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The validity of testicular aspirate cytology and DNA image-analysis of the aspirate in the assessment of infertile men

The validity of testicular aspirate cytology and DNA image-analysis of the aspirate in the... Arab Journal of Urology (2013) 11,91–100 Arab Journal of Urology (Official Journal of the Arab Association of Urology) www.sciencedirect.com ANDROLOGY/SEXUAL MEDICINE ORIGINAL ARTICLE The validity of testicular aspirate cytology and DNA image-analysis of the aspirate in the assessment of infertile men a,c, b a a Safwat E. Abouhashem , Isam Saba , Salah Mostafa , Alaa Abdalla , c a a a Hamdi Almaramhy , Mohamed Mostafa , Deyab Elsayed , Ehab Ibrahim , a a Aref Maroof , Mahmoud Eladl Departments of Urology, Zagazig University, Egypt Departments of Pathology, Zagazig University, Egypt Department of Surgery, Taiba University, Saudi Arabia Received 14 August 2012, Received in revised form 2 December 2012, Accepted 6 December 2012 Available online 29 January 2013 KEYWORDS Abstract Objective: To assess the possibility of using cytological examination and DNA image-analysis of testicular fine-needle aspirates instead of open surgical Cytological examina- biopsy in the investigation of infertile men, as testicular biopsy has long been used tion; for investigating infertility but the interpretation of histological slides is usually sub- DNA image analysis; jective. Testicular fine; Patients and methods: Thirty-three men (aged 22–36 years) were evaluated for Needle aspirate infertility and underwent both open biopsy and fine-needle aspiration of their testes. Subsequently, the needle aspirates were assessed histopathologically and cytologi- ABBREVIATIONS cally, and by DNA image cytometry. The percentages of haploid, diploid and H&E, haematoxylin tetraploid cells were determined for each patient. and eosin; FNA, fine- Results: The cases were divided into four categories: (1) Complete spermatogen- needle aspiration; LH, esis, with a DNA pattern of 1n > 2n > 4n; (2) Maturation arrest, with a DNA Corresponding author. Urology Department, Zagazig University, Zagazig, Egypt. Tel.: +20 122827283. E-mail address: safwat_abouhashem@yahoo.com (S.E. Abouha- shem). Peer review under responsibility of Arab Association of Urology. Production and hosting by Elsevier 2090-598X ª 2013 Arab Association of Urology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aju.2012.12.006 92 Abouhashem et al. luteinising hormone; pattern of 2n > 4n with no haploid cells; (3) Sertoli cell-only syndrome, with a DNA SCO, Sertoli-cell only pattern of only 2n, with no haploid or tetraploid cells; (4) Hypospermatogenesis, with a variable DNA pattern, i.e. mild with 1n > 2n, moderate with 2n > 1n > 4n, and marked where the DNA pattern was 2n > 4n > 1n. From the cytological and DNA image-analysis of the aspirate a diagnosis was possible that had a strong correlation with the histological diagnosis of the same case. From image analysis we could exclude interstitial cells, Sertoli cells and sperms on the static image, and differentiate between spermatozoa and spermatids based on morphological characteristics in the cytological smear. This technique can therefore be used to quantitatively determine the percentages of various cell types within the seminiferous tubules. By coupling image ploidy analysis and cytological examination of a cytological smear, spermato- genesis can be assessed accurately. Conclusion: Image cytometry could be used to exclude interstitial cells, Sertoli cells and sperms on the static image and so produce an accurate assessment of sper- matogenesis. A combination of ploidy and cell morphology characteristics in cyto- logical smears provides an accurate, reproducible and easily used alternative to open testicular biopsy. ª 2013 Arab Association of Urology. Production and hosting by Elsevier B.V. All rights reserved. Introduction a different area of the testis, more representative samples are obtained than by biopsy. Second, the biopsy proce- dure causes many artefacts in histological sections, In cases of infertility due to azoospermia, a finding iden- which has not been seen in samples from FNA. Also, tified in 10% of men evaluated for infertility, paternity as with FNA of other organs, it is a quick, inexpensive can be achieved with spermatozoa retrieved from the and less-invasive method. However, testicular biopsy is testis in combination with in vitro fertilisation and intra- more invasive, painful and requires operating-room cytoplasmic sperm injection [1]. There are many clinical facilities. Moreover, testicular open biopsies have side- features, including testicular size, serum FSH, history of effects like haematoma, inflammation and especially ejaculated sperms or testicular biopsy histology, that can permanent devascularisation of the testis, resulting in predict whether sperm can be retrieved from the testis or testicular atrophy. The reasons that testicular biopsy not [2]. However, among these variables, successful has not become popular and developed into a routine sperm retrieval is most closely related to the findings investigation might be the fear of ‘needle trauma’ and of testicular biopsy [3]. local tumour implantation. However, as in the present However, the histological evaluation of open testicu- study, there were no reported complications such as lar biopsy specimens in male infertility is open to subjec- haemorrhage or trauma from the FNA procedure. tive interpretation [4] and has been restricted by its Ploidy can be analysed by several methods, the first qualitative rather than quantitative nature [5]. Open being flow cytometry, which has been used in an attempt biopsy also has its drawbacks, i.e. it requires a skin inci- to quantify cell types, but has the significant disadvan- sion and some surgical expertise, and it is relatively time- tage of not being able to distinguish between specific consuming and painful. Moreover, in paraffin sections types of haploid cells, the spermatozoa and spermatids. of a testicular biopsy the mature intact spermatozoa Further limitations of flow cytometry include the total are not clearly identified, so it might not be possible to use of the tissue, precluding its further use, the need for differentiate between maturation arrest at the late sper- relatively many cells, inconsistent reproducibility, and matid stage and normal spermatogenesis [6]. Therefore, lack of user discrimination of nuclear artefacts [12–14]. there is a need for a procedure that can accurately diag- Image analysis is a method for quantifying micro- nose the changes in testicular tissue. This is very impor- scopic images of a cell by digital conversion and com- tant for planning the patient for assisted-reproduction puter analysis of the DNA content and morphological programmes or the surgical correction of ductal features of the cell. Image analysis of the testis is partic- obstruction. ularly appealing because of the actively dividing cell Several other quantitative methods were devised but populations and the small amount of material typically never gained popularity [7,8]. Testicular fine-needle aspi- available for examination [15]. ration (FNA) can replace open biopsy as a minimally In the present study we evaluated the correlation be- invasive diagnostic method for assessing