Human Reproduction, Vol. 15, No. 8, 1804-1806,
August 2000
© 2000 European Society of Human Reproduction and Embryology
A 47,XXY fetus conceived after ICSI of spermatozoa from a patient with non-mosaic Klinefelter's syndrome: Case report
1 IVF and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University and 2 Institute of Human Genetics, Kaplan Medical Center, Israel
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Abstract |
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The birth of 12 healthy infants to fathers with non-mosaic Klinefelter's syndrome has been reported so far. The spermatozoa for these pregnancies was obtained from frozen–thawed ejaculate in one pregnancy (twins) and from the testis in the remaining 10 infants. All of them had a normal karyotype. We describe a patient with non-mosaic Klinefelter's syndrome from whom a testicular biopsy was obtained and motile spermatozoa were collected. Of 16 oocytes that were injected, 14 fertilized and cleaved. Three embryos were transferred, resulting in a triplet pregnancy. Karyotype analysis from chorionic villous sampling revealed 46,XX, 46,XY and 46,XXY from the three fetuses. The affected 46,XXY fetus was reduced on the 14th gestational week. The pregnancy culminated with the birth of a healthy male and female, on the 36th gestational week, weighing 3600 and 2660 g respectively. This case report proves the presence of hyperploid spermatozoa in the seminiferous lumen, and strengthens the necessity of genetic diagnosis of the embryos or fetuses in such pregnancies to fathers with non-mosaic Klinefelter's syndrome.
Key words: azoospermia/intracytoplasmic injection/Klinefelter's syndrome/testicular biopsy
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Introduction |
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The most common sex chromosomal aberration among azoospermic patients is 47,XXY known as Klinefelter's syndrome. Up to now, the birth of 12 healthy neonates has been reported following intracytoplasmic sperm injection (ICSI) with the use of spermatozoa from patients with non-mosaic Klinefelter's syndrome (Bourne et al., 1997
; Tournaye et al., 1997; Palermo et al., 1998; Reubinoff et al., 1998; Ron-El et al., 1999; Nodar et al., 1999). All infants had a normal karyotype. In the first reported twin pregnancy, frozen–thawed ejaculated spermatozoa were used (Bourne et al., 1997). In all other cases ICSI was performed with testicular spermatozoa achieved either by testicular biopsy (Tournaye et al., 1997; Palermo et al., 1998; Nodar et al., 1999; Ron-El et al., 1999) or needle aspiration (Reubinoff et al., 1998). Healthy twin males were recently born, the pregnancy being achieved using frozen–thawed testicular spermatozoa (R. Ron-El, unpublished).
Using fluorescence in-situ hybridization (FISH), 2.2% hyperploidy of the spermatozoa was found in a male with mosaic Klinefelter's syndrome (Chevret et al., 1995) and from 2.67% (Guttenbach et al., 1997) to 21.76% (Foresta et al., 1998) in a non-mosaic Klinefelter's syndrome patient. This suggests the relatively low likelihood of having a child with Klinefelter's syndrome when the father has Klinefelter's syndrome.
We report the first case, to our knowledge, of a fetus with Klinefelter's syndrome conceived from a father with apparently non-mosaic Klinefelter's syndrome.
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Case report |
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A 31 year old healthy male with normal appearance presented at our centre because of azoospermia. Orchipexy was performed on the right undescended testis at the age of 13 years. Physical examination revealed normal hair distribution and no gynaecomastia. The volume of the right testis was 12 ml and that of the left testis 6 ml. Three semen analyses with an average volume of 2.8 ± 1.6 ml failed to show spermatozoa. Serum analysis was performed showing follicle-stimulating hormone (FSH) concentration (48.0 IU/l), luteinizing hormone (LH) concentration (24.1 IU/l), low testosterone (15.2 µg/l) and normal prolactin (276 pmol/l). Chromosome analysis showed the 47,XXY karyotype in 34 blood cells.
