Klinefelter syndrome (KS), a chromosomal disorder
due to an extra X chromosome (47,XXY)
Small number of 47,XXY patients have sperm production
that would allow them to benefit from assisted
reproductive techniques (ART) such as micro dissection
testicular sperm extraction (TESE) and intracytoplasmic
sperm injection (ICSI) (
We report on an interesting case of a KS patient with a new type of quintuple mosaicism in peripheral blood lymphocytes.
A 39 year old man was referred to Royan Institute cytogenetic laboratory suffering from infertility. He was born from a full term natural delivery with no apparent complication. The age of his mother at this pregnancy was 35 and his father was 40. The parents were unrelated. Family history of infertility was negative and his only brother fathered a child. On examination he was 180 cm, 82 kg. Each testis volume was 4 ml estimated by Prader’s orchidometer (normal range: 15-25 ml), with normal vas deferens. He had a history of right sided epididymo-orchitis. Stature growth was regular and puberty was normal without testosterone therapy. Motor and mental development of the patient was normal. There were no malformations, no gynecomastia, no diabetes and no reduced muscle strength. Olfaction was normal. The semen analysis showed total azoospermia with low volume (0.3 ml) and normal pH and fructose level. No spermatozoa were found in micro dissection TESE (MD-TESE) and the seminiferous tubules were hyalinized. Histology of testis biopsy specimen showed only Sertoli cells and moderate hyperplasia of the leydig cells. Endocrinological laboratory studies revealed elevated follicle stimulating hormone (FSH=43 mIU/ml, reference 0.9-8.9 mIU/ml) and luteinizing hormone (LH=14.3 mIU/ml, reference 0.8-10 mIU/ml) levels and low testosterone levels (1.4 ng/ml). The results were suggestive for hypergonadotropic hypogonadism and KS was the most probable diagnosis.
Chromosomal analysis was performed on phytohemagglutinin-stimulated peripheral lymphocyte cultures using standard cytogenetic methods. Two different cultures for the sample prepared and two different series of slides from each culture analyzed separately. Half of slides were investigated by GTG and the other half by FISH. 170 GTG banded metaphases from the patient were analyzed at the resolution of 550 bands.
The hybridization on metaphase chromosomes was performed according to standard cytogenetic protocols (
Accordingly, the karyotype of the case was ascertained as: 47,XXY/46,XX/45, X/48,XXXY/46,XY according to The International System for Human Cytogenetic Nomenclature ISCN 2009 (
FISH of interphase cells and mitotic lymphocytes with different probes: Yq Telomere probe (G); X Centromere probe (O) ; 18 Centromere probe (A)
A. 46,XY metaphase and a 45,X interphase cell.
B. 46,XX and a 47,XXY interphase cell.
C. 48,XXXY and a 46,XY interphase cell.
Number of each cell line analyzed by GTG banding and FISH
|Total||FISH Results||GTG Results||Cell line|
GTG banding; G-band by Trypsin using Giemsa and FISH; Fluorescence in situ hybridization.
Mosaicism involving more than three lines is rarely detected and the predominant cell line differs between different cases. The origin of the quintuple mosaicism may be explained by meiotic and mitotic disturbances in the formation of the zygote and embryo. Regarding the possible explanations for mosaicism, the most reasonable hypothesis is a nondisjunctional event. The predominance of the 47,XXY and the presence of 45,X cell lines in our patient strongly suggest the mitotic nondisjunction of the X chromosome in a normal XY zygote results in a 47,XXY and a 45,X clone, with successive nondisjunction of the 47,XXY cell line producing 48,XXXY cells. The XX cell line could be explained by the loss of Y chromosome in a proportion of the XXY cells. It is well known that both the phenotypic sex and gonadal phenotype are influenced by the percentage and distribution of Y-carrying cells in the gonad, but not necessarily in the blood (
The only quintuple mosaicism in patients with Klinefelter’s syndrome (with the cell lines different from ours) has been reported before by Al-Awadi et al. (
A man with KS may wish to reproduce with the aid of modern reproductive technologies. TESE-ICSI procedure has offered a new hope to those patients (
Unfortunately in our patient, there was no sign of spermatogenesis, concluding that he could not profit from assisted reproduction techniques.
Through a combination of GTG-banding and FISH, the karyotype of the patient was determined. FISH is recommended in mosaic forms of KS to exactly define the cytogenetic status of the patient. Generally most individuals with KS have germ cells with sex chromosomal abnormalities and may wish to reproduce with ART, thus it is important that accurate estimation of the frequency of abnormal cells be obtained for fertility counseling, prognosis discussion and risk estimation.