1 Laboratoire de Biologie de la Reproduction, Hôpital Jeanne de Flandre, 59037 Lille cedex, 2 Service de Biologie de la Reproduction, Centre d'AMP, CMCO-SIHCUS, 19 rue Louis Pasteur, 67303 Schiltigheim cedex, 3 Département d'Andrologie, Hôpital Calmette, 59037 Lille cedex, 4 Département d'Histologie et de Biologie de la Reproduction, Faculté de Médecine Cochin Port Royal, Université Descartes, Paris V, 75014 Paris and 5 Département d'Histologie, Faculté de Médecine, Université Warembourg, Lille 2, 59045 Lille cedex, France
6 To whom correspondence should be addressed at: Service de Biologie de la Reproduction, Centre d'AMP, CMCO-SIHCUS, 19 rue Louis Pasteur, 67303 Schiltigheim cedex. Email: isabelle.koscinski{at}chru-strasbourg.fr
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Abstract |
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Key words: azoospermia/flow cytometry/testicular biopsy
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Introduction |
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Materials and methods |
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Patients
Thirty-seven Caucasian infertile patients were diagnosed as NOA based on the following factors: clinical examinations, echographical determination of testis size, multiple semen analyses, seminal concentration of citrate, acid phosphatases, fructose and 14-glucosidase, serum FSH, inhibin B (Serotec; Oxford Bio Innovation, UK) concentrations, as well as karyotype and research of microdeletions of the Y chromosome. Patients were informed about the nature of this study and gave their consent.
Classical cytology
Each patient presented one ejaculate intended exclusively for cytological analysis. Semen was recovered by masturbation after 34 days of sexual abstinence and collected in a sterile container. After liquefaction, 1 h at room temperature, seminal round cells were collected by centrifugation without culture medium (10 min, 500 g). The pellet smears were stained according to HarrisShorr technique and examined for the presence of elongated or round spermatids under a light microscope at x400 magnification.
Flow cytometric analysis
Patients produced a second ejaculate (several weeks after the first) for FCM analysis. Seminal round cells were collected by centrifugation (10 min, 300 g) of the whole ejaculate diluted five times in culture medium (IVFTM; Vitrolife AB, Sweden) and then in 1 x phosphate-buffered saline (PBS) (Dulbecco's; Gibco, Invitrogen Corp., UK) at a concentration of 0.12 x 106 cells/ml. The final pellet was resuspended and 0.51 x 106 round cells in 30 µl of PBS were fixed and permeabilized using the Cytofix-Cytoperm/Cytowash kitTM (BD Biosciences Pharmingen, USA) according to the manufacturer's instructions. Round cells (0.51 x 106) were incubated for 5 min at 4 °C in the dark in 1 ml of PBS 1 x propidium iodide (PI) (1.5 µg/ml).
For haploid control cells, 0.51 x 106 sperm were selected using a two-layer density gradient (PureSpermTM; Nidacon international AB, Sweden). These sperm cells were provided by patients undergoing an IVF attempt for female infertility in our centre. For diploid control cells, 0.51 x 106 lymphocytes from healthy donors were selected using a density gradient (Lymphocyte Separation MediumTM; Eurobio, France). Control cells were fixed, permeabilized, and stained in the same conditions as the round cells. Flow cytometric analysis was performed on a Coulter Epic XL (Beckman Coulter Corp., USA). The cell size (Forward angle light scatter, FSC) and the cell density (90° light scatter, SSC) were simultaneously measured. Instrument settings were adjusted using control cells to observe every event (cells and debris) in the dot-blot diagram. We used a resolution of 1024 channels. FSC detector and SSC photodiode were set in linear mode. A total of 200 000500 000 events were acquired. FCM simultaneously measured the PI fluorescence of stained control cells and patients' seminal round cells. Before staining, control cells and patients' round cells were analysed to assess the absence of spontaneous fluorescence. After staining, the instrument settings were adjusted using the control cells in order to obtain the signal peak of haploid and diploid control cells on the same graph. The same settings were then used for analysing the cell samples of the patients. Fragments of cells contaminating the seminal cell preparations did not allow either quantification or determination of the haploid cell proportion in the semen.
Testicular biopsy
Biopsies were usually bilateral and consisted of a single large biopsy taken from each testis. For each side, the biopsy was divided into two parts for sperm extraction and for histological examination but the part reserved for histological examination was five times smaller than that used for sperm extraction.