spermatogene- tween the results of DNA image analysis of testicular sis [9–11]. FNA has several advantages over open FNA cytological smears and the findings of routine biopsy. First, by using different punctures, each from The validity of testicular aspirate cytology and DNA image-analysis 93 microscopic examination of cytological smears, with his- Spermatozoa have an oval nucleus with a tail opposite to tological slides from open-biopsy specimens of the same the acrosome. In all spermatogenic cells the cytoplasm is testes in the same patients. basophilic and well delineated. An adequate FNA cytolog- ical smear was defined as one that containedP100 clusters Patients and methods ofP20 cells. Patients were classified according to the most mature spermatogenic cells present in the testis, regardless of the number of aspirates in which they appeared, and In a prospective study we assessed 33 infertile men with were thus classified accordingly into four groups; (1) pa- azoospermia, from August 2009 to August 2010, who tients with normal spermatogenesis (smears showed all presented with infertility of at least a 1-year duration. stages of spermatogenesis and a considerable number of Patients who attended the Infertility Clinic were in- mature sperms); (2) hypospermatogenesis (fewer sper- cluded in the study. The procedure was carried out matogenic cells and a few sperms in the smears); (3) matu- regardless of the size of the testis or the hormonal pro- ration arrest (no sperm in the smears); (4) SCO (only file. All selected patients had a complete history taken, Sertoli cells and no spermatogenic cells) [18]. a physical examination, genital examination, a mini- For image analysis we used Feulgen-stained cytologi- mum of two semen analyses on two occasions conducted cal smears from the FNA sample, stained using standard according to WHO guidelines [16] and a standard hor- techniques [9,15,19]. Images were analysed for the DNA monal assessment of luteinising hormone (LH), FSH, index and ploidy histogram type using an image-analysis testosterone and prolactin. The men had no previous system (CAS-200 software. Becton Dickinson Co., hormonal treatment, previous irradiation, history of Immunocytometry Systems, CA, USA) using a ·100 cryptorchidism or any inguinal surgery. magnification objective (numerical aperture 1.30). The nuclear integrated optical density was computed on 256 Procedures densimetric levels; this measures the amount of absorbent material (nuclear DNA content). The DNA index is gen- Open biopsies and needle aspirates were taken simulta- erated from the integrated optical density, the value of neously, with the patient under general anaesthesia, which is 1.00 in the case of a normal G0–G1 diploid pop- from the same testis. The needle aspirates were taken ulation. A special filter suitable for testicular cytological with a 23-G needle and 10 mL syringe attached to a smears was designed using measurements of tens of thou- holder (three punctures from the upper, middle and low- sands of cells obtained from the default automated image er poles of each testis). By using different punctures, analysis filter, which provided individual cellular area, each from a different area of the testis, more representa- shape, DNA content and density. For the quantitative tive sampling is obtained than by biopsy. DNA analysis, each slide was calibrated using Sertoli cells The cell material was placed on two glass slides. The first (diploid) as control cells. The smears were viewed under was allowed to air dry for 24 h, stained with May Grun- oil immersion (10 · 100) and 200 nuclei from each smear wald–Giemsa stain and later examined cytologically. The were analysed for DNA content, randomly, to ensure that other smear was prepared for image DNA analysis. each nucleus was measured only once. The mean DNA Open biopsy specimens were fixed in Bouin’s solution content of the analysed cells was analysed statistically and processed routinely using paraffin embedding. Hae- using the image-analysis quantitative ploidy analysis soft- matoxylin and eosin (H&E)-stained sections from these ware. The cells were analysed by image analysis and clas- cases were evaluated. Based on standard qualitative sified as haploid (1n; cells which have half of the normal interpretation, on H&E-stained sections, biopsies were amount of DNA, i.e. spermatids and spermatozoa), dip- classified into normal spermatogenesis, hypospermato- loid (2n; cells with the normal amount of DNA, i.e. sper- genesis, spermatogenic arrest, or Sertoli cell-only matogonia, primary spermatocytes, Sertoli cells and (SCO) syndrome [17]. Leydig cells); and tetraploid (4n; cells with double the amount of DNA, i.e. cells at the G2/M phase) on the mea- Interpretation of the cytological preparation sured DNA contents (perceived as the optical density). The results were divided into four categories, i.e. complete Two types of cells are found in the testis, Sertoli cells and spermatogenesis, hypospermatogenesis, maturation ar- spermatogenic cells. The Sertoli cells have round vesicular rest and SCO syndrome. No attempt was made to take nuclei with a fine granular chromatin and large nucleolus. a microscopic biopsy because FNA is simpler and the The cytoplasm is abundant, pale and vacuolated, with microscopic biopsy has a poor yield. poorly delineated borders. The spermatogenic cells show The results of the histological examination, FNA transitional forms, from spermatogonia, spermatocyte cytology and image analysis were correlated for each pa- and spermatid, to spermatozoa, characterized by a dimi- tient using Fisher’s exact probability test, and the chi- nution of nuclear size and the condensation of chromatin. square test was used to determine the difference among The spermatogonia have the largest nuclei, followed by the the three methods. primary and secondary spermatocytes and the spermatids. 94 Abouhashem et al. smear, Fig. 2), six showed spermatogenic arrest (all Results spermatogenic cells and no sperms, Fig. 3) and seven had SCO (no spermatogenic cells, no sperm, only Ser- In all, 33 patients (aged 22–36 years) had diagnostic toli cells, Fig. 4). Of the 33 smears analysed by image testicular biopsies for histological, cytological and cytometry analysis, five were normal (1n > 2n > 4n DNA image analysis. All samples had enough mate- pattern, Fig. 5), 11 showed hypospermatogenesis rial for evaluation. Of the 33 specimens examined his- (2n > 1n > 4n pattern, Fig. 6a–c), eight had sper- tologically, six showed normal spermatogenesis, 10 matogenic arrest (2n > 4n pattern, Fig. 7) and nine showed hypospermatogenesis, eight complete sper- had SCO (1n = 0, Fig. 8). The group with hyposper- matogenic arrest and nine had the SCO syndrome. matogenesis was subclassified according to the histo- Of the 33 smears examined cytologically, six showed gram into mild (four), moderate (four) and severe normal spermatogenesis (abundant sperms, Fig. 1), (three) according to the number of haploid cells; the 14 showed hypospermatogenesis (a few sperms per Figure 1 Active spermatogenesis in a case of obstructive azoospermia, with several typical spermatocytes (A), Sertoli cells (B) and spermatozoa (C) visible. Testis FNA, Giemsa stain · 600. Figure 2 Hypospermatogenesis with a few scattered spermatozoa. Testis FNA, Giemsa stain · 600. The