Seminal haploid cell detection by flow cytometry in non-obstructive azoospermia: a good predictive parameter for testicular sperm extraction

I. Koscinski1,2,6, C. Wittemer2, J.M. Rigot3, M. De Almeida4, E. Hermant5 and A. Defossez5

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Testicular sperm extraction (TESE) associated with ICSI gives patients suffering from non-obstructive azoospermia (NOA) the possibility of becoming a father. The success rate of TESE based on sperm recovery is ~50%, and the commonly used non-invasive parameters are not predictive enough. Only the invasive testis biopsy has a good prognostic value. The aim of this study was to evaluate the prognostic value of the detection of seminal haploid cells by flow cytometry (FCM) in order to avoid unnecessary testicular biopsy. METHODS: For 37 NOA patients undergoing testicular biopsy, we measured testis size, serum FSH and inhibin B levels and carried out seminal cytology, seminal FCM analysis and histological examination. RESULTS: Sperm were found in 18 biopsies. These results were correlated with cytology, FCM analysis and the histological examination. FCM was more sensitive than cytology (100 versus 59%) but less specific (67 versus 83.5%) whereas the histological observation of complete spermatogenesis appeared to be less sensitive (50%) but more specific (100%). CONCLUSION: Detection of seminal haploid cells by FCM appears to be an interesting non-invasive technique which can predict TESE results and improve the management of NOA patients.

Key words: azoospermia/flow cytometry/testicular biopsy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In cases of non-obstructive azoospermia (NOA), testicular sperm extraction (TESE) followed by ICSI gives azoospermic patients the possibility of fathering a child (Devroey et al., 1995Go). However, testicular biopsy is an invasive procedure potentially leading to complications such as haematoma, inflammation, fibrosis, and even permanent devascularization and possible androgenous deficiency (Schlegel and Su, 1997Go; Manning et al., 1998Go; Schill et al., 2003Go). Moreover, it is very difficult to predict the success of TESE. Several studies have investigated the predictive value of various non-invasive parameters: testis size, FSH serum concentration (Tournaye et al., 1997Go; Amer et al., 2001Go), inhibin B serum concentration (Foresta et al., 1999Go), seminal anti-Müllerian hormone (AMH) level as well as seminal inhibin B level (Anderson et al., 1998Go; Fenichel et al., 1999Go; Anderson, 2001Go). Besides these non-invasive parameters, testicular histopathology is currently performed (Tournaye et al., 1996Go, 1997Go) and is regarded as the best predictor for successful TESE (Su et al., 1999Go) especially in cases where spermatids are observed (Ezeh et al., 1999Go). However, the invasive character of this procedure can lead to the previously described complications and hence it is no longer used by the majority of fertility centres. TESE success seems to be correlated with the detection of round spermatids in seminal fluid (Amer et al., 2001Go), but the identification of immature germinal cells in semen using classical cytological methods is difficult. To facilitate cytological analysis, several staining methods have been proposed, sometimes paired with a discontinuous density gradient separation step (Angelopoulos et al., 1997Go; Gandini et al., 1999Go; Johanisson et al., 2000Go; Amer et al., 2001). Immunocytochemistry techniques using monoclonal antibodies recognizing acrosomal antigens have also been proposed (Kurth et al., 1991Go; Gallo et al., 1991Go; Mendoza et al., 1996Go) but the sensitivity and the specificity of this method are insufficient. Flow cytometry (FCM) can analyse the size and density of cells and has been used to identify murine round spermatids in testis (Lassalle et al., 1999Go). This method uses only physical cell parameters and thus hampers the identification of human spermatids in semen (Ziyyat et al., 1999Go). The haploid status of spermatids can be inferred from observation of a single spot for each of the two probes used in fluorescence in situ hybridization (FISH) experiments (Mendoza et al., 1996Go). DNA content can also be stained with a fluorescent intercalating agent and analysed by FCM. The intensity of the fluorescent signal is related to DNA content and accessibility which depends on chromatin condensation (Evenson et al., 1980Go; Evenson and Melamed, 1983Go). For each semen cell, FCM analyses the size, the granular cytoplasmic structure, and the intensity of the fluorescence signal, which identifies any haploid cells in semen (Spano and Evenson, 1993Go). FCM analysis has already been used on human seminal cells to study the differentiation state of germ cells and to evaluate the most current spermatogenesis abnormalities (Hacker-Klom et al., 1999Go). FCM has not yet been applied to the analysis of NOA semen samples. The aim of the present work was to test the value of FCM identification of haploid semen round cells as a predictive parameter for TESE in cases of NOA. Our goal was to avoid useless testicular biopsies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The local ethics committee gave its consent for the use of human products according to the needs of the study.

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 {alpha}1–4-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 3–4 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 Harris–Shorr 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 0.1–2 x 106 cells/ml. The final pellet was resuspended and 0.5–1 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 (0.5–1 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, 0.5–1 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, 0.5–1 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 200 000–500 000 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 {chi}2-test or the Fisher exact test if necessary. Sensitivities and specificities were expressed with their 95% confidence intervals.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients' mean values
The mean age of patients was 32.9 ± 4.6 (range 23–42) years. The mean volume of both testes combined was 16.1 ± 8.1 (range 2.7–33.0) ml and the mean volume of the largest testis was 8.9 ± 4.1 (range 1.5–17.0) ml. The mean FSH and inhibin B serum concentration was respectively 22.9 ± 18.6 IU/l (range 1.9–89.4; normal range 2.0–10.0) and 47.5 ± 57.4 pg/ml (range 14–264; normal range 285 ± 32). For all patients, the karyotype was normal and no microdeletion of the Y chromosome was found.

