Can biological or clinical parameters predict testicular sperm recovery in 47,XXY Klinefelter’s syndrome patients?

V. Vernaeve1, C. Staessen, G. Verheyen, A. Van Steirteghem, P. Devroey and H. Tournaye

Centre For Reproductive Medicine, University Hospital, Dutch-speaking Brussels Free University (Vrije Universiteit Brussel), Laarbeeklaan 101, B-1090, Belgium

1 To whom correspondence should be addressed. e-mail: valerievernaeve{at}yahoo.fr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Contradictory results are available regarding prediction of testicular sperm extraction in 47,XXY patients. This study, therefore, aimed at assessing the availability of testicular sperm and evaluates clinical parameters predicting successful sperm retrieval in azoospermic 47,XXY Klinefelter’s syndrome patients. METHODS: Sperm recovery procedures were performed in 50 non-mosaic azoospermic Klinefelter patients. The facial hair pattern and the presence of gynaecomastia in men with successful and unsuccessful sperm recovery were compared using Fisher’s exact test. The predictive value of clinical parameters such as age, testicular volume, FSH, FSH:LH ratio, testosterone and androgen sensitivity index (LHxtestosterone) for successful testicular sperm retrieval was evaluated using the receiver operating characteristics (ROC) curve analysis. RESULTS: In 24 patients (48%) testicular sperm were recovered. Ninety-four per cent of the men in whom sperm was found had a normal facial hair pattern compared to 93% in whom no sperm was recovered (not significant, NS). Seventeen percent of the men with successful testicular sperm extraction had gynaecomastia compared to 31% of the men with failed testicular sperm extraction (NS). The mean testicular volume of the largest testis in patients with sperm found was 4.2 ml compared to 3.6 ml in patients with no sperm found (NS). The mean FSH and testosterone values in patients with sperm recovered were 31.2 IU/l and 3.1 ng/ml versus 40.4 IU/l (P = 0.04) and 3.2 ng/ml (NS) in patients without sperm recovered. All examined clinical and biological parameters failed to predict the outcome of the testicular sperm extraction using ROC curve analysis. CONCLUSION: As in the general population of men with non-obstructive azoospermia, there are currently no clinical parameters predicting successful sperm retrieval in the subpopulation of patients with non-mosaic Klinefelter syndrome.

Key words: azoospermia/ICSI/Klinefelter/prediction/TESE


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The clinical treatment of azoospermic males suffering from testicular failure changed dramatically after the introduction of testicular sperm extraction (TESE) and ICSI (Devroey et al., 1995Go; Tournaye et al., 1995Go). In 13% of these azoospermic patients, testicular failure was caused by Klinefelter’s syndrome (Matsumiya et al., 1994Go). About 80% of these patients have a uniform 47,XXY karyotype. The other 20% are either 47,XXY/46,XY mosaics or have higher-grade sex chromosomal aneuploidy or structurally abnormal X chromosomes (Nieschlag et al., 2000Go). The most important features of Klinefelter’s syndrome include azoospermia, small firm testes, gynaecomastia, elevated urinary gonadotrophin titres and varying degrees of eunuchoidism (Paulsen et al., 1968Go).

In general, patients with no evidence of mosaicism are considered sterile. However, occasionally, single foci of spermatogenesis may exist in their testes (Steinberger et al., 1965Go; Skakkebaek et al., 1969Go; Tournaye et al., 1996Go) explaining the presence of sperm in their ejaculate (Ferguson-Smith et al., 1957Go; Foss and Lewis, 1971Go; Tournaye et al., 1996Go; Palermo et al., 1998Go).