validity of testicular aspirate cytology and DNA image-analysis 95 Figure 3 Arrest of spermatogenesis at the primary spermatocyte stage. The cell has a round nucleus, deeply stained, threaded chromatin, and an eccentric nucleolus. The cytoplasm is scanty and basophilic. Testis FNA, Giemsa stain · 600. Figure 4 SCO syndrome; only Sertoli cells are shown in the smear with no other cells from the spermatocytic series. Cells have a round or kidney-shaped nucleus with an eccentric nucleolus. The cytoplasm is very large, pale or slightly basophilic, with a triangular or elongated shape and ill-defined borders. Testis FNA, Giemsa stain · 600. DNA pattern was 1n P 2n in mild hypospermatogen- togenesis showed spermatogenic arrest in two patients esis (Fig. 6a), 2n > 1n in moderate (Fig. 6b) and and germ cell aplasia in another two on histological 2n > 4n > 1n in severe hypospermatogenesis examination. (Fig. 6c; Table 1). There was also a strong correlation between the find- Comparing the findings of the cytological smears ings of the image cytometry histograms and the results with those of the histological examination, there was a of the histological examination (P = 0.009) in cases of good correlation, using Fisher’s exact probability test spermatogenic arrest and SCO syndrome, while one of (P = 0.001) in cases of normal spermatogenesis, while 11 diagnosed as hypospermatogenesis showed normal four of 14 cytological smears diagnosed as hyposperma- spermatogenesis on histological examination. The dif- 96 Abouhashem et al. Figure 5 An image-cytometry histogram showing three peaks in an obstructive azoospermic case. There is a predominant haploid peak denoting active spermatogenesis (1n > 2n > 4n). ference between the results of the histological examina- smears with histological slides from open biopsy speci- tion, cytological smears and image cytometry histo- mens of the same testes. grams was statistically insignificant (P= 0.35). FNA has emerged as a simple, minimally invasive, reliable and relatively well-tolerated alternative to open Discussion testicular biopsy for investigating azoospermic patients [21]. In the present study, of 33 specimens examined his- tologically, six showed normal spermatogenesis and 10 The recently updated European Association of Urology showed hypospermatogenesis. The procedure was guidelines (2011) [20] recommended that a testicular carried out regardless of the size of the testis or the hor- biopsy is the best procedure not only to distinguish be- monal profile, so patients with azoospermia had normal tween obstructive azoospermia and unobstructive azoo- spermatogenesis or hypospermatogenesis due to a sec- spermia, but also to predict the possibility of sperm ondary obstructive cause, and not primary testicular retrieval in patients with the latter condition. The pres- failure, i.e., eight with complete spermatogenic arrest ent study is the first to be reported in Egyptian patients, and nine with SCO syndrome. Of the 33 smears exam- and compared to Western studies. ined cytologically, six showed normal spermatogenesis, Until recently, open testicular biopsy was the stan- 14 had hypospermatogenesis, six spermatogenic arrest dard method for ascertaining the cause of azoospermia. and seven SCO. Of the 33 smears analysed by image However, the evaluation of a testicular biopsy specimen cytometry analysis five were normal, 11 showed hypo- and aspirate in male infertility has been hampered by the spermatogenesis, eight spermatogenic arrest and nine qualitative rather than quantitative nature of routine SCO. histological and cytological techniques, and the subjec- In the present study, the FNA-derived stained cyto- tivity in interpretation, resulting in a wide inter-observer logical smears showed a good correlation with the histo- discrepancy in the interpretation of samples from rou- logical findings of the open testicular biopsy. Our results tine testicular biopsies taken to assess azoospermia. This agree with those of previous studies comparing FNA wide inter-observer discrepancy was attributed to many and open testicular biopsy [22–24]. In four of the present factors, i.e. a poorly prepared biopsy, insufficient size of patients diagnosed with hypospermatogenesis by FNA, the biopsy, and that no particular method for evaluating the histological diagnosis might suggest no active sper- a biopsy is uniformly applied and serves as a standard matogenesis, i.e. spermatogenic arrest in two and SCO procedure for pathologists [4]. Because it is important in two. This finding could be explained in that active to evaluate testicular spermatogenesis in azoospermic spermatogenesis might be focal or limited to some sem- patients, we correlated the results of DNA image analy- iniferous tubules, and an open testicular biopsy (which is sis of testicular cytological smears and the findings of a single-site biopsy) might miss these areas, while FNA routine microscopic examination of FNA cytological The validity of testicular aspirate cytology and DNA image-analysis 97 Figure 6 An image-cytometry histogram showing three peaks with; nearly equal diploid and haploid peaks denoting less active spermatogenesis in a case of mild hypospermatogenesis (1nP 2n); b, with a predominant diploid peak denoting moderate hypospermatogenesis (2n > 1n > 4n); c, a large diploid peak, small tetraploid, and a much smaller haploid peak in a case with marked hypospermatogenesis (2n > 4n > 1n). can be taken from the upper, middle and lower poles of The advantages of image analysis are many, e.g. the each testis, and can precisely evaluate testicular ability of the operator to select the cells to be measured spermatogenesis [25,26]. for ploidy, and the ability to distinguish between sper- 98 Abouhashem et al. Figure 7 An image-cytometry histogram showing two peaks, a diploid and tetraploid peak in a case of complete maturation arrest (2n > 4n). Figure 8 An image-cytometry histogram showing only one diploid peak in a case of the SCO syndrome. Only one type of cell is present in the smear (2n). matids and spermatozoa, a distinction which is usually The technique using image analysis requires only an air- difficult in routine paraffin sections [6]. It can also be dried smear, which is prepared immediately with the applied to cytological smears [27] and tissue embedded routine smears from the testicular FNA. These can be in paraffin blocks [15] after homogenisation of paraffin prepared easily, even in outpatient clinics. There is no sections. Image analysis is widely used as a method to time limit on when these smears must be stained or ana- evaluate the ploidy of cells, especially of malignant cells. lysed. In addition, the smears remain intact after analy- The validity of testicular aspirate cytology and DNA image-analysis 99 Table 1 The histological, cytological and DNA image analysis results of the 33 patients. Category Open biopsy FNA cytology DNA image analysis Normal spermatogenesis 6 6 5 Hypospermatogenesis 10 14 11 Spermatogenic arrest 8 6 8 SCO syndrome 9 7 9 sis, so that image analysis can always be repeated and Conflict of interest reviewed. In the present study there was an excellent correlation None. between DNA image analysis of FNA cytology and the histological diagnosis in cases of spermatogenic arrest Funding and SCO syndrome, while one of the 11 histograms