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ABSTRACT: Klinefelter syndrome, with an incidence of 1:600 male newborns, is the most frequent form of male hypogonadism. However, despite its relatively high frequency, the syndrome is often overlooked. To prevent such oversights, the clinical features should be better characterized, and simple screening tests should be used more frequently. In a cohort of 309 patients suspected of having Klinefelter syndrome, we evaluated the clinical symptoms as well as the diagnostic value of the Barr body test for screening procedures. On the basis of chromosome analysis, 85 patients (group I) were diagnosed as having Klinefelter syndrome, and 224 patients had a 46,XY karyotype (group II). Barr body analysis revealed a specificity of 95% and a sensitivity of 82% for the diagnosis of Klinefelter syndrome. General features (eg, reason for admission, age, age of the parents, body weight, and frequency of maldescended testes) were not different between the groups, except that group I had a higher proportion of patients with a lower educational background. Compared to group II, patients with Klinefelter syndrome were taller (P < .001); had smaller testis volumes (P < .0001), higher follicle-stimulating hormone (FSH) and luteinizing hormone (LH) values; and carried a tendency for less androgenic phenotype and secondary hair distribution. Testosterone, estradiol, sex hormone—binding globulin (SHBG), and prostate-specific antigen (PSA) serum levels as well as prostate volume were not significantly different between the groups. In patients who provided an ejaculate, azoospermia was found in 54% of the patients in group II and in 93% of the patients with Klinefelter syndrome. Although not exclusively characteristic for Klinefelter syndrome, the combination of low testicular volume and azoospermia, together with elevated gonadotropins, is highly indicative for a Klinefelter syndrome and should stimulate further clinical investigations. Barr body analysis provides a quick and reliable screening test, which, however, must be confirmed by karyotyping.
With a prevalence of 1:600 in the male population, Klinefelter syndrome, first described in 1942, is the most frequent form of male hypogonadism (Klinefelter et al, 1942). About 80% of patients with 47,XXY bear a congenital numerical chromosome aberration. The other 20% are represented either by 47,XXY/46,XY mosaics or higher-grade sex chromosomal aneuploidy or structurally abnormal × chromosomes (Nieschlag et al, 2000).
Clinically, the syndrome is characterized in adolescents and adults by the constellation of small, firm testes and symptoms of androgen deficiency. Other often-associated clinical features are azoospermia, tall stature, and bilateral painless gynecomastia. Diagnosis is confirmed by chromosome analysis performed in lymphocytes (Jacobs and Strong, 1959). In clinical routine, the occurrence of Barr bodies in a buccal smear (Moore and Barr, 1955) has been used as a rapid and simple diagnostic method in suspected Klinefelter syndrome. However, the diagnostic accuracy of screening for Barr bodies has never been evaluated systematically, and this simple test has been neglected in recent years. Since the symptoms of the Klinefelter syndrome are not exclusive and the syndrome may be overlooked during clinical diagnosis, we evaluated the sensitivity and specificity of this simple laboratory technique in the diagnostic workup of patients suspected to suffer from Klinefelter syndrome.
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- Subjects and Methods
Klinefelter syndrome is the most common cause of gonosomal aberration in all ethnic groups, and in about 3% of our patients, a Klinefelter syndrome was suspected. A definite diagnosis of Klinefelter syndrome was established in 38% of these 309 patients on the basis of karyotype analysis in lymphocytes, which is the diagnostic gold standard (Zang, 1984). Klinefelter syndrome was diagnosed in 1.5% of our patients, which is in agreement with other infertility clinics (Chiang et al, 2000). The higher incidence of patients with Klinefelter syndrome in infertility clinics (Nielsen and Wohlert, 1991) can be explained by the severe impairment of spermatogenesis and the high incidence of signs of androgen deficiency, which accumulate in a tertiary andrology center.
We and others (Filippi, 1986) used Barr body analysis as a quick screening test, which is performed together with blood donation for karyotyping and other laboratory tests. With simple staining methods that are available in every andrology laboratory, Barr body analysis can be processed within 1 hour. In contrast, at our university, as in most other settings, karyotype analysis is performed by a genetics department. At our university, the mean time required for the results of karyotype analysis is 5 to 10 days, depending on the clinical indication. In not-urgent situations like those in this study, it took 17 days (±3 days SEM), whereas Barr body results were available within 1 hour. Barr body analysis was not only quick, but it also showed a very good specificity of 95% and a satisfying sensitivity of 82%. This diagnostic accuracy was also reported by other groups, which, however, found a higher sensitivity of Barr body analysis (97%) in a preselected collective of 64 patients with known Klinefelter syndrome (Grabski et al, 1979). However, in any case, a karyotype analysis has to be performed to obtain definite results, although karyotype analysis may also miss tissue-specific mosaic Klinefelter syndrome. In our 11 patients with false-positive results, it is notable that they also presented with a reduced mean testicular volume of 10 mL and that 7 of 10 patients also were azoospermic. In the patients with false-negative results, no obviously different characteristics compared to the patients correctly diagnosed could be observed. However, it cannot be excluded that a false-positive Barr body test may occur in a mosaic individual with a Klinefelter karyotype in buccal epithelia cells but a normal karyotype in peripheral lymphocytes, and a false-negative Barr body test may occur in a patient with mosaic Klinefelter syndrome having a normal karyotype in a buccal smear but a Klinefelter karyotype in lymphocytes.