The first pregnancy after ICSI with ejaculated sperm from a non-mosaic 47,XXY man was reported by Hinney et al. (1997Go). But even in cases of azoospermia, these men can father their own genetic children by performing ICSI with sperm extracted from their testes (Tournaye et al., 1996Go). From small series it appears that, although considered sterile in general, in about half of 47,XXY patients surgical sperm retrieval will reveal sperm (Tournaye et al., 1997aGo; Reubinoff et al., 1998Go; Palermo et al., 1999Go; Friedler et al., 2001Go). Since unsuccessful sperm recovery procedure has important emotional and financial implications, objective counselling based on predictive factors is of utmost importance. To date, different parameters such as testicular volume, serum FSH, serum testosterone levels, testicular ultrasonography, fluorescence in situ hybridization (FISH) of peripheral lymphocytes and histopathological findings have been studied on a small number of non-mosaic Klinefelter patients (Westlander et al., 2001Go; Madgar et al., 2002Go). However, contradictory conclusions have been reported. Therefore, the present study aimed at assessing the value of clinical and biological parameters for predicting successful testicular sperm retrieval in a large consecutive series of patients with non-mosaic Klinefelter’s syndrome.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Between May 1994 and December 2002, 50 non-mosaic Klinefelter patients, not receiving any androgen substitution therapy, underwent a surgical sperm recovery procedure. A physical examination was carried out to determine the typical features of the disorder. The facial hear pattern, testicular volume and presence of gynaecomastia was recorded. A normal facial hear pattern was defined as the presence of beard growth at physical examination, or, if shaving was needed, at least three times a week in order to have no beard. The testicular size was determined by palpation and compared to testis-shaped models of defined sizes (Prader orchidometer) at the outpatient clinic and during the general anaesthesia for TESE. Chromosome analysis was carried out on cultured peripheral lymphocytes using standard techniques. The routine analysis was performed on G-banded chromosome preparations of ≥15–30 metaphases.

Serum LH and FSH were measured with the automated Elecsys 2010 immunoanalyser (Roche Diagnostics, Germany). Intra-assay and inter-assay coefficients of variation were <3% and <4% for LH and <3% and <6% for FSH respectively.

Testosterone was measured using the Spectria Testosterone coated tube radioimmunoassay (Orion Diagnostica, Finland).

The androgen sensitivity index (LHxtestosterone) was calculated according to Kamischke et al. (2003Go).

Testicular sperm recovery
Open excisional testicular biopsies were taken under general anaesthesia as described earlier by Tournaye et al. (1997aGo), either on the day of ovum retrieval or during preliminary surgery aiming for cryopreservation. When sperm or late elongated spermatids (stage Sd2) according to Clermont (1963Go) were found on wet preparations of the biopsies or when a representative number of samples (i.e. at least four biopsies on each side) were taken randomly, surgery was discontinued. Wet preparation of testicular tissue was performed by mechanical shredding as described earlier (Verheyen et al., 1995Go). Since 1998, enzymatic digestion of the testicular tissue with collagenase type IV has been performed whenever sperm was not found after mechanical shredding (Crabbé et al., 1998Go). During surgery, a randomly taken single small biopsy was sent for histopathological examination.

Statistical analysis
The predictive power of clinical parameters such as age, testicular volume, FSH, FSH:LH ratio, testosterone and androgen sensitivity index for the two groups, i.e. successful and unsuccessful sperm recovery, were evaluated by receiver operating characteristics (ROC) analysis (Zweig et al., 1993Go) using Medcalc (Medcalc Software, Belgium). ROC graphs are plots of all the sensitivity (y-axis) and specificity (x-axis) pairs which are possible for all levels of a particular parameter. They are constructed by plotting the false positive rate defined as (number of false positive results)/(number of true negative + number of false positive results) or 1 – specificity on the x-axis. The y-axis shows the true positive rate or sensitivity, i.e. (number of true positive results)/(number of true positive + number of false negatives). The best discriminating threshold value between two conditions, e.g. the presence or absence of testicular sperm, is the value located at the greatest distance from the diagonal. Calculation of the area under the curve provides a quantitative measure of accuracy, i.e. the ability of this parameter to discriminate between two conditions. A test with perfect discrimination has a ROC plot that passes through the upper left-hand corner, where the true positive fraction is 1.0 or 100% (maximal sensitivity), and the false-positive fraction is 0 (maximal specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left-hand corner (0% true positive rate and 0% false positive rate) to the upper right-hand corner (100% true positive rate and 100% false positive rate) with an area under the curve of 0.5. Thus, in ROC curve analysis, many efficiencies for all decision levels can be computed, resulting in an overall quantification of accuracy not affected by the prevalence of a condition, e.g. the presence or absence of sperm in a wet preparation. But ROC curve analysis also provides a qualitative measure since, for each index parameter, it provides the best threshold value with the highest clinical usefulness (Zweig et al., 1993Go).