that suggested hypospermatogenesis was from a patient who No funding. had normal spermatogenesis on histological examina- tion. However, this difference was statistically insignifi- References cant. These results are in agreement with previously published studies [15,27]. [1] Schlegel PN, Palermo GD, Goldstein M, Menendez S, Zaninovic Although DNA image analysis cannot provide vital LL, Veeck LL, et al. Testicular sperm extraction with intracyto- plasmic sperm injection for nonobstructive azoospermia. Urology information on the architecture of the testis, it is useful 1997;49:435–40. in providing an objective and quantitative assessment of [2] Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber the degree of spermatogenesis. S, et al. Are there any predictive factors for successful testicular Our data showed that a cytological examination cou- sperm recovery in azoospermic patients? Hum Reprod pled with DNA image cytometry of the FNA cytological 1997;12:80–6. [3] Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, smears can evaluate accurately the classically defined Schlegel PN. Testicular sperm extraction with intracytoplasmic histological types, and might have the potential to re- sperm injection for nonobstructive azoospermia. Testicular his- place testicular biopsy in the assessment of spermato- tology can predict success of sperm retrieval. J Urol genesis in infertile patients with azoospermia. The 1999;161:112–6. value of FNA in sperm retrieval is also confirmed [28]. [4] Cooperberg MR, Chi T, Jad A, Cha Turek PJ. Variability in testis biopsy interpretation: implications for male infertility care in the In centres like ours, where cryopreservation is not avail- era of intracytoplasmic sperm injection. Fertil Steril able, patients with azoospermia can be evaluated using 2005;84:672–7. FNA cytology. [5] Cerilli LA, Kuang W, Rogers DA. Practical approach to The histological difference between late maturation testicular biopsy interpretation for male infertility. Arch Pathol Lab Med 2010;134:1197–204. arrest and normal spermatogenesis can be difficult to [6] Kim E, Greer JA, Abranis J. Testicular touch preparation discern. We used other clinical features, including a cytology. J Urol 1996;156:1412–4. physical examination, semen analysis, and hormone [7] Johnsen SG. Testicular biopsy score count-a method for regis- evaluation, to differentiate between the two. Therefore, tration of spermatogenesis in human testes: normal values and patients with numerous spermatids on histology but results in 335 hypogonadal males. Hormones 1970;1:2–25. [8] Silber SJ, Rodriguez-Rigau LJ. Quantitative analysis of testicle with no clinical evidence of ejaculated spermatozoa or biopsy: determination of partial obstruction and prediction of confirmed obstruction, were deemed to have late matu- sperm count after surgery for obstruction. Fertil Steril ration arrest. As the aspiration of interstitial tissue is dif- 1981;36:480–5. ficult, Leydig cells were detected in only two patients. [9] Khadra A, Abdulhadi I, Ghunain S, Kilani Z. Efficiency of It was also suggested that testicular tissue from men percutaneous testicular sperm aspiration as a mode of sperm with idiopathic azoospermia undergoing intracytoplas- collection for intracytoplasmic sperm injection in nonobstructive azoospermia. J Urol 2003;169:603–5. mic sperm injection should be evaluated histologically [10] Meng MV, Cha I, Ljung BM, Turek PJ. Testicular fine-needle for carcinoma in situ of the testis, especially in countries aspiration in infertile men. Correlation of cytologic pattern with where the testicular malignancy rate is high. biopsy histology. Am J Surg Pathol 2001;25:71–9. In conclusion, image cytometry can be used to ex- [11] Jain M, Kumari N, Rawat A, Gupta RK. Usefulness of testicular clude interstitial cells, Sertoli cells and sperm on the sta- fine needle aspiration cytology in cases of infertility. Indian J Pathol Microbiol 2007;50:851–4. tic image, and so produce an accurate assessment of [12] Suter L, Koch E, Bechter R, Bobadilla M. Three-parameter flow spermatogenesis. The combination of ploidy and cell cytometric analysis of rat spermatogenesis. Cytometry morphology characteristics in cytological smears repre- 1997;27:161–8. sents a feasible, accurate and reproducible alternative [13] Hirsch IH, McCue P, Kulp-Hugues D, Sedor J, Flanigan M. to open testicular biopsy for evaluating the testes of Validation of flow cytometry analysis in the objective assessment of spermatogenesis comparison to the quantitative testicular infertile men. biopsy. J Urol 1993;150:342–6. 100 Abouhashem et al. [14] Lee SE, Choo MS. Flow-cytometric analysis of testes in infertile comparative study of cytology and histology. Cytopathology men a comparison of the ploidy to routine histopathologic study. 2008;19:363–8. Eur Urol 1991;20:33–8. [23] Srivastava A, Raghavendran M, Jain M, Gupta S, Chaudhary H. [15] Kim ED, Lin WW, Abrams J, Lipshultz LI. Image analysis Fine-needle aspiration cytology of the testis: can it be a single assessment of the abnormal testis biopsy in male infertility. J Urol diagnostic modality in azoospermia? Urol Int 2004;73:23–7. 1997;158:82–4. [24] Qublan HS, Al-Jader KM, Al-Kaisi NS, Alghoweri AS, Abu- [16] World Health Organization. Laboratory Manual for the Exami- Khait SA, Abu-Qamar AA, et al. Fine needle aspiration cytology nation of Human Semen and Semen Cervical Mucus Interaction. compared with open biopsy histology for the diagnosis of 3rd ed. Cambridge: Cambridge University Press; 1992. azoospermia. J Obstet Gynaecol 2002;22:527–31. [17] Levin HS. Testicular biopsy in the study of male infertility. Its [25] Beliveau ME, Turek PJ. The value of testicular ‘mapping’ in men current usefulness, histological techniques, and prospects for the with non-obstructive azoospermia. Asian J Androl future. Hum Pathol 1979;10:569–84. 2011;13:225–30. [18] Schenck U, Schill WB. Cytology of the human seminiferous [26] Plas E, Riedl CR, Engelhardt PF, Muhlbauer H, Pfluger H. epithelium. Acta Cytol 1988;32:689–96. Unilateral or bilateral testicular biopsy in the era of intracyto- [19] Odabas O, Ugras S, Aydin S, Yilmaz Y, Atilla MK. Assessment plasmic sperm injection. J Urol 1990;162:2010–3. of testicular cytology by fine-needle aspiration and the imprint [27] Gottschalk-Sabag S, Weiss DB, Sherman Y. Assessment of technique: are they reliable diagnostic modalities? Br J Urol spermatogenic process by deoxyribonucleic acid image analysis. 1997;79:445–8. Fertil Steril 1995;64:403–7. [20] European Association of Urology. <http://www.uroweb.org/ [28] Arı¨dogan IA, Bayazı¨t Y, Yaman M, Erso¨ z C, Doran S. professional-resources/guidelines>; 2011 [accessed April 2011]. Comparison of fine-needle aspiration and open biopsy of testis [21] Carpi A, Agarwal A, Sabanegh E, Todeschini G, Balercia G. in sperm retrieval and histopathologic diagnosis. Andrologia Percutaneous biopsy of the testicle: a mini review with a proposal 2003;35:121–5. flow chart for non-obstructive azoospermia. Ann Med 2011;43:83–9. [22] Mehrotra R, Chaurasia D. Fine needle aspiration cytology of the testis as the first-line diagnostic modality in azoospermia: a http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arab Journal of Urology Taylor & Francis

The validity of testicular aspirate cytology and DNA image-analysis of the aspirate in the assessment of infertile men

