Analysis of the sex chromosomeconstitution of sperm in men with a47,XYY mosaic karyotype by fluorescencein situ hybridization

Alvin Soon Tiong Lim, B.Sc. (Hon.), Yang Fong, M.D., and Su Ling Yu, M.D.

Department of Obstetrics and Gynecology, Singapore General Hospital, Singapore

Objective: To determine the incidence of sex chromosome aneuploidy in the sperm of two men with a47,XYY/46,XY karyotype.

Design: Case report.

Setting: Infertility clinic in a teaching hospital.

Patient(s): One patient with near normal semen parameters whose wife had a history of miscarriages and onepatient with primary infertility and severe oligoasthenozoospermia.

Intervention(s): Cytogenetic analysis of peripheral lymphocytes and three-color X/Y/18 fluorescence in situhybridization analysis of sperm.

Main Outcome Measure(s): Analysis of sex chromosome disomy and diploidy rates in sperm.

Result(s): Both patients had a 47,XYY/46,XY karyotype. The hyperdiploidy rate of patient 1 was 19% andthat of patient 2 was 90%. The incidence of disomy XY was significantly elevated in both patients comparedwith the controls (0.23% and 1.02%, respectively, versus 0.10%). The incidence of disomy YY (0.44% versus0.10%) was increased only in patient 2, as was the incidence of disomy 18 (0.49% versus 0.09%) and the rateof diploidy (0.83% versus 0.13%). The rate of 24,XX sperm in both patients was not different from that in thecontrols.

Conclusion(s): Patients with a 47,XYY mosaic karyotype may be at risk of producing offspring with ahyperdiploid sex constitution. These patients should have their sperm investigated by fluorescence in situhybridization to determine their particular risks before they undergo intracytoplasmic sperm injection. (FertilSterilt 1999;72:121–3. ©1999 by American Society for Reproductive Medicine.)

Key Words: Diploidy, disomy, FISH, mosaicism, oligoasthenozoospermia, XYY

Sex chromosome aneuploidy is by far themost common chromosome abnormality inadults and often is associated with infertility.Men with a 47,XYY karyotype can have semenparameters ranging from severe oligozoosper-mia to normozoospermia because the conditionappears to have a deleterious effect on somebut not on others. Intracytoplasmic sperm in-jection may be the most viable option for se-verely affected men.

It has been suggested that the extra Y chro-mosome in these men is lost before the forma-tion of spermatocytes, leaving an XY bivalentat diakinesis to proceed with normal meiosis(1). More recent reports, however, have indi-cated that there is an increase in the incidenceof chromosomally abnormal spermatozoa in

the semen of men with a 47,XYY karyotype(2, 3). It is therefore important to perform morestudies on these men, as well as those who aremosaic for the condition, to determine the in-cidence of sex chromosome aneuploidy in theirsperm. An increased incidence of sex chromo-some hyperhaploid sperm would mean thatthey have an increased risk of transmitting theextra sex chromosome to their offspring.

A triple-color X(FITC)/Y(Cy3)/18(FITC-Cy3)fluorescence in situ hybridization protocol (Cy-tocell Ltd., United Kingdom) was used to eval-uate the sperm of two men (patients 1 and 2)with a 47,XYY/46,XY karyotype. The probeswere all specific for alphoid sequences. Insti-tutional review board approval was obtainedfor this study.

Received November 18,1998; revised andaccepted February 25,1999.Supported by theSingapore General HospitalResearch Fund, Singapore,Singapore.Reprint requests: AlvinSoon Tiong Lim, B.Sc.,Department of Obstetricsand Gynecology,Singapore GeneralHospital, Outram Road,Singapore 169608(FAX: 65-2253464; E-mail:stalvinlim@hotmail.com).

REPRODUCTIVE BIOLOGYFERTILITY AND STERILITY tVOL. 72, NO. 1, JULY 1999Copyright ©1999 American Society for Reproductive MedicinePublished by Elsevier Science Inc.Printed on acid-free paper in U.S.A.

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Nineteen percent of patient 1’s peripheral lymphocytesbelonged to the abnormal cell line. Seminal analysis showedmild teratozoospermia but sperm densities were normal,averaging 603 106/mL. The 35-year-old man’s wife hadthree consecutive miscarriages before being delivered of ahealthy girl with a 46,XX karyotype. Patient 2 was a 37-year-old man with infertility in whom 90% of the peripherallymphocytes belonged to the abnormal cell line. His spermcount was only 0.63 106/mL.

Two karyotypically normal fertile men in their late 20swho had normal semen parameters served as controls.

Sperm were scored as disomic when the distance betweentwo sex chromosome signals was at least one signal domainapart, the signals were of the same size and intensity, andthere was only one signal for chromosome 18. Sperm wereconsidered diploid when there were two sex chromosomesignals and two chromosome 18 signals. Cells were consid-ered sperm when the tails were observed.

The data were analyzed by thex2 test and by Fisher’s

exact test. AP value of ,.05 was considered statisticallysignificant.

Semen smears from patient 2 revealed an abundance ofimmature sperm cells (Table 1). The semen profile showedimpaired spermatogenesis, with many germ cells that wereunable to complete meiosis. In contrast, no immature germcells were found in the semen of patient 1, suggesting thatspermatogenesis was not affected. More than 10,000 spermwere analyzed for patient 1 (Table 2) but only 2,000 spermwere scored for patient 2.

