Multicolor Fluorescence In Situ HybridizationAnalysis of Meiotic Chromosome Segregation in a47,XYY Male and a Review of the Literature

Qinghua Shi and Renee H. Martin*Department of Medical Genetics, Faculty of Medicine, University of Calgary, and Genetics Department, AlbertaChildren’s Hospital, Calgary, Canada

The frequencies of aneuploid and diploidsperm were determined in a 47,XYY male us-ing multi-color fluorescence in situ hybrid-ization (FISH) analysis, and compared withthose from 10 control donors. A total of30,078 sperm from the patient was scored,15,044 by two-color FISH for chromosomes13 and 21, and 15,034 by three-color FISHfor the sex chromosomes using chromosome1 as an internal autosomal control for dip-loidy and lack of hybridization. The fre-quencies of X-bearing (49.73%) and Y-bearing sperm (49.46%) in control maleswere not significantly different from the ex-pected 50% (x2-test for goodness of fit). Theratio of 24,X (50.60%) to 24,Y sperm (48.35%)in the patient, however, was significantlydifferent from the controls (P = 0.0144, x2-test for independence) and from the ex-pected 1:1 ratio (P = 0.0055, x2-test for good-ness of fit). There was no significantincrease in the frequency of diploid spermwhen compared with the controls (x2-testfor independence). Significantly increasedfrequencies were found for 24,YY (0.07% vs.0.02%, P = 0.0009) and 24,XY (0.44% vs. 0.29%,P = 0.0025), but not for 24,XX (0.05% vs. 0.05%,P > 0.05), 24,+13 (0.07% vs. 0.07%, P > 0.05) or24,+21 sperm (0.21% vs. 0.18%, P > 0.05) in the47,XYY male when compared with controldonors (x2-test for independence). Our re-sults support the theory that loss of the ex-tra Y chromosome occurs during spermato-genesis in most cells. In this XYY patientthere was a significant increase in the fre-quency of sperm with sex chromosomal ab-

normalities but no suggestion of an inter-chromosomal effect on autosomes. All3-color FISH studies in the literature dem-onstrate a significantly increased risk ofgonosomal aneuploidy in XYY males, withthe risk being on the order of 1%. Am. J.Med. Genet. 93:40–46, 2000.© 2000 Wiley-Liss, Inc.

KEY WORDS: 47,XYY; aneuploidy; fluores-cence in situ hybridization;spermatozoa

INTRODUCTIONSince the first report of a 47,XYY man [Hauschka et

al., 1962], numerous studies on the chromosome con-stitution of germ cells and testicular histology of thesepatients have been made, using conventional cytoge-netic methods (Table I), histological approaches[Tettenborn et al., 1970; Skakkebæk et al., 1970,1973a,b; Baghdassarian et al., 1975; Faed et al., 1976;Muller et al., 1995], and recently fluorescence in situhybridization (FISH) techniques (Table II). The majorfindings of these studies are as follows: 1) persistenceof the extra Y chromosome in germ cells can impairtesticular tubules and result in low sperm counts in47,XYY males; 2) the extra Y chromosome is lost frommost germ cells of 47,XYY men; 3) some XYY cells cansurvive meiosis and result in gonosomally aneuploidsperm; and 4) frequencies of germ cells with gonosomalabnormality, assessed by conventional cytogeneticmethods, FISH, or electron microscopically synaptone-mal complex observation [Speed et al., 1991; Gabriel-Robez et al., 1996; Solari and Rey Valzacchi, 1997; Ber-thelsen et al., 1981], were very different among studiesan between patients studied by the same group [Chev-ret et al., 1997; Martini et al., 1996]. Some questionsremain unanswered: a) is there a significant differencein the frequency of disomic sperm between 47,XYYmales with normal sperm counts and those with lowersperm counts, as has been observed in normal men;and b) does the persistence of the extra Y chromosomedisturb disjunction of other chromosomes during meio-sis? To address these questions, studies on more

Grant sponsor: Medical Research Council of Canada; Grantnumber: MA 7961.

*Correspondence to: Renee H. Martin, Ph.D., Department ofMedical Genetics, Alberta Children’s Hospital, 1820 RichmondRoad SW, Calgary, Alberta T2T 5C7, Canada. E-mail:rhmartin@ucalgary.ca

Received 20 October 1999; Accepted 25 February 2000

American Journal of Medical Genetics 93:40–46 (2000)

© 2000 Wiley-Liss, Inc.