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Abstract

Arab Journal of Urology (2013) 11,91–100 Arab Journal of Urology (Official Journal of the Arab Association of Urology) www.sciencedirect.com ANDROLOGY/SEXUAL MEDICINE ORIGINAL ARTICLE The validity of testicular aspirate cytology and DNA image-analysis of the aspirate in the assessment of infertile men a,c, b a a Safwat E. Abouhashem , Isam Saba , Salah Mostafa , Alaa Abdalla , c a a a Hamdi Almaramhy , Mohamed Mostafa , Deyab Elsayed , Ehab Ibrahim , a a Aref Maroof , Mahmoud Eladl Departments of Urology, Zagazig University, Egypt Departments of Pathology, Zagazig University, Egypt Department of Surgery, Taiba University, Saudi Arabia Received 14 August 2012, Received in revised form 2 December 2012, Accepted 6 December 2012 Available online 29 January 2013 KEYWORDS Abstract Objective: To assess the possibility of using cytological examination and DNA image-analysis of testicular fine-needle aspirates instead of open surgical Cytological examina- biopsy in the investigation of infertile men, as testicular biopsy has long been used tion; for investigating infertility but the interpretation of histological slides is usually sub- DNA image analysis; jective. Testicular fine; Patients and methods: Thirty-three men (aged 22–36 years) were evaluated for Needle aspirate infertility and underwent both open biopsy and fine-needle aspiration of their testes. Subsequently, the needle aspirates were assessed histopathologically and cytologi- ABBREVIATIONS cally, and by DNA image cytometry. The percentages of haploid, diploid and H&E, haematoxylin tetraploid cells were determined for each patient. and eosin; FNA, fine- Results: The cases were divided into four categories: (1) Complete spermatogen- needle aspiration; LH, esis, with a DNA pattern of 1n > 2n > 4n; (2) Maturation arrest, with a DNA Corresponding author. Urology Department, Zagazig University, Zagazig, Egypt. Tel.: +20 122827283. E-mail address: safwat_abouhashem@yahoo.com (S.E. Abouha- shem). Peer review under responsibility of Arab Association of Urology. Production and hosting by Elsevier 2090-598X ª 2013 Arab Association of Urology. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aju.2012.12.006 92 Abouhashem et al. luteinising hormone; pattern of 2n > 4n with no haploid cells; (3) Sertoli cell-only syndrome, with a DNA SCO, Sertoli-cell only pattern of only 2n, with no haploid or tetraploid cells; (4) Hypospermatogenesis, with a variable DNA pattern, i.e. mild with 1n > 2n, moderate with 2n > 1n > 4n, and marked where the DNA pattern was 2n > 4n > 1n. From the cytological and DNA image-analysis of the aspirate a diagnosis was possible that had a strong correlation with the histological diagnosis of the same case. From image analysis we could exclude interstitial cells, Sertoli cells and sperms on the static image, and differentiate between spermatozoa and spermatids based on morphological characteristics in the cytological smear. This technique can therefore be used to quantitatively determine the percentages of various cell types within the seminiferous tubules. By coupling image ploidy analysis and cytological examination of a cytological smear, spermato- genesis can be assessed accurately. Conclusion: Image cytometry could be used to exclude interstitial cells, Sertoli cells and sperms on the static image and so produce an accurate assessment of sper- matogenesis. A combination of ploidy and cell morphology characteristics in cyto- logical smears provides an accurate, reproducible and easily used alternative to open testicular biopsy. ª 2013 Arab Association of Urology. Production and hosting by Elsevier B.V. All rights reserved. Introduction a different area of the testis, more representative samples are obtained than by biopsy. Second, the biopsy proce- dure causes many artefacts in histological sections, In cases of infertility due to azoospermia, a finding iden- which has not been seen in samples from FNA. Also, tified in 10% of men evaluated for infertility, paternity as with FNA of other organs, it is a quick, inexpensive can be achieved with spermatozoa retrieved from the and less-invasive method. However, testicular biopsy is testis in combination with in vitro fertilisation and intra- more invasive, painful and requires operating-room cytoplasmic sperm injection [1]. There are many clinical facilities. Moreover, testicular open biopsies have side- features, including testicular size, serum FSH, history of effects like haematoma, inflammation and especially ejaculated sperms or testicular biopsy histology, that can permanent devascularisation of the testis, resulting in predict whether sperm can be retrieved from the testis or testicular atrophy. The reasons that testicular biopsy not [2]. However, among these variables, successful has not become popular and developed into a routine sperm retrieval is most closely related to the findings investigation might be the fear of ‘needle trauma’ and of testicular biopsy [3]. local tumour implantation. However, as in the present However, the histological evaluation of open testicu- study, there were no reported complications such as lar biopsy specimens in male infertility is open to subjec- haemorrhage or trauma from the FNA procedure. tive interpretation [4] and has been restricted by its Ploidy can be analysed by several methods, the first qualitative rather than quantitative nature [5]. Open being flow cytometry, which has been used in an attempt biopsy also has its drawbacks, i.e. it requires a skin inci- to quantify cell types, but has the significant disadvan- sion and some surgical expertise, and it is relatively time- tage of not being able to distinguish between specific consuming and painful. Moreover, in paraffin sections types of haploid cells, the spermatozoa and spermatids. of a testicular biopsy the mature intact spermatozoa Further limitations of flow cytometry include the total are not clearly identified, so it might not be possible to use of the tissue, precluding its further use, the need for differentiate between maturation arrest at the late sper- relatively many cells, inconsistent reproducibility, and matid stage and normal spermatogenesis [6]. Therefore, lack of user discrimination of nuclear artefacts [12–14]. there is a need for a procedure that can accurately diag- Image analysis is a method for quantifying micro- nose the changes in testicular tissue. This is very impor- scopic images of a cell by digital conversion and com- tant for planning the patient for assisted-reproduction puter analysis of the DNA content and morphological programmes or the surgical correction of ductal features of the cell. Image analysis of the testis is partic- obstruction. ularly appealing because of the actively dividing cell Several other quantitative methods were devised but populations and the small amount of material typically never gained popularity [7,8]. Testicular fine-needle aspi- available for examination [15]. ration (FNA) can replace open biopsy as a minimally In the present study we evaluated the correlation be- invasive diagnostic method for assessing spermatogene- tween the results of DNA image analysis of testicular sis [9–11]. FNA has several advantages over open FNA cytological smears and the findings