Testicular sperm extraction (TESE)
TESE was performed by mincing and shredding the testicular tissue in culture medium (IVFTM; Vitrolife AB, Sweden). An aliquot was examined under an inverted microscope using x400 magnification. If no sperm cells were found, the preparation was centrifuged at 300 g for 10 min and the pellet re-examined for the presence of sperm. TESE was considered positive when at least one living sperm cell was observed (this was either spontaneously motile or was shown to be viable by the hypo-osmotic swelling test).
Histological analysis
Histopathological examination was performed on the second part of the biopsy after fixation in Bouin's solution and embedding in paraffin. Sections 6 µm thick were stained (Trichrome of Masson) and examined under a light microscope at x400 magnification. Four types of pathology were quantified: SCO: Sertoli cells only, or total absence of germ cells; MA: maturation arrest (often at the state of primary spermatocyte); FCS: focal complete spermatogenesis (a complete spermatogenesis is observed only in a small proportion of the seminiferous tubules with a focused distribution); and DS: diffused diminution of normal spermatogenesis (a complete spermatogenesis is observed in most of the seminiferous tubules, but with a low density of germinal cells within each tubule).
Statistical analysis
Comparisons of means were performed using a Wilcoxon test (non-parametric analysis). Comparisons of frequencies were performed using the 2-test or the Fisher exact test if necessary. Sensitivities and specificities were expressed with their 95% confidence intervals.
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Results |
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TESE results and non-invasive classical parameters
Biopsies were taken from both testes in 36 patients, and from only the right testis in one patient (who had no left testis). Sperm were retrieved in 18 patients (49%); from only the largest testis in three patients (with testis size of 4, 9 and 12 ml). TESE results according to the non-invasive parameters are presented in Table I. The age of patients in the positive or negative sperm TESE groups was similar (31.7 ± 4.3 for positive TESE and 34.1 ± 4.61 for negative TESE, not significant). No significant differences were observed with the other parameters: volume of both testes or of the largest testis, FSH and inhibin B levels.
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The cytometry graph shown in Figure 1 displays the level of IP fluorescence according to the size of the analysed cells. Several fluorescence levels could be determined: the lowest fluorescence level corresponded to the cell fragments and debris, a higher fluorescence level to the haploid control cells (sperm); the diploid control cells showed the highest fluorescence level on this graph. The spermatids, which share the ploidia of sperm, with a lower level of condensation in the chromatin, should be detected between haploid and diploid control cells. Spermatocytes I, having a quadriploidia, were distinguished at an upper level in some samples (not shown). These graphs were obtained for each patient. When an FCM result was positive, several aspects of the graphs could be observed: either there was an important and/or very individualized cell contingent with the same fluorescent signal as sperm cells (10 cases), or the fluorescent signal of round cells was intermediate between sperm and lymphocytes (eight cases). Sometimes a small number of intact cells was analysed in comparison with the amount of debris and the presence of the haploid peak was not so evident (six cases).
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Discussion |
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In order to avoid useless TESE, different non-invasive parameters have been proposed to predict the outcome of TESE. In our study, we confirmed that the combined volume of both testes or of the largest testis did not predict TESE outcome (Devroey et al., 1995; Amer et al., 2001
; Seo and Ko, 2001
). Similarly, FSH or inhibin B serum concentrations did not predict TESE results (Kim et al., 1997
; Mulhall et al., 1997
; Amer et al., 2001
; Seo and Ko, 2001
; Vernaeve et al., 2002
). The seminal inhibin B level (not measured in this study) has been described to be a more reliable predictor (Anderson et al., 1998
; Frydelund-Larsen et al., 2002
; Bailly et al., 2003
) but the wide range of concentrations complicates the interpretation. Moreover, the regulation of the seminal concentration of inhibin B secreted by Sertoli cells seems to be complex with a probable contribution of accessory sex glands (Garem et al., 2002
).
As reported in previous literature, the histology was correlated with the TESE outcome. The best prognosis was observed with complete spermatogenesis (focal spermatogenesis or diffused diminution in the degree of normal spermatogenesis). Maturation arrest had a better prognostic value (positive TESE in 57% of cases) than germinal cell aplasia (positive TESE in only 24% of cases) which agrees with other studies (Tournaye et al., 1997; Silber et al., 1997
; Su et al., 1999
; Sousa et al., 2000
; Amer et al., 2001
; Seo and Ko, 2001
). For some authors, the observation of testicular round spermatids (Silber et al., 1997
) or late spermatid forms (Mulhall et al., 1997
) was considered as the best predictive element of a positive TESE.