TA

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Meiotic Chromosome Segregation in XYY Males 41

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42 Shi and Martin

47,XYY men are required. In this communication, theincidence of diploid sperm and disomic sperm for chro-mosomes X, Y, 13 and 21 was investigated in a 47,XYYman, using multi-color FISH and compared with thosefrom other 47,XYY men reported in the literature.

MATERIAL AND METHODS

An investigation into the karyotype of a 26-year-oldpatient was initiated when his wife (46,XX) had onespontaneous abortion and gave birth to two childrenwith ambiguous genitalia who died at ages of 30 to 50days. Chromosomes of the two deceased children werenot analyzed. Analysis of 100 metaphase lymphocytesshowed all to have a karyotype of 47,XYY. At the age of28 years, after fathering a healthy 10-month-old girl,his karyotype was confirmed by analyzing another 100metaphase lymphocytes and his semen was collectedfor this study. Seminal analysis of two ejaculatesshowed oligospermia, with a mean volume of 2.5 ml, amean sperm density of 5 × 105/ml and 55% motility.Both semen samples contained approximately 30%white blood cells.

Non-smoking, non alcohol-drinking men with nofamily history of genetic disease and no known expo-

sure to radiation or mutagens were selected as controldonors. Their chromosome complements had neverbeen examined before. After liquifaction, 5–10 ml of se-men from control donors were smeared as a thin layeronto ethanol-cleaned microscopic slides and air-driedfor about 24 hr at room temperature. Samples from thepatient were centrifuged at 1,000 g for 5 min. The pel-lets were resuspended in 100 ml supernatant and 8 mlof the concentrated semen was used per slide. Slideswere stored at −20°C until used for FISH analysis (twoto five months).

Two multi-color hybridizations were performed onthe patient. Three-color FISH analysis with DNAprobes specific to chromosomes X (FITC-labeled a-sat-ellite probe), Y (SpectrumOrange Yq probe, Vysis,Downers Grove, IL) and 1 (AMCA-labeled satellite IIIprobe) was carried out as previously described byKinakin et al. [1997]. Details of two-color FISH analy-sis with commercially-available DNA probes specific tochromosome 13 and 21 (13q14 and 21q22.13–q22.2, Vy-sis) were described in McInnes et al. [1998]. Only slideswith sperm decondensed to 1.5–2.5 times the size ofundecondensed sperm were scored. The hybridizationefficiency was at least 99.4% for all the slides scored.

Using a 2 × 2 x2-test for independence, results from

TABLE II. Frequencies of Haploid, Disomic and Diploid Sperm as Determined by FISH and Sperm Counts in the Semen of47,XYY Patients and the Concurrent Controls From the Literature

Patient no.Control(number) Reference

No. ofspermscored

% of sperm with different chromosome constitution Spermcounts

(×106/ml)

Sex ratio(X-bearing/Y-bearing

sperm)23,X 23,Y 24,XX 24,YY 24,XY 46,XX 46,YY 46,XY Others (frequency)

Patient–1 Hans et al., 2006 46.4 44.6 0.3 0.4 0.25 0.3 0.35 2.7e 24, +17 (0.49) – 150–350 1:1Control (12) 1994 12636 47.7 46.8 0.28 0.21 0.21 0.18 0.17 0.62 24, +17 (0.33) – Normal 1:1Patient–2 Martini et 1933† 43.5 37.5 2.0e 0.8e 2.3e – – – – – 2 >1 (P 4 0.003)Patient–3 al., 1996 2334† 45.4 35.0 2.7e 2.3e 5.4e – – – – – 3.6 >1 (P 4 0.000)Control (2) 5806† 50.1 48.6 0.05 0.1 0.4 – – – – – Normal 1:1Patient–4 Mercier et 95179 37.37 48.00 0.34e 4.65e 9.37e – – – 24, +8 (0.18) Diploid Normal 0.78:1

al., 1996 24, +18 (0.17) (0.11)Control (?) 30122 49.94 49.81 0.04 0.05 0.16 – – – – Diploid No data 1:1