Comparisons for qualitative variables were performed using Fisher’s exact test. A Mann–Whitney test was used when data were not normally distributed. P < 0.05 was considered to be statistically significant.

This study was approved by our institutional review board.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 50 Klinefelter patients examined, nine (18%) showed complete or incomplete germ-cell aplasia (Sertoli cell-only), three (6%) showed complete or incomplete maturation arrest and 38 (76%) showed tubular sclerosis and atrophy at their testicular histopathology.

In 24 out of these 50 patients (48%) testicular sperm were recovered. The mean (95% confidence interval) age of the patients with sperm found was 29.5 (26.9–32.1) versus 32.8 (29.5–36.1) for those with no sperm found and the area under the curve (AUC) for the age of the patients at TESE was 0.67 (Figure 1). Ninety-four percent of the men with sperm found had a normal facial hair pattern compared to 93% with no sperm recovered (NS). Seventeen percent of the men with successful testicular sperm extraction had gynaecomastia compared to 31% of the men with failed testicular sperm extraction (NS). The mean testicular volume of the largest testis of patients with sperm found was 4.2 versus 3.6 ml in patients with no sperm found (NS) and the AUC for the testicular volume was 0.65 (Figure 1). The mean FSH value for patients with sperm found was 31.2 versus 40.4 IU/l for patients with no sperm found (P = 0.04) and the AUC for the FSH value was 0.68 (Figure 1). The mean testosterone value for patients with sperm found was 3.1 versus 3.2 ng/ml for patients with no sperm found (NS) and the AUC for the testosterone value was 0.51 (Figure 1). The AUC for the FSH:LH ratio was 0.59 and the AUC for the androgen sensitivity index was 0.61 (Figure 1). In 44 patients detailed information was available as to the presence or absence of sperm or spermatids on histopathology. In all patients in whom sperm were found at histology, sperm were also found on wet preparation (n = 6). There were no cases of patients with sperm found on histolopathology but not on wet preparation. Fifteen patients had no sperm found on histopathology but had a successful TESE. For 23 patients there were no sperm on histology and none after TESE. Thus, histology has a sensitivity of 29%, specificity of 100%, positive predictive value of 100% and negative predictive value of 6%.



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Figure 1. Receiver operating characteristics (ROC) curves of clinical and biological parameters for discriminating successful and failed testicular sperm extraction in Klinefelter patients. The best discriminating values are indicated. TESTO = Testosterone; LHXTESTO = Androgen sensitivity index.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recovery of testicular sperm for ICSI has become a useful therapeutic strategy in non-mosaic Klinefelter patients (Tournaye et al., 1996Go). However, only in half of the patients is this procedure successful in terms of finding sperm that can be used for ICSI (Tournaye et al., 1997aGo). An unsuccessful sperm recovery procedure has important emotional and financial implications as well as medical complications such as devascularization and fibrosis of the testis (Friedler et al., 1997Go; Schlegel and Su, 1997Go; Ron-El et al., 1998Go). Therefore, an objective counselling based on predictive parameters may be useful for these patients.