It has been suggested (Ezeh et al., 1998) that the presence of spermatids in the seminal fluid may reflect their presence in the testis and could be a non-invasive predictive factor before TESE. Therefore, there may be a threshold level of spermatogenesis below which no spermatid could be detected in semen (Ezeh et al., 1998
) in analogy with the histopathological threshold of six mature spermatids per seminiferous tubule that is correlated with the presence of sperm in the seminal fluid (Silber et al., 1997
).
In this study, a significant correlation was found between the detection of seminal spermatids by HarrisShorr staining and TESE results, as in the studies using MayGrünwaldGiemsa (MGG) staining (Amer et al., 2001). Immunostaining with acrosome-specific monoclonal antibodies reaches a sensitivity of 75%, and a specificity of 69% (Ezeh et al., 1998
) which is similar to the use of the anti-proacrosin antibody (4D4). The detection of this antigen from the spermatocyte I stage onwards leads to false positive results and the loss of this antigen by the cell seems to be usual (Mendoza et al., 1996
).
FCM can analyse several thousand events quickly with a good reproducibility. The ploïdia analysis requires cell fixation and permeabilization. After a stay of several days in the genital tractus, however, round cells were weakened by the fixation/permeabilization procedure. This probably explains the large amount of cellular fragments and debris in the samples. Therefore the proportion of haploid cells among all seminal round cells could not be quantified.
In the present study, the sensitivity of FCM was 100% and the specificity 68%. This mild specificity was due to six patients having a negative TESE despite a positive FCM. These six false positive FCM presented a germ cell aplasia in three cases and a maturation arrest in three cases. The apparent absence of spermatogenesis could reflect a partial or incomplete defect, which is why one single large biopsy may not be representative of the functionality of the whole of the testis (Amer et al., 1999). Patients with positive FCM and negative TESE could present a very focal spermatogenesis which could be identified only by multifocal biopsy. This is why more and more teams practice a multifocal needle aspiration. Using such a technique, it seems possible to exhibit a very great heterogeneity in histological patterns observed in the aspiration products at different sites of the testes and to establish a histological mapping of the testes (Meng et al., 2000
). So, it was possible to find sperm cells in one or several sites (Turek et al., 1999
) which is in accordance with our observation that sperm could be retrieved in 24% of patients presenting an SCO.
Moreover, repeated or multiple needle or open testicular biopsies required for such mapping, diagnosis and TESE, could subject the patients to potential risks of vascular injuries and further to androgenic defect or testis atrophy (Harrington et al., 1996; Friedler et al., 1997
; Schlegel and Su, 1997
). The defenders of the needle aspiration therefore considered that the amount of valuable tissue removed from the failing testes was minimized, and they observed a high clinical retrieval rate of sperm obtained by post-needle aspiration TESE (Turek et al., 1999
).
The comparison of classical cytology with the FCM showed a better sensitivity for FCM (100 versus 59% for cytology) and a better specificity for classical cytology (83 versus 68% for FCM). Classical cytology is a cheap and simple technique which does not require any special high cost equipment: in this study, this technique revealed the presence of spermatids in the majority of positive FCM cases. But a negative classical cytology agreed with the results of the FCM in only 50% of the cases.
These observations have led us to propose a strategy for the management of NOA patients. The detection of round spermatids in semen must be initially conducted by classical cytology. If this detection is positive, then the TESE is proposed without doing any FCM assay. In case of a negative result, the FCM assay should be carried out: if FCM is also negative, the indication for TESE would be reconsidered and perhaps abandoned. If FCM is positive for the presence of haploid round cells, we would propose to carry out a TESE, accepting a risk of 22% of having an unsuccessful TESE. This proposition for the management of NOA patients is summarized in Figure 2.
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In conclusion, we have evaluated the detection of seminal spermatids as a prognostic factor of TESE in cases of NOA. The detection of seminal haploid round cells using FCM ploïdia analysis offers a good predictive parameter for successful TESE compared with testicular size, serum FSH and inhibin B concentrations as well as histopathological findings. FCM appeared to be more sensitive than classical cytology with HarrisShorr staining but less specific. By combining these two techniques, cytology and FCM analysis, an accurate management of non-obstructive azoospermic patients could be conducted. This strategy would allow us to prevent some unnecessary biopsies with their possible deleterious effects for the patient.
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Acknowledgements |
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References |
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---|
Amer M, Abd El-Nasser T, El Haggar S, Mostafa T, Abdel-Malak G and Zohdy W (2001) May-GrunwaldGiemsa stain for detection of spermatogenic cells in the ejaculate: a simple predictive parameter for successful testicular sperm retrieval. Hum Reprod 16, 14271432.