of routine biopsy. First, by using different punctures, each from The validity of testicular aspirate cytology and DNA image-analysis 93 microscopic examination of cytological smears, with his- Spermatozoa have an oval nucleus with a tail opposite to tological slides from open-biopsy specimens of the same the acrosome. In all spermatogenic cells the cytoplasm is testes in the same patients. basophilic and well delineated. An adequate FNA cytolog- ical smear was defined as one that containedP100 clusters Patients and methods ofP20 cells. Patients were classified according to the most mature spermatogenic cells present in the testis, regardless of the number of aspirates in which they appeared, and In a prospective study we assessed 33 infertile men with were thus classified accordingly into four groups; (1) pa- azoospermia, from August 2009 to August 2010, who tients with normal spermatogenesis (smears showed all presented with infertility of at least a 1-year duration. stages of spermatogenesis and a considerable number of Patients who attended the Infertility Clinic were in- mature sperms); (2) hypospermatogenesis (fewer sper- cluded in the study. The procedure was carried out matogenic cells and a few sperms in the smears); (3) matu- regardless of the size of the testis or the hormonal pro- ration arrest (no sperm in the smears); (4) SCO (only file. All selected patients had a complete history taken, Sertoli cells and no spermatogenic cells) [18]. a physical examination, genital examination, a mini- For image analysis we used Feulgen-stained cytologi- mum of two semen analyses on two occasions conducted cal smears from the FNA sample, stained using standard according to WHO guidelines [16] and a standard hor- techniques [9,15,19]. Images were analysed for the DNA monal assessment of luteinising hormone (LH), FSH, index and ploidy histogram type using an image-analysis testosterone and prolactin. The men had no previous system (CAS-200 software. Becton Dickinson Co., hormonal treatment, previous irradiation, history of Immunocytometry Systems, CA, USA) using a ·100 cryptorchidism or any inguinal surgery. magnification objective (numerical aperture 1.30). The nuclear integrated optical density was computed on 256 Procedures densimetric levels; this measures the amount of absorbent material (nuclear DNA content). The DNA index is gen- Open biopsies and needle aspirates were taken simulta- erated from the integrated optical density, the value of neously, with the patient under general anaesthesia, which is 1.00 in the case of a normal G0–G1 diploid pop- from the same testis. The needle aspirates were taken ulation. A special filter suitable for testicular cytological with a 23-G needle and 10 mL syringe attached to a smears was designed using measurements of tens of thou- holder (three punctures from the upper, middle and low- sands of cells obtained from the default automated image er poles of each testis). By using different punctures, analysis filter, which provided individual cellular area, each from a different area of the testis, more representa- shape, DNA content and density. For the quantitative tive sampling is obtained than by biopsy. DNA analysis, each slide was calibrated using Sertoli cells The cell material was placed on two glass slides. The first (diploid) as control cells. The smears were viewed under was allowed to air dry for 24 h, stained with May Grun- oil immersion (10 · 100) and 200 nuclei from each smear wald–Giemsa stain and later examined cytologically. The were analysed for DNA content, randomly, to ensure that other smear was prepared for image DNA analysis. each nucleus was measured only once. The mean DNA Open biopsy specimens were fixed in Bouin’s solution content of the analysed cells was analysed statistically and processed routinely using paraffin embedding. Hae- using the image-analysis quantitative ploidy analysis soft- matoxylin and eosin (H&E)-stained sections from these ware. The cells were analysed by image analysis and clas- cases were evaluated. Based on standard qualitative sified as haploid (1n; cells which have half of the normal interpretation, on H&E-stained sections, biopsies were amount of DNA, i.e. spermatids and spermatozoa), dip- classified into normal spermatogenesis, hypospermato- loid (2n; cells with the normal amount of DNA, i.e. sper- genesis, spermatogenic arrest, or Sertoli cell-only matogonia, primary spermatocytes, Sertoli cells and (SCO) syndrome [17]. Leydig cells); and tetraploid (4n; cells with double the amount of DNA, i.e. cells at the G2/M phase) on the mea- Interpretation of the cytological preparation sured DNA contents (perceived as the optical density). The results were divided into four categories, i.e. complete Two types of cells are found in the testis, Sertoli cells and spermatogenesis, hypospermatogenesis, maturation ar- spermatogenic cells. The Sertoli cells have round vesicular rest and SCO syndrome. No attempt was made to take nuclei with a fine granular chromatin and large nucleolus. a microscopic biopsy because FNA is simpler and the The cytoplasm is abundant, pale and vacuolated, with microscopic biopsy has a poor yield. poorly delineated borders. The spermatogenic cells show The results of the histological examination, FNA transitional forms, from spermatogonia, spermatocyte cytology and image analysis were correlated for each pa- and spermatid, to spermatozoa, characterized by a dimi- tient using Fisher’s exact probability test, and the chi- nution of nuclear size and the condensation of chromatin. square test was used to determine the difference among The spermatogonia have the largest nuclei, followed by the the three methods. primary and secondary spermatocytes and the spermatids. 94 Abouhashem et al. smear, Fig. 2), six showed spermatogenic arrest (all Results spermatogenic cells and no sperms, Fig. 3) and seven had SCO (no spermatogenic cells, no sperm, only Ser- In all, 33 patients (aged 22–36 years) had diagnostic toli cells, Fig. 4). Of the 33 smears analysed by image testicular biopsies for histological, cytological and cytometry analysis, five were normal (1n > 2n > 4n DNA image analysis. All samples had enough mate- pattern, Fig. 5), 11 showed hypospermatogenesis rial for evaluation. Of the 33 specimens examined his- (2n > 1n > 4n pattern, Fig. 6a–c), eight had sper- tologically, six showed normal spermatogenesis, 10 matogenic arrest (2n > 4n pattern, Fig. 7) and nine showed hypospermatogenesis, eight complete sper- had SCO (1n = 0, Fig. 8). The group with hyposper- matogenic arrest and nine had the SCO syndrome. matogenesis was subclassified according to the histo- Of the 33 smears examined cytologically, six showed gram into mild (four), moderate (four) and severe normal spermatogenesis (abundant sperms, Fig. 1), (three) according to the number of haploid cells; the 14 showed hypospermatogenesis (a few sperms per Figure 1 Active spermatogenesis in a case of obstructive azoospermia, with several typical spermatocytes (A), Sertoli cells (B) and spermatozoa (C) visible. Testis FNA, Giemsa stain · 600. Figure 2 Hypospermatogenesis with a few scattered spermatozoa. Testis FNA, Giemsa stain · 600. The validity of testicular aspirate cytology and DNA image-analysis 95 Figure 3 Arrest of spermatogenesis at the primary spermatocyte stage. The cell has a