Birth of a healthy girl after ICSI with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome: Case report

D. Crüger1,3, B. Toft2, I. Agerholm2, J. Fedder2, F. Hald2 and G. Bruun-Petersen1

1 Department of Clinical Genetics, Vejle Hospital, DK-7100 Vejle and 2 The Fertility Clinic, Braedstrup Hospital, DK-8740 Brædstrup, Denmark


    Abstract
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
Klinefelter's syndrome is a major contributor to male infertility. Recent reports of births after ICSI with especially testicular spermatozoa from infertile men with this syndrome are promising. The birth of a healthy girl after ICSI treatment with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome is reported. The non-mosaic karyotype was confirmed by chromosome analysis of both peripheral blood leukocytes and fibroblasts from a skin biopsy. In conclusion, in a very few cases, men with apparently non-mosaic Klinefelter's syndrome have ejaculated spermatozoa that can result in a birth of a healthy child following ICSI.

Key words: chromosome analysis/fertility/ICSI/Klinefelter's syndrome/non-mosaicism


    Introduction
 Top
 Abstract
 Introduction
 Case report
 Discussion
 References
 
Klinefelter's syndrome is a chromosomal disorder affecting ~1 in 600 liveborn boys (Nielsen and Wohlert, 1991Go). The majority (85%) have the abnormal karyotype 47,XXY whereas 15% have a mosaic form, often 46,XY/47,XXY. There is great variability in the clinical findings, with fewer manifestations in the mosaic men. The most prominent finding is the small hypoplastic testes, but the clinical picture also includes gynaecomastia. As a general rule, Klinefelter's syndrome is associated with sterility, but reports of exceptions exist (Terzoli et al.1992Go). However, men who have fathered offspring naturally usually have mosaicism (Cozzi et al.1994Go).

Since many men with Klinefelter's syndrome have few or non-apparent clinical findings, most are unrecognized until the man presents with infertility. In fertility clinics, ~10% of males with azoospermia are karyotyped as having Klinefelter's syndrome.

To date, several reports of successful ICSI treatment of infertility in men with Klinefelter's syndrome have been published, but only one paper reports fertilization with freshly ejaculated spermatozoa (Bourne et al.1997Go). This report describes the birth of a healthy girl after ICSI treatment with ejaculated spermatozoa from a man with non-mosaic Klinefelter's syndrome.


    Case report
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
A healthy couple, both 28 years old, were evaluated after several years of primary infertility. The woman had regular ovulatory cycles and a normal hysterosalpingogram.

The man was 185 cm tall and weighed 102 kg. Both testes were located in the scrotum, they were firm and had ultrasound determined volumes of 1.0 ml and 1.1 ml respectively. No gynaecomastia was found. Analysis of a semen sample showed only five spermatozoa, of which three were motile and two immotile with abnormal morphology.

As a routine analysis of all men presenting for ICSI, a karyotype was made from peripheral blood leukocytes. All 15 metaphases analysed showed a 47,XXY karyotype. The woman had a normal 46,XX female karyotype. As a result of the abnormal karyotype, the couple was referred for genetic counselling and was informed of the findings and their very poor chances of a naturally occurring pregnancy. Their chance of a pregnancy after ICSI was also discussed. They were informed that only very few centres had successfully achieved pregnancy by ICSI with spermatozoa from men with Klinefelter's syndrome and that an increased risk of sex chromosome aneuploidies in the offspring could not be excluded. Prenatal diagnosis was therefore highly recommended.

As confirmation of the non-mosaic karyotype found in the chromosome analysis of peripheral blood leukocytes from the man, a skin biopsy was taken for tissue culture and subsequent chromosome analysis. We found 24 metaphases with 47,XXY and a single metaphase with the normal female karyotype 46,XX. The investigation was therefore extended in this tissue with a fluorescence in-situ hybridization (FISH) analysis with X and Y centromeric probes to reveal a possible mosaicism. This analysis showed the XXY karyotype in all of the 82 metaphases examined. It was concluded that the male was non-mosaic, and the single cell found with 46,XX primary could be due to an artefact.

After counselling, the couple decided to have ICSI performed and were referred back to the fertility clinic for oocyte retrieval and ICSI.

The woman's hormone profile on the third day of the cycle was FSH 5.4 IU/l and LH 3.7 IU/l. Ovarian stimulation was carried out by standard procedures. Four oocytes were retrieved.

The concentration and motility in the ejaculated semen were analysed. Prior to the ICSI procedure, three semen analyses had shown very few motile spermatozoa (3, 6 and 19 respectively). On the day of ICSI, nine motile spermatozoa were found. Only motile spermatozoa were used for injection, and all four metaphase II oocytes were injected. Examination on the next day showed 2-pronuclei in each of them. Two embryos were transferred on day 2, resulting in a singleton pregnancy.