We examined a consecutive series of 50 non-mosaic Klinefelter patients who did not receive any hormonal substitution therapy for ≥4 months before sperm retrieval, since testosterone is known to block spermatogenesis at the stage of spermatogonial differentiation (McLachlan et al., 2002Go). In 24 out of 50 patients (48%) testicular sperm were retrieved by TESE. This recovery rate is similar to that of the general population of non-obstructive azoospermic men (Tournaye et al., 2002Go).

The physical appearance of our Klinefelter patients differs from that described earlier (Paulsen et al., 1968Go). Retarded facial hair growth was found in 81% and gynaecomastia in 50% of patients. This may be explained by referral bias: our series may represent a selected group of patients virile enough to find female partners.

None of the examined clinical and biological parameters, i.e. age of the patients, testicular volume of the largest testis, FSH value, FSH:LH ratio, testosterone value and androgen sensitivity index, had an acceptable predictive power. The presence of sperm at histology was the best predictive parameter, although the absence of sperm in a single testicular biopsy did not preclude the presence of sperm at wet preparation. This corroborates the findings in the population of non-obstructive azoospermic men not suffering from 47,XXY Klinefelter’s syndrome (Gottschalk-Sabag et al., 1995Go; Tournaye et al., 1997bGo; Ezeh et al., 1999Go). In a population of 47,XXY Klinefelter patients (n = 19), the study by Westlander et al. (2001Go) also found no predictive value of parameters such as testicular volume, serum FSH, FISH of peripheral lymphocytes and buccal tissue and testicular ultrasonography. Only the histopathological finding had a limited predictive value as in our study. Madgar et al. (2002Go) found testicular volume, testosterone levels and hCG test to be important predictive factors of spermatogenesis in 20 patients with a non-mosaic Klinefelter’s syndrome.

When no sperm are found after TESE, the only alternatives for the couple to fulfil their desire to have a child are artificial insemination with donor sperm or adoption. If sperm are found, ICSI with fresh or frozen–thawed testicular sperm can be proposed. Whether TESE–ICSI treatment in men with Klinefelter’s syndrome should be combined with preimplantation genetic diagnosis (PGD) by FISH is still under debate. Most groups do not perform PGD because of the large percentage of normal (23,X or 23,Y) sperm produced by these Klinefelter patients and because the procedure is not carried out at their centre (Guttenbach et al., 1997Go; Estop et al., 1998Go; Levron et al., 2000Go). We prefer to perform PGD because of the significant increase in aneuploidy in embryos of couples with Klinefelter syndrome (46.0%) compared with a control group of patients on whom PGD was performed because of a X-linked disease (22.8%) (Staessen et al., 2003Go). To date, a total of 36 healthy children have been born after ICSI with testicular sperm from non-mosaic Klinefelter patients (Palermo et al., 1998Go; Reubinoff et al., 1998Go; Nodar et al., 1999Go; Levron et al., 2000Go; Friedler et al., 2001Go; Greco et al., 2001Go; Poulakis et al., 2001Go; Westlander et al., 2001Go; Bergère et al., 2002Go; Rosenlund et al., 2002Go; Yamamoto et al., 2002Go; Staessen et al., 2003Go; Tachdjian et al., 2003Go) and only one triplet pregnancy was reduced to a twin pregnancy because of the presence of a 47,XXY fetus (Ron-El et al., 2000Go) (Table I).


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Table I. Overview of the reported pregnancies obtained after ICSI with testicular sperm in non-mosaic azoospermic Klinefelter patients
 
In conclusion, testicular sperm can be found in about half of the azoospermic non-mosaic Klinefelter patients but, unfortunately, to date no clinical predictive factors for successful testicular sperm extraction are available.


    Acknowledgements
 
We would like to thank the clinical, paramedic and laboratory staff of the Centre for Reproductive Medicine and the Centre for Medical Genetics. Furthermore, we are very grateful to Mr Michael Whitburn of the Language Education Centre of our University for editing our paper. The work was supported by grants from the Fund for Scientific Research–Flanders (FWO–Vlaanderen).


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Submitted on January 14, 2004; accepted on March 16, 2004.