round nucleus, deeply stained, threaded chromatin, and an eccentric nucleolus. The cytoplasm is scanty and basophilic. Testis FNA, Giemsa stain · 600. Figure 4 SCO syndrome; only Sertoli cells are shown in the smear with no other cells from the spermatocytic series. Cells have a round or kidney-shaped nucleus with an eccentric nucleolus. The cytoplasm is very large, pale or slightly basophilic, with a triangular or elongated shape and ill-defined borders. Testis FNA, Giemsa stain · 600. DNA pattern was 1n P 2n in mild hypospermatogen- togenesis showed spermatogenic arrest in two patients esis (Fig. 6a), 2n > 1n in moderate (Fig. 6b) and and germ cell aplasia in another two on histological 2n > 4n > 1n in severe hypospermatogenesis examination. (Fig. 6c; Table 1). There was also a strong correlation between the find- Comparing the findings of the cytological smears ings of the image cytometry histograms and the results with those of the histological examination, there was a of the histological examination (P = 0.009) in cases of good correlation, using Fisher’s exact probability test spermatogenic arrest and SCO syndrome, while one of (P = 0.001) in cases of normal spermatogenesis, while 11 diagnosed as hypospermatogenesis showed normal four of 14 cytological smears diagnosed as hyposperma- spermatogenesis on histological examination. The dif- 96 Abouhashem et al. Figure 5 An image-cytometry histogram showing three peaks in an obstructive azoospermic case. There is a predominant haploid peak denoting active spermatogenesis (1n > 2n > 4n). ference between the results of the histological examina- smears with histological slides from open biopsy speci- tion, cytological smears and image cytometry histo- mens of the same testes. grams was statistically insignificant (P= 0.35). FNA has emerged as a simple, minimally invasive, reliable and relatively well-tolerated alternative to open Discussion testicular biopsy for investigating azoospermic patients [21]. In the present study, of 33 specimens examined his- tologically, six showed normal spermatogenesis and 10 The recently updated European Association of Urology showed hypospermatogenesis. The procedure was guidelines (2011) [20] recommended that a testicular carried out regardless of the size of the testis or the hor- biopsy is the best procedure not only to distinguish be- monal profile, so patients with azoospermia had normal tween obstructive azoospermia and unobstructive azoo- spermatogenesis or hypospermatogenesis due to a sec- spermia, but also to predict the possibility of sperm ondary obstructive cause, and not primary testicular retrieval in patients with the latter condition. The pres- failure, i.e., eight with complete spermatogenic arrest ent study is the first to be reported in Egyptian patients, and nine with SCO syndrome. Of the 33 smears exam- and compared to Western studies. ined cytologically, six showed normal spermatogenesis, Until recently, open testicular biopsy was the stan- 14 had hypospermatogenesis, six spermatogenic arrest dard method for ascertaining the cause of azoospermia. and seven SCO. Of the 33 smears analysed by image However, the evaluation of a testicular biopsy specimen cytometry analysis five were normal, 11 showed hypo- and aspirate in male infertility has been hampered by the spermatogenesis, eight spermatogenic arrest and nine qualitative rather than quantitative nature of routine SCO. histological and cytological techniques, and the subjec- In the present study, the FNA-derived stained cyto- tivity in interpretation, resulting in a wide inter-observer logical smears showed a good correlation with the histo- discrepancy in the interpretation of samples from rou- logical findings of the open testicular biopsy. Our results tine testicular biopsies taken to assess azoospermia. This agree with those of previous studies comparing FNA wide inter-observer discrepancy was attributed to many and open testicular biopsy [22–24]. In four of the present factors, i.e. a poorly prepared biopsy, insufficient size of patients diagnosed with hypospermatogenesis by FNA, the biopsy, and that no particular method for evaluating the histological diagnosis might suggest no active sper- a biopsy is uniformly applied and serves as a standard matogenesis, i.e. spermatogenic arrest in two and SCO procedure for pathologists [4]. Because it is important in two. This finding could be explained in that active to evaluate testicular spermatogenesis in azoospermic spermatogenesis might be focal or limited to some sem- patients, we correlated the results of DNA image analy- iniferous tubules, and an open testicular biopsy (which is sis of testicular cytological smears and the findings of a single-site biopsy) might miss these areas, while FNA routine microscopic examination of FNA cytological The validity of testicular aspirate cytology and DNA image-analysis 97 Figure 6 An image-cytometry histogram showing three peaks with; nearly equal diploid and haploid peaks denoting less active spermatogenesis in a case of mild hypospermatogenesis (1nP 2n); b, with a predominant diploid peak denoting moderate hypospermatogenesis (2n > 1n > 4n); c, a large diploid peak, small tetraploid, and a much smaller haploid peak in a case with marked hypospermatogenesis (2n > 4n > 1n). can be taken from the upper, middle and lower poles of The advantages of image analysis are many, e.g. the each testis, and can precisely evaluate testicular ability of the operator to select the cells to be measured spermatogenesis [25,26]. for ploidy, and the ability to distinguish between sper- 98 Abouhashem et al. Figure 7 An image-cytometry histogram showing two peaks, a diploid and tetraploid peak in a case of complete maturation arrest (2n > 4n). Figure 8 An image-cytometry histogram showing only one diploid peak in a case of the SCO syndrome. Only one type of cell is present in the smear (2n). matids and spermatozoa, a distinction which is usually The technique using image analysis requires only an air- difficult in routine paraffin sections [6]. It can also be dried smear, which is prepared immediately with the applied to cytological smears [27] and tissue embedded routine smears from the testicular FNA. These can be in paraffin blocks [15] after homogenisation of paraffin prepared easily, even in outpatient clinics. There is no sections. Image analysis is widely used as a method to time limit on when these smears must be stained or ana- evaluate the ploidy of cells, especially of malignant cells. lysed. In addition, the smears remain intact after analy- The validity of testicular aspirate cytology and DNA image-analysis 99 Table 1 The histological, cytological and DNA image analysis results of the 33 patients. Category Open biopsy FNA cytology DNA image analysis Normal spermatogenesis 6 6 5 Hypospermatogenesis 10 14 11 Spermatogenic arrest 8 6 8 SCO syndrome 9 7 9 sis, so that image analysis can always be repeated and Conflict of interest reviewed. In the present study there was an excellent correlation None. between DNA image analysis of FNA cytology and the histological diagnosis in cases of spermatogenic arrest Funding and SCO syndrome, while one of the 11 histograms that suggested hypospermatogenesis was from a patient who No funding. had normal spermatogenesis on histological examina- tion. However, this difference