(0.07)Control (1) 48399 50.16 49.77 0.004 0.01 0.05 – – – – – Normal 1:1Patient–5 Mennicke 1000 50.5 47.5 0.7 0.5 0.8 0 0 0 0 – 10 1:1Patient–6 et al., 410 51.7 45.1 1.2 1.0 1.0 0 0 0 0 – 12 1:1Patient–7 1997* 9099 52.15 40.76 0.31 1.02 3.11 0.02 0.01 0.05 24, +18 (0.14) – 16.2 >1 (P < 0.000)Patient–8 Chevret 24315 43.11 55.20 0.02 0.08 0.24 0.02 0.04e 0.17e 24, +1 (0.23) – 110 <1 (P < 0.001)Patient–9 et al., 10827 42.76 56.21 0 0.19b 0.52b 0 0.02 0.11 24, +1 (0.18) – 75 <1 (P < 0.001)Control (5) 1997 142050 50.10 48.22 0.04 0.01 0.36 0.06 0.005 0.09 24, +1 (0.27) – 50–70 1:1Patient–10 Blanco et 1926 50.30 45.38 0.15 0.01d 0.30a – – – 24, +18 (0.20) Diploid 3 >1 (P 4 0.025)

(0.30)Control (5) al., 1997 51368 49.98 48.70 0.10 0.16 0.11 – – – 24, +18 (0.10) Diploid Normal 1:1 for 4 and >1

(0.24) for a male(P < 0.0001)

Patient–11 Akrai et 59 41.7 56.9 0 0 0 0 0 0 – – Oligo- 1:1al., 1997 spermia

Control (1) 748 54.0 46.0 0 0 0 0 0 0 – – Normal >1 (P 4 0.028)Patient–12 Martin et 10002 49.4 49.8 0.08 0.03 0.55f – – – 24, +13 (0.40)f 24, +21 42.5 1:1

(0.43)Control (18) al., 1999 181556 49.79 49.51 0.05 0.06 0.30 – – – 24, +13 (0.13) 24, +21 Normal 1:1 for 17 and

(0.37) >1 for a man(P < 0.05)

Patient–13 Morel et 26675 41.58 47.79 1.00 1.64 3.01 24, +8 (0.26) Diploid Normal 0.86:1al., 1999* 24, +18 (0.20) (0.15)

aP < 0.05.bP < 0.01.cP < 0.001.dP < 0.0001.eP < 0.00001 (compared with the concurrent controls, x2-test).fP < 0.001 (compared with the concurrent controls, two-tailed Z-test).*No data from control donors were reported.†No information available.

Meiotic Chromosome Segregation in XYY Males 43

the 47,XYY patient were compared with the data onmore than 200,000 sperm from 10 control donors withnormal sperm parameters according to WHO stan-dards [World Health Organization, 1992], who were21–37 (mean 29.6) years old.

RESULTS

Summaries of the results obtained from the 47,XYYpatient are shown in Tables III and IV. In total, 30,078sperm were scored, with 15,044 by two-color FISH forchromosome 13 and 21 and 15,034 by three-color FISHfor the sex chromosomes using chromosome 1 as aninternal autosomal control for diploidy/disomy and lackof hybridization. The ratio of X-bearing (50.60%) to Y-bearing sperm (48.35%) was significantly differentfrom the expected 1:1 ratio (P 4 0.0055, x2-test forgoodness of fit). In addition, the patient’s sex ratio alsodiffered significantly from that of control donors (P 40.0144, x2-test for independence), where X-bearing(49.73%) and Y-bearing sperm (49.46%) were not sig-nificantly different from the expected 50% value (49681vs. 49413, P > 0.05, x2-test for goodness of fit). Com-parisons of the incidence of disomic and diploid spermbetween the patient and the controls were performedusing a 2 × 2 x2-test for independence. Only frequenciesof 24,YY and 24,XY sperm in the patient were signifi-cantly higher than those in the controls (Tables III andIV).