Anderson RA (2001) Clinical studies: inhibin in the adult male. Mol Cell Endocrinol 180, 109116.[CrossRef][ISI][Medline]
Anderson RA, Irvine DS, Balfour C, Groome NP and Riley SC (1998) Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis. Hum Reprod 13, 920926.[Abstract]
Angelopoulos T, Krey L, McCullough A, Adler A and Grifo JA (1997) A simple and objective approach to identifying human round spermatids. Hum Reprod 12, 22082216.[Abstract]
Bailly M, Guthauser B, Bergere M, Wainer R, Lombroso R, Ville Y and Selva J (2003) Effects of low concentrations of inhibin B on the outcomes of testicular sperm extraction and intracytoplasmic sperm injection. Fertil Steril 79, 905908.[CrossRef][ISI][Medline]
Devroey P, Liu J, Nagy Z, Goossens A, Tournaye H, Camus M, Van Steirteghem A and Silber S (1995) Pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. Hum Reprod 10, 14571460.[Abstract]
Evenson DP and Melamed MR (1983) Rapid analysis of normal and abnormal cell types in human semen and testis biopsies by flow cytometry. J Histochem Cytochem 31, 248253.[ISI][Medline]
Evenson DP, Darzynkiewicz Z and Melamed MR (1980) Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210, 11311133.[ISI][Medline]
Ezeh UI, Martin M, Cooke ID and Moore HD (1998) Correlation of testicular pathology and sperm extraction in azoospermic men with ejaculated spermatids detected by immunofluorescent localization. Hum Reprod 13, 30613065.[Abstract]
Ezeh UI, Taub NA, Moore HD and Cooke ID (1999) Establishment of predictive variables associated with testicular sperm retrieval in men with non-obstructive azoospermia. Hum Reprod 14, 10051012.
Fenichel P, Rey R, Poggioli S, Donzeau M, Chevallier D and Pointis G (1999) Anti-Müllerian hormone as a seminal marker for spermatogenesis in non-obstructive azoospermia. Hum Reprod 14, 20202024.
Foresta C, Bettella A, Petraglia F, Pistorello M, Luisi S and Rossato M (1999) Inhibin B levels in azoospermic subjects with cytologically characterized testicular pathology. Clin Endocrinol (Oxf) 50, 695701.[CrossRef][ISI][Medline]
Friedler S, Raziel A, Strassburger D, Soffer Y, Komarovsky D and Ron-El R (1997) Testicular sperm retrieval by percutaneous fine needle sperm aspiration compared with testicular sperm extraction by open biopsy in men with non-obstructive azoospermia. Hum Reprod 12, 14881493.[Abstract]
Frydelund-Larsen L, Krausz C, Leffers H, Andersson AM, Carlsen E, Bangsboell S, McElreavey K, Skakkebaek NE and Rajpert-De Meyts E (2002) Inhibin B: a marker for the functional state of the seminiferous epithelium in patients with azoospermia factor C microdeletions. J Clin Endocrinol Metab 87, 56185624.
Gallo JM, Escalier D, Grellier P, Precigout E, Albert M, David G and Schrevel J (1991) Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation. J Histochem Cytochem 39, 273282.[Abstract]
Gandini L, Lenzi A, Lombardo F, Pacifici R and Dondero F (1999) Immature germ cell separation using a modified discontinuous Percoll gradient technique in human semen. Hum Reprod 14, 10221027.
Garem YF, Arini AF, Beheiry AH, Zeid SA and Comhaire FH (2002) Possible relationship between seminal plasma inhibin B and spermatogenesis in patients with azoospermia. J Androl 23, 825829.
Hacker-Klom UB, Gohde W, Nieschlag E and Behre HM (1999) DNA flow cytometry of human semen. Hum Reprod 14, 25062512.
Harrington TG, Schauer D and Gilbert BR (1996) Percutaneous testis biopsy: an alternative to open testicular biopsy in the evaluation of the subfertile man. J Urol 156, 16471651.[CrossRef][ISI][Medline]
Johanisson E, Campana A, Luthi R and de Agostini A (2000) Evaluation of round cells in semen analysis: a comparative study. Hum Reprod Update 6, 404412.
Kim ED, Gilbaugh JH, 3rd, Patel VR, Turek PJ and Lipshultz LI (1997) Testis biopsies frequently demonstrate sperm in men with azoospermia and significantly elevated follicle-stimulating hormone levels. J Urol 157, 144146.[CrossRef][ISI][Medline]
Kurth BE, Klotz K, Flickinger CJ and Herr JC (1991) Localization of sperm antigen SP-10 during the six stages of the cycle of the seminiferous epithelium in man. Biol Reprod 44, 814821.