In general clinical features, no obvious differences between the patients with confirmed Klinefelter syndrome and the group with normal karyotypes existed. In contrast to Carothers and Filippi (1988), we found no increased risk of procreating a child with Klinefelter syndrome at an advanced paternal or maternal age. This is in agreement with our finding that older men have no increase in sperm aneuploidies over younger men (Bernardini et al, 1998; Luetjens et al, 2002). The intelligence of some but not all Klinefelter patients may be slightly reduced, and deficits are observed in verbal and cognitive abilities (Rovet et al, 1995; Nieschlag et al, 2000). Moreover, in our population, the proportion of men with professions involving higher educational levels was significantly reduced in the Klinefelter patients compared to group II (Table 1).
Characteristic clinical features of adults with Klinefelter syndrome are small, firm testes, hypergonadotropic azoospermia, gynecomastia, and tall eunuchoid stature (Klinefelter et al, 1942; Nieschlag et al, 2000). However, diagnosis is often delayed because of the substantial variation in clinical presentation in adults (Smyth and Bremner, 1998; Amory et al, 2000; Wilkes, 2000) and the relatively discrete symptoms, especially prior to midpuberty (Salbenblatt et al, 1985). Moreover, according to our study, 60% of the patients with Klinefelter syndrome were not suspected of having Klinefelter syndrome in the referring secondary or primary center, despite previous external clinical investigations. In addition, 5% of the patients with a normal karyotype were referred for suspected Klinefelter syndrome, although the initial diagnosis could not be confirmed on the basis of karyotype analysis.
Compared to our group with normal karyotypes, our patients with Klinefelter syndrome were significantly taller, had azoospermia more often, and had higher gonadotropin concentrations. However, body height, presence of azoospermia, gonadotropin levels, and secondary body hair distribution, as well as the comparable incidence of gynecomastia, showed a surprising overlap between both groups in whom Klinefelter syndrome was suspected and were of low specificity. However, the group with normal karyotypes represents a heterogeneous group with various diagnoses, which might explain this missing difference. They were considered eligible for the Barr body test and karyotype analysis if they showed at least 1 clinical feature (eg, decreased testicular volume, gynecomastia, or azoospermia) of Klinefelter syndrome, while in most of these patients, hormone values were not known at the time of the Barr body test and karyotype analysis. On the other hand, not all patients with Klinefelter syndrome show all or at least some of the classical signs of Klinefelter syndrome, and the quick, cheap, and simple Barr body test in this situation can increase the diagnostic accuracy. It can be used for screening a larger proportion of men at the first visit, of whom those with a positive Barr body test and those with characteristic hormone profiles and clinical features (regardless of the result of the Barr body test) will be karyotyped at the second visit in the clinic. In the meantime, results of the Barr body test, together with hormone and ejaculate values and the results of the physical examination, would allow more precise handling of the patient.
Of the general and clinical features, bitesticular volume appears to be the most sensitive parameter (Table 2), showing the smallest overlap between the groups, and all Klinefelter patients had subnormal sonographic bitesticular volumes, with a mean of 4.7 mL. Among the patients with suspected Klinefelter syndrome but normal karyotypes, many also had reduced testicular volumes, despite a mean sonographic testicular volume (13.7 mL) in the normal range. However, the magnitude of the reduction in testicular volume was mostly not comparable to the patients with Klinefelter syndrome. Testosterone levels between the groups were not significantly different (Table 2). However, the proportion of adult men with hypogonadal testosterone values (<12 nmol/L) was nearly double (61%) in the Klinefelter patients compared to group II (36%). The androgen sensitivity index (Table 2) was significantly increased in the patients with Klinefelter syndrome, which was due to the markedly increased LH serum levels. However, in contrast to patients with androgen receptor mutations and in view of the similar testosterone serum levels, this probably does not reflect different androgen sensitivities compared to patients with a normal karyotype.
FSH is generally inversely correlated with total testicular volume and Sertoli cell—only tubuli (Pierik et al, 1998; von Eckardstein et al, 1999). Whereas in group II FSH was weakly correlated with total testicular volume, this correlation was absent in the patients with Klinefelter syndrome (Figure 1), reflecting the severe exocrine testicular failure. Interestingly, in both groups, a weak correlation between testosterone and total testicular volume was evident (Figure 2). Such a correlation cannot be found in normal men. In the patients with Klinefelter syndrome, this correlation might be explained by the low testicular volume, which, to a large extent, is occupied by the hyperplastic Leydig cells.
In 4 (ie, 7%) of the investigated Klinefelter patients, spermatozoa were found in the ejaculate (Table 3) that could be used for intracytoplasmic sperm injection. However, it may not be excluded that, in addition, individual Klinefelter patients with sperm could have been identified if repeated semen analysis had been performed in all patients. In all patients with repeated semen samples, the diagnosis of azoospermia was subsequently confirmed. Testicular sperm retrieved from patients with Klinefelter syndrome demonstrate a normal pattern of sex chromosome segregation comparable with the rates found in other severe male factor infertility patients (Levron et al, 2000), and successful pregnancies from patients with Klinefelter syndrome have been reported (Palermo et al, 1998). Westlander et al (2001) have found no exclusive clinical features of patients with Klinefelter syndrome, azoospermia, and sperm in the testis. Nor was any clinical feature predictive for sperm in the ejaculate in our Klinefelter patients. However, they were younger compared to the cohort's mean age. Therefore, it might well be that, in some patients with Klinefelter syndrome, the depletion of germ cells appears later and that therefore the chance of finding sperm in younger Klinefelter patients is greater than in older ones. Early diagnosis would offer these patients the possibility of cryopreserving their spermatozoa for later use in assisted reproduction. However, to confirm this hypothesis, longitudinal examinations including semen analysis in pubertal boys with Klinefelter syndrome would be required.