DISCUSSION

The sex ratio in our 47,XYY patient was significantlydifferent from the controls and the expected 50%, withan elevated level of X-bearing sperm. This is not thecase for the patient previously reported by this labora-tory, where the frequencies of X-bearing and Y-bearingsperm were not significantly different from 50%, eitherby sperm karyotype analysis [Benet and Martin, 1984]or using a similar three-color FISH technique [Martinet al., 1999]. Of 12 other 47,XYY patients studied byFISH, four had less 23,X sperm than 23,Y sperm[Chevret et al., 1997; Mercier et al., 1996] and eightwere stated to have an equal frequency of X- and Y-bearing sperm (Table II)[ Han et al., 1994; Martini etal., 1996; Ariki et al., 1997; Blanco et al., 1997]. Statis-tical analysis of the data reported (Table II) using a 2 ×2 x2-test, however, indicated that four patients had sig-nificantly more 23,X than 23,Y sperm, rather than anequal frequency of X- and Y-bearing sperm as stated bythe reports [Martini et al., 1996; Blanco et al., 1997]. Asex ratio of 1:1 has been consistently found in more

than 53 concurrent controls in this and previous re-ports, with only three exceptions showing more 24,Xthan 24,Y sperm (Table II). Out of fourteen 47,XYYmales studied by FISH so far, the five men with moreX-bearing sperm than Y-bearing sperm, including ourpatient, were consistently oligospermic; the six menwho had an equal or less frequency of 24,X sperm than24,Y sperm had normal sperm counts; the remainingthree were oligospermic but had a sex ratio of 1:1(Table II). Therefore, it is reasonable to suggest thatthe deviation of sex ratio from 1:1 in sperm in some47,XYY males is not due to chance. Distribution of thesecond Y chromosome during meiosis as well as cellline selection based on chromosome constitution mayaccount for the distortion of sex ratio. Chevret et al.[1997] suggested that preferential YY pairing with sub-sequent loss of the X univalent may lead to an excess of24,Y sperm. Observations on synaptonemal complexesin 47,XYY males have revealed that the majority ofspermatocytes showed a Y-Y bivalent plus a univalentX [Speed et al., 1991; Solari and Valzacchi, 1997]. Thereason for having more 24,X than 24,Y sperm in47,XYY men, however, remains puzzling. As men-tioned above, in FISH studies to date, all 47,XYY menwho had an excess of 24,X sperm were oligospermic.Numerous meiotic and histological studies of oligosper-mic 47,XYY patients have indicated that in the absenceof other causes (e.g., undescended testis, etc.), two Ychromosomes persist through spermatogonia, meioticprophase and metaphase, with a variable degree of de-generation of the germ cells through spermatogoniauntil as late as the spermatid stage (Table I). Also insemen of these men, a relatively large number of im-mature germ cells (IGC) was observed. No evidence ofmore degenerative Y-bearing IGC than X-bearing IGC,however, was found after FISH analysis [Han et al.,1994; Martini et al., 1996; Blanco et al., 1997]. There-fore, to answer the question as to why oligospermic47,XYY men had an excess of 24,X sperm, more meioticand sperm chromosome constitution studies areneeded.

Several studies on sex chromosomal aneuploidy insperm of 47,XYY men have been published (Table II).No increase in the frequency of any category of sexchromosomal aneuploidy was found in 47,XYY patientsby Han et al. [1994] or Araki et al. [1997], but these twogroups studied only 2006 and 59 sperm, respectively.In contrast, Martini et al. [1996], Mercier et al. [1996],and Morel et al. [1999] reported that between 5.65%and 15% of sperm had an extra sex chromosome (TableII). It should be noted, however, that two-color FISH

TABLE III. Frequencies (%) of Disomic Sperm

Chromosome47,XYY

male

Controls

P-value*Mean ± SD (range)

13 0.07 0.07 ± 0.03 (0.02–0.12) 0.875921 0.21 0.18 ± 0.05 (0.09–0.27) 0.4598XX 0.05 0.05 ± 0.03 (0.01–0.09) 0.9211YY 0.07 0.02 ± 0.02 (0.01–0.06) 0.0009XY 0.44 0.29 ± 0.09 (0.13–0.49) 0.0025

*P-value was calculated using a 2 × 2 x2-test for independence.