The ratio of X-bearing sperm to Y-bearing sperm in boththe patients, as well as the controls, was 1:1. No differencewas noted in the incidence of karyotypically abnormal spermin patient 1 compared with the controls, except for a slightbut significant increase in sperm with a 24,XY karyotype(0.23% versus 0.10%,P,.05). This increase was moresignificant in patient 2 compared with the controls (1.02%versus 0.10%,P,.0001), aswere the incidences of 24,YY(0.44% versus 0.02%,P,.0001), disomy 18 (0.49% ver-sus 0.09%,P,.001), anddiploidy (0.83% versus 0.13%,P,.0001).However, there was no increase in the incidenceof sperm with a 24,XX karyotype in either patient.

The differences in the incidence of disomy between thetwo patients appeared to be related to their relative levels ofmosaicism. The low level of XYY cells apparently did notaffect spermatogenesis in patient 1, except for the slightincrease in sperm with a 24,XY karyotype, whereas the 90%incidence of XYY cells resulted in severe oligozoospermiain patient 2. The high-level mosaicism also resulted in karyo-typically abnormal sperm, especially in regard to disomy XYand YY. The elevated levels of disomy 18 and diploidy mayhave been a consequence of severe oligozoospermia.

Elevated rates of disomy YY in men with a 47,XYY

T A B L E 1

Germ cells in semen samples from men with a mosaicXYY karyotype.

Cell type

No. (%) of germ cells of indicated cell type

Patient 1(47,XYY/46,XY)

Patient 2(47,XYY/46,XY)

Sperm 200 (100.0) 103 (51.5)Spermatids 0 62 (31.0)Spermatocytes 0 38 (17.5)

T A B L E 2

Fluorescence in situ hybridization analysis of sperm from men with a 47,XYY/46,XY karyotype.

Karyotype byFISH analysis

No. (%) of sperm from indicated patient group

Mean of controls(46,XY)

Patient 1(47,XYY/46,XY)

Patient 2(47,XYY/46,XY)

23,X 4,973 (49.73) 4,844 (48.44) 1,012 (49.22)23,Y 4,989 (49.89) 5,225 (52.25) 986 (47.96)24,XY 10 (0.10) 23 (0.23)* 21 (1.02)†24,XX 4 (0.04) 0‡ 1 (0.05)‡24,YY 2 (0.02) 2 (0.02)‡ 9 (0.44)†24,X/Y,118 9 (0.09) 5 (0.05)‡ 10 (0.49)§Diploid 13 (0.13) 13 (0.13)‡ 17 (0.83)†

Total 10,000 10,112 2,056

Note: FISH 5 fluorescence in situ hybridization.* P,.05 (versus controls).† P,.0001(versus controls).‡ No statistically significant difference versus controls.§ P,.001 (versus controls).

122 Lim et al. Analysis of sperm from mosaic 47,XYY men Vol. 72, No. 1, July 1999

karyotype suggest that some of these hyperdiploid cells canundergo meiotic division. It is postulated that disomy YYsperm arise through YY bivalent pairing at meiosis I, leavinga free X univalent within the sex vesicle that subsequently iseliminated through anaphase lag (3). An increased incidenceof disomy XY spermatozoa theoretically also could have arisenas a result of XY bivalent pairing in the primary spermatocyte.

These findings support the hypothesis that men with a47,XYY karyotype, as well as those with its mosaic form,are at risk of producing offspring with an extra sex chromo-some. This is especially true because the origin of 47,XYYmale offspring is determined completely by the father.

Similar findings of sperm with an increased incidence ofsex chromosome disomy have been recorded in patients withKlinefelter syndrome and its mosaic forms. Guttenbach et al.(4) and Kruse et al. (5) noted increased incidences of disomyXY and XX sperm, providing strong evidence that some47,XXY cells also can undergo meiotic division.

These findings suggest that oligozoospermic patients withsex chromosome aneuploidies who are registered for anintracytoplasmic sperm injection program should first bescreened for their sperm sex chromosome constitution todetermine their particular risks.

References1. Chandley AC, Fletcher J, Robinson JA. Normal meiosis in two 47,XYY

men. Hum Genet 1976;33:231–40.2. Blanco J, Rubio C, Simon C, Egozcue J, Vidal F. Increased incidence of

disomic sperm nuclei in a 47,XYY male assessed by fluorescent in situhybridization (FISH). Hum Genet 1997;99:413–6.

3. Chevret E, Rousseaux S, Monteil M, Usson Y, Cozzi J, Pelletier R, et al.Meiotic behaviour of sex chromosomes investigated by three-colourFISH on 35,142 sperm nuclei from two 47,XYY males. Hum Genet1997;99:407–12.

4. Guttenbach M, Michelmann HW, Hinney B, Engel W, Schmid M.Segregation of sex chromosomes into sperm nuclei in a man withKlinefelter’s karyotype: a FISH analysis. Hum Genet 1997;99:474–7.

5. Kruse R, Guttenbach M, Schartmann B, Schubert R, van-der-Ven H,Schmid M, et al. Genetic counseling in a patient with XXY/XXXY/XY mosaic Klinefelter’s syndrome: estimate of sex chromo-some aberrations in sperm before intracytoplasmic sperm injection.Fertil Steril 1998;69:482–5.

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