The couple was offered prenatal diagnosis but they did not accept. The pregnancy was uneventful and at term the woman had a normal vaginal delivery of a healthy girl, weight 3950 g, length 53 cm. Chromosome analysis of peripheral leukocytes from the baby showed a normal 46,XX female karyotype.


    Discussion
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 Abstract
 Introduction
 Case report
 Discussion
 References
 
Since 1997, births after ICSI with spermatozoa from apparently non-mosaic men with Klinefelter's syndrome have been described in a number of papers. Most confirm ICSI of testicular spermatozoa (Tournaye et al.1997Go; Palermo et al.1998Go; Reubinoff et al.1998Go; Nodar et al.1999Go; Ron-El et al.1999Go, 2000aGo; Kitamura et al.2000Go) and only two reports describe delivery after ICSI with ejaculated spermatozoa (Bourne et al.1997Go; Ron-El et al.2000bGo). This report confirms that in a few cases motile spermatozoa are present in ejaculates from males with Klinefelter's syndrome. Ejaculated spermatozoa should be the first choice for ICSI before testicular biopsy is considered.

It is common knowledge that most males with Klinefelter's syndrome are sterile. Only a few reports of naturally occurring pregnancies exist. Mosaicism could be the explanation for the few spermatozoa found in some men with Klinefelter's syndrome. In all the above-mentioned reports of birth after ICSI treatment, the males are described to be non-mosaic. However only chromosome analysis of peripheral blood leukocytes is reported and to minimize the possibility of a mosaic karyotype at least one other tissue should be analysed. In one of the reports (Ron-El et al.1999Go), a buccal smear was analysed by X chromatin analysis, but this is an inadequate and out-dated analysis, which is by no means effective in revealing mosaicism. We suggest that all men with Klinefelter's syndrome who have either ejaculated or testicular spermatozoa have a chromosome analysis of both peripheral blood leukocytes and another tissue (skin) to clarify if a mosaic karyotype is present, since it must be stated that the presence of spermatozoa in the ejaculate of men with Klinefelter's syndrome is nearly always due to mosaicism.

In a previous report (Reubinoff et al.1998Go), the non-mosaic karyotype was further confirmed by FISH analysis of 100–200 peripheral blood lymphocytes. They did not mention if the analysis was performed with metaphase or interphase nuclei. To be of any value it should be a metaphase analysis, as interphase nuclei analysis is not recommended for diagnosis of mosaicism (Klinger et al.1992Go; Feldman et al.2000Go). A metaphase analysis by FISH of the same tissue as the traditional chromosome analysis is a further statement of a non-mosaic karyotype. However, it cannot replace analysis of another tissue when a mosaic karyotype should be excluded.

We recommend genetic counselling and further genetic analysis of all men with Klinefelter's syndrome and their partners. Close collaboration between the genetic departments and the fertility clinics is of great value to the patients, who can receive information about their genetic disorder and the risk in their offspring.

Many reports of an increased ratio of sex-chromosomal disomy in spermatozoa from males with Klinefelter's syndrome have been published (Cozzi et al.1994Go; Chevret et al.1996Go; Guttenbach et al.1997Go; Foresta et al.1998Go; Rives et al.2000Go). All authors find that the ratio of 24,XX bearing spermatozoa in particular is higher in males with a 47,XXY or a 46,XY/47,XXY karyotype, supporting the hypothesis that a few 47,XXY spermatogonias are able to complete meiosis and produce mature spermatozoa (Foresta et al., 1999Go). The risk of an unbalanced karyotype in an offspring is therefore thought to be increased. However, Levron et al. (2000) found that most of the testicular spermatozoa retrieved from men with Klinefelter's syndrome demonstrated a normal pattern of sex chromosome segregation (Levron et al., 2000Go).

Even though all couples are recommended to have prenatal diagnosis after ICSI, a considerable number do not want this procedure because of the risk of unwanted abortion. Therefore preimplantion genetic diagnosis could be considered. Staessen et al. (1996) performed preimplantation genetic diagnosis in five embryos prior to transfer (Staessen et al., 1996Go). All were found normal regarding the X and Y chromosomes, but no on-going pregnancies occurred. Bielanska et al. (2000) carried out preimplantation genetic diagnosis in 10 spare embryos after ICSI with spermatozoa from men with Klinefelter's syndrome (Bielanska et al. 2000Go). However no embryos showed sex-chromosomal aneuploidies, but 70% had other abnormalities including chaotic chromosome patterns (Staessen et al., 1996Go).

It therefore seems that the majority of spermatozoa retrieved from males with Klinefelter's syndrome that are effective in achieving fertilization and embryo development have a normal chromosome pattern. However, couples need genetic counselling regarding the fact that they have an increased risk of a child with an unbalanced karyotype. They must then decide whether to take that risk or have conventional prenatal or preimplantation genetic diagnosis.


    Notes
 
3 To whom correspondence should be addressed. E-mail: dgc{at}vs.vejleamt.dk Back


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 Abstract
 Introduction
 Case report
 Discussion
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Submitted on November 30, 2000; accepted on May 14, 2001.