The patient's wife was a 26 year old healthy woman with normal ovulatory cycles and a normal hysterosalpingography. Ovarian stimulation was achieved by the combination of gonadotrophin-releasing hormone (GnRH) agonist (Decapeptyl®, microcapsules, 3.75 mg; Ferring, Malmo, Sweden) and urinary FSH (Metrodin®; Teva, Petah-Tiqua, Israel). Human chorionic gonadotrophin (Chorigon®; Teva), 10 000 units, was administered when the leading follicle reached a mean diameter of 20 mm and oestradiol concentration was 1965 pg/ml. Oocyte collection was carried out 37 h later and luteal phase was supplemented with micronized progesterone (Utrogestan®; Besins-Iscovesco, Paris, France), 100 mg three times per day, intravaginally.
On the oocyte retrieval day, two biopsies were taken from the right testis under general anaesthesia in which an immediate evaluation revealed no spermatozoa. The sample included a large area of scar tissue, presumably as a consequence of the orchipexy. A single biopsy was taken from the left testis, in which motile spermatozoa were detected. The wet preparation was elaborated as previously described (Friedler et al., 1997).
After 3 h of incubation, aliquots of the suspension from the homogenate of the testicular tissue were distributed in dozens of droplets, each of 8 µl, for a careful search for spermatozoa. Sixteen motile spermatozoa were found and injected into 16 MII oocytes; of these, 15 oocytes fertilized, 14 cleaved, three of which were replaced 44 h after oocyte retrieval at the 4-cell stage. The transferred embryos were judged to be of grade I, I-II and II morphology (I – best; IV – worst).
The remaining testicular tissue was frozen after removing a small specimen for histology. The histological findings in the right testis were of diffuse hyalinization of seminiferous tubules and the interstitium. No Leydig cells were present. In the left testis thick membranes of the seminiferous tubules were seen with sporadic sperm cells in different stages of spermatogenesis in the lumen.
The couple consistently refused any pre-implantation genetic diagnosis which was extensively discussed on several occasions before starting their treatment.
Pregnancy was achieved, and the first ß human chorionic gonadotrophin (HCG) concentration was 525 IU at 2 weeks after the embryo transfer. Three sacs with visible fetal poles and heart pulsations could be seen in the sixth gestational week. Since the couple considered reducing one fetus, the possibility of performing chorionic villous sampling (CVS) was discussed with them at the 10th gestational week. CVS was guided by abdominal ultrasound (ATL 3000, HDI, Bothell, WA, USA). The location of the three sacs and their respective placentae were noted and recorded. Sampling was performed with a spinal needle, 19 gauge, by single aspiration from each placenta. The samples were analysed by FISH and subsequently the cytogenetic karyotype was determined by G-banding. Both methods disclosed a normal 46,XX and a normal 46,XY chromosome in all cells of the two placentae of two fetuses and a 47,XXY karyotype in all the examined cells of the third placenta of the third fetus. In the 13th gestational week, reduction of the fetus with Klinefelter's syndrome was performed by direct cardiac injection of KCl (15% solution) after informed consent and institutional board approval. Heart movements stopped within 1–2 min. Amniotic fluid was aspirated while retracting the needle from the sac, to confirm the 47,XXY karyotype of the fetus. The cytogenetic analysis from the amniotic fluid disclosed a 47,XXY karyotype. The woman delivered in the 36th week of her pregnancy by Caesarean section, giving birth to a healthy boy and girl of 3600 and 2660 g respectively.
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Discussion |
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Patients with oligoteratoasthenozoospermia have a rate of sex chromosome disomy of 0.64% in the spermatozoa and an incidence of 1.07% diploid spermatozoa (Aran et al., 1999
). Both numbers are significantly higher than those found in spermatozoa of the normal population, which are 0.37 and 0.25% respectively. The rate of haploid spermatozoa among patients with mosaic Klinefelter's syndrome as evaluated by FISH varied from 90.2–96.3% (Cozzi et al., 1994; Chevret et al., 1995; Martini et al., 1996; Kruse et al., 1998; Lim et al., 1999; Okada et al., 1999). Among patients with non-mosaic Klinefelter's syndrome the rate of haploid spermatozoa varied from 92.25% – analysis of 2206 spermatozoa from one case (Guttenbach et al., 1997) to 84.63 and 76.47% – scoring of 10 000 spermatozoa from each of two patients (Foresta et al., 1998). While sex chromosomal hyperploidy was found at an incidence of 0.9–2.5% in the mosaic form, its incidence in the non-mosaic form varied from 2.52% (Okada et al., 1999), 3.48% (Guttenbach et al., 1997) to 21.76% (Foresta et al., 1998). Estop et al. (1998) found a correlation between sperm morphology and sex chromosome abnormality in a Klinefelter male. The percentage of spermatozoa with an abnormal number of sex chromosomes increased from 1/6 among spermatozoa with normal morphology to 11/18 in spermatozoa with abnormal morphology. This discrepancy in the frequency of sex chromosome hyperploidy in non-mosaic Klinefelter's males has still to be clarified. It may be due to the small number of patients examined, although the number of spermatozoa scored by FISH was high. To date, all of these studies investigating chromosome abnormalities in spermatozoa from patients with Klinefelter's syndrome were performed on ejaculated spermatozoa.
