia from 43±13% (>20 mill/ejaculate) (n=7) to 55±16% ( 20 and >20 mill/ejaculate) (n=6) to 68±8% (<5 mill/ejaculate) (n=11) and a significant (P=0.01) decrease of score count from 7.0±2.7 to 2.0±1.8. In this group the increase of labelled spermatogonia was correlated with sperm concentrations in the ajaculate (correlation coefficient: r=-0.6). In biopsies of azoospermic patients showing maturation arrest at the level of spermatocytes or spermatids (n=5) the percentage of labelled spermatogonia was within the range of 24% to 59%. Immunoreactivity in Sertoli cells was only found in single degenerating cells and in tubules showing Sertoli Cell Only Syndrome (SCO) without lumen formation. Sertoli cells within immature seminiferous cords were immunonegative, indicating that Sertoli cell SOx immunoreactivity is rather a sign of physiological alterations in degenerating cells than dependent on the stage of differentiation. Leydig cells did not show changes of immunoreactivity in any biopsy. It is concluded that SOx expression in spermatogonia may serve as a marker for spermatogenic efficiency.     

Ploidy and nuclear area as a predictive factor of histologic grade in primary breast cancer

OBJECTIVE: To compare ploidy and nuclear area with histologic grade in breast cancer using cytologic samples. STUDY DESIGN: Fine needle aspirates from 85 patients with primary breast cancer were analyzed to identify ploidy and nuclear area. The Feulgen technique was used to stain the material. We used the SAMBA 4000 image analysis system (Grenoble, France) for analyzing ploidy and nuclear area. Each patient underwent a biopsy, and the histologic grade was analyzed. RESULTS: A significant association was found between ploidy and nuclear area, between histologic grade and nuclear area, and between ploidy and histologic grade. As ploidy became aneuploid and polyploid and nuclear area became larger, histologic grade became higher. CONCLUSION: A reliable and rapid evaluation of variables for breast cancer can be achieved using cytologic preparations by measuring ploidy and nuclear area of malignant cells with an image analysis system. Ploidy and nuclear area have a significant association with histologic grade.     

Gastric xanthoma: a diagnostic problem on brushing cytology smears

OBJECTIVE: To describe the cytologic findings of gastric xanthomas and to compare them with the findings of signet-ring adenocarcinoma because atypical xanthoma cells can be easily confused with signet-ring adenocarcinoma cells. STUDY DESIGN: Five cases of gastric xanthoma that were confirmed by biopsy reports were selected for study. The patients' ages ranged between 50 and 58 years; 4 were men and 1 was a woman. Twenty-one cases of signet-ring adenocarcinoma confirmed by biopsy reports were selected for comparison. Special staining was performed. RESULTS: The brushing cytology smears of the 5 cases of xanthoma revealed atypical cells, so initially they were reported as suspicious for signet-ring adenocarcinoma and biopsy examination advised. After learning of the histologic diagnosis of xanthoma, we performed special staining. The xanthoma cells were negative with periodic acid-Schiff (PAS) stain but showed a positive reaction with Oil red 0 and weakly positive reaction with Masson trichrome. Signet-ring adenocarcinoma cells showed a strongly positive reaction with PAS stain. CONCLUSION: Gastric brushing cytology findings of xanthomas have not been described before. At times the differentiation of atypical xanthoma cells from signet-ring adenocarcinoma cells is very difficult on brushing cytology smears. In this study the nuclear changes and special stains helped differentiate the 2 lesions.     

Retroperitoneal extramedullary anaplastic plasmacytoma masquerading as sarcoma: Report of a case with an unusual presentation and imprint smears

BACKGROUND: Extramedullary plasmacytoma of the retroperitoneum is rare. Furthermore, plasmacytoma with anaplastic features can be confused with high grade sarcoma clinically and histologically, particularly when the initial immunohistochemical tumor markers are negative. However, paying attention to cytologic imprint smears can give valuable clues to the correct diagnosis. CASE: A 73-year-old male was admitted to our hospital with a recent history of back pain. Abdominal computed tomography revealed a large retroperitoneal mass (6.8 x 5.1 cm). The initial pathologic evaluation revealed a high grade pleomorphic neoplasm that failed to express multiple epithelial, mesenchymal, lymphoid and melanoma immunohistochemical markers. Subsequent fresh tissue evaluation with touch imprints and immunophenotypic characterization confirmed the plasma cell origin of the tumor. Thorough retrospective review of the touch imprint smears clearly showed the plasmacytic cytologic features. Features of multiple myeloma were essentially absent. CONCLUSION: Performing cytologic imprint smears on fresh tissue material may help in making the correct diagnosis and is highly recommended.     

In vivo role of alpha-mannosidase IIx: ineffective spermatogenesis resulting from targeted disruption of the Man2a2 in the mouse

Alpha-mannosidase IIx (MX) is an enzyme closely related to the Golgi N-glycan processing enzyme alpha-mannosidase II (MII). The enzymatic activity of MX in vitro is minimal. Therefore, the in vivo role of MX in N-glycan processing is as yet unclear. The targeted disruption of the gene encoding MX in the mouse resulted in an obvious phenotype, i.e., MX-deficient males were found to be infertile. Testes from homozygous mutant male mice are smaller than those from wild-type or heterozygous littermates. Histology of the MX null mouse testis showed significant reduction of spermatogenic cells in the seminiferous tubules. Electron microscopy showed that prominent intercellular spaces surround MX-deficient spermatogenic cells, suggesting a failure of germ cell adhesion to Sertoli cells. Quantitative structural analyses of N-glycans from wild-type and MX-deficient mouse testis showed that wild-type testes contain GlcNAc-terminated complex type N-glycans, while they are significantly reduced in MX-deficient mutant testis. An in vitro assay for adhesion of spermatogenic cells to Sertoli cells was carried out. By testing the effect of each purified N-glycan oligosaccharide, it was demonstrated that a GlcNAc-terminated tri-antennary, fucosylated N-glycan has an activity on the adhesion between germ cells and Sertoli cells. Thus, the targeted disruption of the gene encoding MX uncovered a novel carbohydrate recognition system in a biologically important process, spermatogenesis.