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
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Abstract |
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Key words: azoospermia/ICSI/Klinefelter/prediction/TESE
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Introduction |
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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., 1965; Skakkebaek et al., 1969
; Tournaye et al., 1996
) explaining the presence of sperm in their ejaculate (Ferguson-Smith et al., 1957
; Foss and Lewis, 1971
; Tournaye et al., 1996
; Palermo et al., 1998
).
The first pregnancy after ICSI with ejaculated sperm from a non-mosaic 47,XXY man was reported by Hinney et al. (1997). 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., 1996
). 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., 1997a
; Reubinoff et al., 1998
; Palermo et al., 1999
; Friedler et al., 2001
). 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., 2001
; Madgar et al., 2002
). 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 Klinefelters syndrome.
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Materials and methods |
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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. (2003).
Testicular sperm recovery
Open excisional testicular biopsies were taken under general anaesthesia as described earlier by Tournaye et al. (1997a), 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 (1963
) 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., 1995
). 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., 1998
). 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., 1993) 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., 1993
).
Comparisons for qualitative variables were performed using Fishers exact test. A MannWhitney 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.
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Results |
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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 (26.932.1) versus 32.8 (29.536.1) 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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Discussion |
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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., 2002
). 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., 2002
).
The physical appearance of our Klinefelter patients differs from that described earlier (Paulsen et al., 1968). 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 Klinefelters syndrome (Gottschalk-Sabag et al., 1995; Tournaye et al., 1997b
; Ezeh et al., 1999
). In a population of 47,XXY Klinefelter patients (n = 19), the study by Westlander et al. (2001
) 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. (2002
) found testicular volume, testosterone levels and hCG test to be important predictive factors of spermatogenesis in 20 patients with a non-mosaic Klinefelters 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 frozenthawed testicular sperm can be proposed. Whether TESEICSI treatment in men with Klinefelters 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., 1997; Estop et al., 1998
; Levron et al., 2000
). 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., 2003
). To date, a total of 36 healthy children have been born after ICSI with testicular sperm from non-mosaic Klinefelter patients (Palermo et al., 1998
; Reubinoff et al., 1998
; Nodar et al., 1999
; Levron et al., 2000
; Friedler et al., 2001
; Greco et al., 2001
; Poulakis et al., 2001
; Westlander et al., 2001
; Bergère et al., 2002
; Rosenlund et al., 2002
; Yamamoto et al., 2002
; Staessen et al., 2003
; Tachdjian et al., 2003
) and only one triplet pregnancy was reduced to a twin pregnancy because of the presence of a 47,XXY fetus (Ron-El et al., 2000
) (Table I).
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Acknowledgements |
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References |
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Submitted on January 14, 2004; accepted on March 16, 2004.