A chromosomal analysis of intratesticular cells from two patients with non-mosaic Klinefelter's syndrome who exhibited intratesticular residual spermatogenesis showed 100% hyperploidy (47,XXY) in the 630 Sertoli cells and the 30 spermatogonia and primary spermatocytes, 30% hyperploidy (24,XY and 24,XX) among the 51 secondary spermatocytes and spermatids and 20–25% hyperploidy among the 45 spermatozoa (Foresta et al., 1999). Despite the low number of spermatogenic cells retrieved for analysis, this study demonstrates that 47,XXY spermatogonia are able to complete the spermatogenic process leading to the formation of mature spermatozoa with a normal sex chromosome pattern. On the other hand, the relatively high proportion of spermatids and mature spermatozoa carrying a normal sex chromosome pattern demonstrates the difficult progression of the 47,XXY germ cells through the meiotic process. This is probably associated with a high incidence of death of those spermatocytes affected by chromosomal abnormalities in Klinefelter patients. These data coincide with the fact that all 12 infants (four twin sets and four singletons) born up to now to fathers with non-mosaic Klinefelter's syndrome have a normal karyotype. Pre-implantation genetic diagnosis was performed in only three of the eight pregnancies (Tournaye et al., 1997; Reubinoff et al., 1998). In two cases embryos were biopsied and a chaotic chromosome segregation was noted (Reubinoff et al., 1998). A recent study of FISH in spare embryos from a mosaic Klinefelter patient revealed that only three of 10 embryos tested had normal 18, X and Y chromosome complements. This patient had a 3.9% incidence of sex chromosome hyperploidy of the analysed sperm nuclei (Bielanska et al., 2000). Four of the abnormal embryos were found to have chaotic mosaicism and the remaining three had diploid mosaicism. With such a low rate of embryos with normal karyotype, one would probably have expected a higher number of chromosomally affected newborns so far. However, the present case is the first, to the best of our knowledge, in which the karyotype of the fetus from a father with non-mosaic Klinefelter's syndrome was 47,XXY. Since the couple wished to reduce one fetus, it was thus logical to choose the affected fetus for reduction.
Until now, studies showing a low rate of spermatozoa with sex chromosomal abnormalities were encouraging. Therefore, it is understandable why parents of at least three of the 12 infants born to fathers with non-mosaic Klinefelter's syndrome to date refused any antenatal genetic investigation. In our case, the likelihood that the 47,XXY fetus was paternally derived is high. However, the genetic anomaly could have been caused by maternal non-disjunction.
Couples who seek treatment when the male partner has Klinefelter's syndrome should be provided with comprehensive information about the risk of producing chaotic embryos or chromosomally affected children.
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Acknowledgments |
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The authors would like to acknowledge the collaboration of M. Schachter and the embryologists O. Bern, E. Kasterstein, D. Komarovsky from the IVF Unit, Assaf Harofeh Medical Center, and G. Rosenzaft from the Institute of Human Genetics, Kaplan Medical Center.
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Notes |
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3 To whom correspondence should be addressed at: IVF and Infertility Unit, Assaf Harofeh Medical Center, Tel Aviv University, Israel. E-mail: rronel{at}asaf.health.gov.il
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References |
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Submitted on February 4, 2000; accepted on May 10, 2000.
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