Lassalle B, Ziyyat A, Testart J, Finaz C and Lefevre A (1999) Flow cytometric method to isolate round spermatids from mouse testis. Hum Reprod 14, 388394.
Manning M, Junemann KP and Alken P (1998) Decrease in testosterone blood concentrations after testicular sperm extraction for intracytoplasmic sperm injection in azoospermic men. Lancet 352, 37.[CrossRef][ISI][Medline]
Meng MV, Cha I, Ljung BM and Turek PJ (2000) Relationship between classic histological pattern and sperm findings on fine needle aspiration map in infertile men. Hum Reprod 15, 19731977.
Mendoza C, Benkhalifa M, Cohen-Bacrie P, Hazout A, Menezo Y and Tesarik J (1996) Combined use of proacrosin immunocytochemistry and autosomal DNA in situ hybridisation for evaluation of human ejaculated germ cells. Zygote 4, 279283.[ISI][Medline]
Mulhall JP, Burgess CM, Cunningham D, Carson R, Harris D and Oates RD (1997) Presence of mature sperm in testicular parenchyma of men with nonobstructive azoospermia: prevalence and predictive factors. Urology 49, 9196.[CrossRef][ISI][Medline]
Schill T, Bals-Pratsch M, Kupker W, Sandmann J, Johannisson R and Diedrich K (2003) Clinical and endocrine follow-up of patients after testicular sperm extraction. Fertil Steril 79, 281286.[CrossRef][ISI][Medline]
Schlegel PN and Su LM (1997) Physiological consequences of testicular sperm extraction. Hum Reprod 12, 16881692.[Abstract]
Seo JT and Ko WJ (2001) Predictive factors of successful testicular sperm recovery in non-obstructive azoospermia patients. Int J Androl 24, 306310.[CrossRef][ISI][Medline]
Silber SJ, Nagy Z, Devroey P, Tournaye H and Van Steirteghem AC (1997) Distribution of spermatogenesis in the testicles of azoospermic men: the presence or absence of spermatids in the testes of men with germinal failure. Hum Reprod 12, 24222428.[Abstract]
Sousa M, Fernandes S and Barros A (2000) Prognostic factors for successful testicle spermatid recover. Mol Cell Endocrinol 166, 3743.[CrossRef][ISI][Medline]
Spano M and Evenson DP (1993) Flow cytometric analysis for reproductive biology. Biol Cell 78, 5362.[CrossRef][ISI][Medline]
Su LM, Palermo GP, Goldstein M, Veeck LL, Rosenwaks Z and Schlegel PN (1999) Testicular sperm extraction with intracytoplasmic sperm injection for nonobstructive azoospermia: testicular histology can predict success of sperm retrieval. J Urol 161, 112116.[ISI][Medline]
Tournaye H, Liu J, Nagy PZ, Camus M, Goossens A, Silber S, Van Steirteghem AC and Devroey P (1996) Correlation between testicular histology and outcome after intracytoplasmic sperm injection using testicular spermatozoa. Hum Reprod 11, 127132.[Abstract]
Tournaye H, Verheyen G, Nagy P, Ubaldi F, Goossens A, Silber S, Van Steirteghem AC and Devroey P (1997) Are there any predictive factors for successful testicular sperm recovery in azoospermic patients? Hum Reprod 12, 8086.[CrossRef][ISI][Medline]
Turek PJ, Givens CR, Schriock ED, Meng MV, Pedersen RA and Conaghan J (1999) Testis sperm extraction and intracytoplasmic sperm injection guided by prior fine-needle aspiration mapping in patients with nonobstructive azoospermia. Fertil Steril 71, 552557.[CrossRef][ISI][Medline]
Vernaeve V, Tournaye H, Schiettecatte J, Verheyen G, Van Steirteghem A and Devroey P (2002) Serum inhibin B cannot predict testicular sperm retrieval in patients with non-obstructive azoospermia. Hum Reprod 17, 971976.
Ziyyat A, Lassalle B, Testart J, Briot P, Amar E, Finaz C and Lefevre A (1999) Flow cytometry isolation and reverse transcriptasepolymerase chain reaction characterization of human round spermatids in infertile patients. Hum Reprod 14, 379387.
Submitted on September 8, 2004; resubmitted on February 21, 2005; accepted on February 25, 2005.
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