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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Table I. Testicular sperm extraction (TESE) results according to non-invasive parameters

 
Histology and TESE results
Table II presents the results of the histological examination according to TESE results. Usually, the histological pattern was the same in both testes. When it was not the case, the histology reported in Table II was taken from the side of the positive TESE. When the TESE was negative, the histology was homogeneous in both testes. According to the previously described histological classification, 17 SCO, 11 MA, two FCS and seven DS were observed. In the 17 patients with SCO, sperm were found in four patients (23.5%). In the 11 cases of MA, sperm were retrieved from five biopsies (45.4%). The histological evidence of a complete spermatogenesis even reduced (DS) or very focalized (FCS) was confirmed by the presence of sperm in all the nine biopsies (100%). There was a significant difference in the results of TESE between patients with complete spermatogenesis (FCS and DS) and patients with an absence of MA (100 and 32.14% of positive TESE respectively, P=0.0012).


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Table II. Testicular sperm extraction (TESE) results according to histopathological analysis

 
Seminal cytology and TESE results
Cytology was carried out on 35 patients and detected spermatids in 13 cases (37.1%). The results according to the TESE outcome are presented in Table III. There was a statistically significant correlation between the detection of spermatids with classical cytology and the result of TESE (P=0.01, Table I). This technique had a low sensitivity: 58% (95% CI 33–82), but a high specificity: 83% (95% CI 59–96).


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Table III. Seminal spermatid detection by classical cytology or flow cytometry (FCM) and testicular sperm extraction (TESE) results

 
Seminal FCM analysis and TESE results
FCM was carried out on 37 patients and detected haploid cells in 24 patients. In 13 cases only were diploid cells detected. Positive FCM analysis positively correlated with successful TESE (P=0.0001). Table III also presents the FCM results according to the TESE: FCM had a high sensitivity: 100% (95% CI 81–100), and a lower specificity: 68% (95% CI 43–87).

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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Figure 1. IP fluorescence levels according to the cell size. Haploid c = control haploid cells (sperm cells); diploid c = control diploid cells (lymphocytes); patient round c = patient seminal round cells; debris = cell debris.

 
FCM and seminal cytology
The correlation between FCM and classical cytology results (Table IV) was statistically significant (P=0.0034): when cytology analysis was positive, the FCM was always positive (13 cases) and when FCM was negative, the cytology was always negative (13 cases). The combination of negative cytology and negative FCM predicted a negative TESE in all nine cases (100%). The combination of positive cytology and positive FCM predicted a successful TESE in seven out of nine cases (78%).


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Table IV. Comparison of flow cytometry (FCM) and cytology for the detection of seminal spermatids

 
FCM and histopathology
The comparison of FCM and histopathology (Table V) showed that the presence of complete spermatogenesis (FCS or DS) in histopathology was always associated with a positive FCM and a positive TESE (nine cases). The six positive FCM followed by a negative TESE corresponded with SCO in three cases and MA in three cases. Moreover, whatever the result of histopathological analysis, a negative FCM result always predicted a negative TESE (13 cases), leading to a negative predictive value of 100% for FCM.


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Table V. Testicular sperm extraction (TESE) results according to flow cytometry (FCM) result and histopathology

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
For patients presenting an NOA, ICSI using TESE has become a standard therapy (Devroey et al., 1995Go). But TESE is an invasive procedure with potential complications, especially when TESE is linked to multiple extensive testicular biopsies as is often necessary for these patients (Schlegel and Su, 1997Go; Manning et al., 1998Go).

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., 1995Go; Amer et al., 2001Go; Seo and Ko, 2001Go). Similarly, FSH or inhibin B serum concentrations did not predict TESE results (Kim et al., 1997Go; Mulhall et al., 1997Go; Amer et al., 2001Go; Seo and Ko, 2001Go; Vernaeve et al., 2002Go). The seminal inhibin B level (not measured in this study) has been described to be a more reliable predictor (Anderson et al., 1998Go; Frydelund-Larsen et al., 2002Go; Bailly et al., 2003Go) 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., 2002Go).

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., 1997Go; Silber et al., 1997Go; Su et al., 1999Go; Sousa et al., 2000Go; Amer et al., 2001Go; Seo and Ko, 2001Go). For some authors, the observation of testicular round spermatids (Silber et al., 1997Go) or late spermatid forms (Mulhall et al., 1997Go) was considered as the best predictive element of a positive TESE.

It has been suggested (Ezeh et al., 1998Go) 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., 1998Go) 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., 1997Go).

In this study, a significant correlation was found between the detection of seminal spermatids by Harris–Shorr staining and TESE results, as in the studies using May–Grünwald–Giemsa (MGG) staining (Amer et al., 2001Go). Immunostaining with acrosome-specific monoclonal antibodies reaches a sensitivity of 75%, and a specificity of 69% (Ezeh et al., 1998Go) 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., 1996Go).

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., 1999Go). 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., 2000Go). So, it was possible to find sperm cells in one or several sites (Turek et al., 1999Go) 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., 1996Go; Friedler et al., 1997Go; Schlegel and Su, 1997Go). 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., 1999Go).

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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Figure 2. Proposition of non-obstructive azoospermia (NOA) for the management of NOA patients.

 
It would also be interesting to reconsider the method of TESE in patients having a negative classical cytology and a positive FCM. Indeed, these patients, presenting an SCO histology, may have some very localized spots of complete spermatogenesis justifying a multifocal TESE or a fine needle aspiration mapping before TESE which could be more efficient than a direct single large biopsy in each testis.

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 Harris–Shorr 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We cordially thank C.Grussmacher for cytometry technical assistance, P.Devos for helpful statistical suggestions, R.Dolan and Gita for language editing.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on September 8, 2004; resubmitted on February 21, 2005; accepted on February 25, 2005.





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