was statistically insignifi- References cant. These results are in agreement with previously published studies [15,27]. [1] Schlegel PN, Palermo GD, Goldstein M, Menendez S, Zaninovic Although DNA image analysis cannot provide vital LL, Veeck LL, et al. Testicular sperm extraction with intracyto- plasmic sperm injection for nonobstructive azoospermia. Urology information on the architecture of the testis, it is useful 1997;49:435–40. in providing an objective and quantitative assessment of [2] Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber the degree of spermatogenesis. S, et al. Are there any predictive factors for successful testicular Our data showed that a cytological examination cou- sperm recovery in azoospermic patients? Hum Reprod pled with DNA image cytometry of the FNA cytological 1997;12:80–6. [3] Su LM, Palermo GD, Goldstein M, Veeck LL, Rosenwaks Z, smears can evaluate accurately the classically defined Schlegel PN. Testicular sperm extraction with intracytoplasmic histological types, and might have the potential to re- sperm injection for nonobstructive azoospermia. Testicular his- place testicular biopsy in the assessment of spermato- tology can predict success of sperm retrieval. J Urol genesis in infertile patients with azoospermia. The 1999;161:112–6. value of FNA in sperm retrieval is also confirmed [28]. [4] Cooperberg MR, Chi T, Jad A, Cha Turek PJ. Variability in testis biopsy interpretation: implications for male infertility care in the In centres like ours, where cryopreservation is not avail- era of intracytoplasmic sperm injection. Fertil Steril able, patients with azoospermia can be evaluated using 2005;84:672–7. FNA cytology. [5] Cerilli LA, Kuang W, Rogers DA. Practical approach to The histological difference between late maturation testicular biopsy interpretation for male infertility. Arch Pathol Lab Med 2010;134:1197–204. arrest and normal spermatogenesis can be difficult to [6] Kim E, Greer JA, Abranis J. Testicular touch preparation discern. We used other clinical features, including a cytology. J Urol 1996;156:1412–4. physical examination, semen analysis, and hormone [7] Johnsen SG. Testicular biopsy score count-a method for regis- evaluation, to differentiate between the two. Therefore, tration of spermatogenesis in human testes: normal values and patients with numerous spermatids on histology but results in 335 hypogonadal males. Hormones 1970;1:2–25. [8] Silber SJ, Rodriguez-Rigau LJ. Quantitative analysis of testicle with no clinical evidence of ejaculated spermatozoa or biopsy: determination of partial obstruction and prediction of confirmed obstruction, were deemed to have late matu- sperm count after surgery for obstruction. Fertil Steril ration arrest. As the aspiration of interstitial tissue is dif- 1981;36:480–5. ficult, Leydig cells were detected in only two patients. [9] Khadra A, Abdulhadi I, Ghunain S, Kilani Z. Efficiency of It was also suggested that testicular tissue from men percutaneous testicular sperm aspiration as a mode of sperm with idiopathic azoospermia undergoing intracytoplas- collection for intracytoplasmic sperm injection in nonobstructive azoospermia. J Urol 2003;169:603–5. mic sperm injection should be evaluated histologically [10] Meng MV, Cha I, Ljung BM, Turek PJ. Testicular fine-needle for carcinoma in situ of the testis, especially in countries aspiration in infertile men. Correlation of cytologic pattern with where the testicular malignancy rate is high. biopsy histology. Am J Surg Pathol 2001;25:71–9. In conclusion, image cytometry can be used to ex- [11] Jain M, Kumari N, Rawat A, Gupta RK. Usefulness of testicular clude interstitial cells, Sertoli cells and sperm on the sta- fine needle aspiration cytology in cases of infertility. Indian J Pathol Microbiol 2007;50:851–4. tic image, and so produce an accurate assessment of [12] Suter L, Koch E, Bechter R, Bobadilla M. Three-parameter flow spermatogenesis. The combination of ploidy and cell cytometric analysis of rat spermatogenesis. Cytometry morphology characteristics in cytological smears repre- 1997;27:161–8. sents a feasible, accurate and reproducible alternative [13] Hirsch IH, McCue P, Kulp-Hugues D, Sedor J, Flanigan M. to open testicular biopsy for evaluating the testes of Validation of flow cytometry analysis in the objective assessment of spermatogenesis comparison to the quantitative testicular infertile men. biopsy. J Urol 1993;150:342–6. 100 Abouhashem et al. [14] Lee SE, Choo MS. Flow-cytometric analysis of testes in infertile comparative study of cytology and histology. Cytopathology men a comparison of the ploidy to routine histopathologic study. 2008;19:363–8. Eur Urol 1991;20:33–8. [23] Srivastava A, Raghavendran M, Jain M, Gupta S, Chaudhary H. [15] Kim ED, Lin WW, Abrams J, Lipshultz LI. Image analysis Fine-needle aspiration cytology of the testis: can it be a single assessment of the abnormal testis biopsy in male infertility. J Urol diagnostic modality in azoospermia? Urol Int 2004;73:23–7. 1997;158:82–4. [24] Qublan HS, Al-Jader KM, Al-Kaisi NS, Alghoweri AS, Abu- [16] World Health Organization. Laboratory Manual for the Exami- Khait SA, Abu-Qamar AA, et al. Fine needle aspiration cytology nation of Human Semen and Semen Cervical Mucus Interaction. compared with open biopsy histology for the diagnosis of 3rd ed. Cambridge: Cambridge University Press; 1992. azoospermia. J Obstet Gynaecol 2002;22:527–31. [17] Levin HS. Testicular biopsy in the study of male infertility. Its [25] Beliveau ME, Turek PJ. The value of testicular ‘mapping’ in men current usefulness, histological techniques, and prospects for the with non-obstructive azoospermia. Asian J Androl future. Hum Pathol 1979;10:569–84. 2011;13:225–30. [18] Schenck U, Schill WB. Cytology of the human seminiferous [26] Plas E, Riedl CR, Engelhardt PF, Muhlbauer H, Pfluger H. epithelium. Acta Cytol 1988;32:689–96. Unilateral or bilateral testicular biopsy in the era of intracyto- [19] Odabas O, Ugras S, Aydin S, Yilmaz Y, Atilla MK. Assessment plasmic sperm injection. J Urol 1990;162:2010–3. of testicular cytology by fine-needle aspiration and the imprint [27] Gottschalk-Sabag S, Weiss DB, Sherman Y. Assessment of technique: are they reliable diagnostic modalities? Br J Urol spermatogenic process by deoxyribonucleic acid image analysis. 1997;79:445–8. Fertil Steril 1995;64:403–7. [20] European Association of Urology. <http://www.uroweb.org/ [28] Arı¨dogan IA, Bayazı¨t Y, Yaman M, Erso¨ z C, Doran S. professional-resources/guidelines>; 2011 [accessed April 2011]. Comparison of fine-needle aspiration and open biopsy of testis [21] Carpi A, Agarwal A, Sabanegh E, Todeschini G, Balercia G. in sperm retrieval and histopathologic diagnosis. Andrologia Percutaneous biopsy of the testicle: a mini review with a proposal 2003;35:121–5. flow chart for non-obstructive azoospermia. Ann Med 2011;43:83–9. [22] Mehrotra R, Chaurasia D. Fine needle aspiration cytology of the testis as the first-line diagnostic modality in azoospermia: a

Journal

Arab Journal of Urology – Taylor & Francis

Published: Mar 1, 2013

Keywords: Cytological examination; DNA image analysis; Testicular fine; Needle aspirate; H&E, haematoxylin and eosin; FNA, fine-needle aspiration; LH, luteinising hormone; SCO, Sertoli-cell only

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