TABLE IV. Frequencies (%) of Diploid Sperm

Hybridization47,XYY

male

Controls

P-value*Mean ± SD (range)

Probes X, Y and 1X-X-1-1 0.06 0.05 ± 0.04 (0.00–0.13) 0.4654Y-Y-1-1 0.07 0.04 ± 0.04 (0.01–0.13) 0.1275X-Y-1-1 0.20 0.23 ± 0.12 (0.04–0.45) 0.4561Total 0.33 0.32 ± 0.19 (0.07–0.70) 0.8288

Probes 13 and 2113-13-21-21 0.39 0.38 ± 0.19 (0.22–0.73) 0.8195

*P-value was calculated using a 2 × 2 x2-test for independence.

44 Shi and Martin

was used in these studies, that does not distinguishdiploid sperm from disomic sperm, although Mercier etal. [1996] and Morel et al. [1999] attempted to deter-mine the diploidy frequency in general by using twoautosome probes in sperm from the same ejaculate.Mennicke et al. [1997] demonstrated frequencies ofgonosomal disomy in three patients, ranging from0.31–1.2% for 24,XX, 0.5–1.02% for 24,YY and 0.8–3.11% for 24,XY, but this group also used a two-colorFISH procedure and did not present data from the con-current controls nor the statistical results between pa-tients and controls. In our patient, elevated frequencieswere found for 24,YY and 24,XY sperm, but not 24,XXsperm (Table III). Similar results have been reportedby several other studies using three-color FISH (TableII). This means that XYY cells are able to progressthrough meiosis and produce 24,YY or 24,XY sperm[Hulten, 1970; Hulten and Pearson, 1971]. The birth of47,XYY children to 47,XYY males also supports thishypothesis [Boucharlat and Jalbert, 1969; Sundquistand Hellstrom, 1969]. Most children of 47,XYY males,however, are normal and only 1% or less of 24,YY and24,XY sperm were found in 47,XYY men after three-color FISH (Table II and present study). This is consis-tent with the results from direct observations on mei-otic chromosomes in 47,XYY males (Table I). Theseresults indicate that the extra Y chromosome is lostduring meiosis in most XYY cells because the majorityof sperm are chromosomally normal. Whether the extraY chromosome was actively eliminated from XYY cells[Thompson et al., 1967] or was randomly lost from aprimitive germ cell or spermatogonium followed by se-lective proliferation of the resulting XY cell line [Evanset al., 1970] remains unknown. To address these ques-tions, more studies, particularly longitudinal studieson meiosis in 47,XYY men are needed.

Comparisons of this study to others shows that thereare significant variations in the estimations of the fre-quencies of sperm exhibiting an abnormal number ofsex chromosomes in both controls and 47,XYY males(Tables II and III). Both methodological and inter-individual variations may explain these differences.Higher incidences of disomy are apparently present in2-colour FISH studies than in 3-colour FISH, due to theinability of 2-colour FISH to distinguish between diso-mic and diploid cells [Han et al., 1994; Martini et al.,1996; Mercier et al. 1996; Blanco et al., 1997; Chevretet al., 1997; Martin et al., 1999] (Table II). Han et al.[1994] and Mercier et al. [1996] used EDTA (ethylenediamine tetraacetic acid)/DTT (dithiothreitol) andDTeT (dithioerythreitol) for decondensation, respec-tively; while other groups used either DTT only [Blancoet al., 1994; Martini et al., 1996; Chevret et al., 1997] orDTT/LIS (lithium-3,5-diiodosalicylic acid)[this study:Mennicke et al., 1997; Martin et al., 1999; Morel et al.,1999]. To detect hybridized DNA probes, Martini et al.[1996] applied avidin-peroxidase/diaminobenzidineand viewed with a bright-field microscope, whereas allother groups used immunofluorescence or fluorescenceand viewed with a fluorescence microscope. Large in-ter-individual variations were found within studies:the frequency of gonosomal disomy in control donorsperm ranged from 0.22–0.63% in this study (data not

shown), 0.18–0.51% in the study by Blanco et al. [1994]and 0.116–0.90% in the study by Chevret et al. [1997].An approximately two-fold difference in the disomy fre-quency between 47,XYY patients in the same study hasbeen observed [Martini et al., 1996; Chevret et al.,1997; Mennicke et al., 1997]. Meiotic analysis and syn-aptonemal complex observations have also shown dif-ferences among patients in the proportions of sper-matogonia and spermatocytes at pachytene ormetaphase I containing two Y chromosomes (Table I).

It has been documented by meiotic studies that per-sistence of the extra Y chromosome in germ cells canimpair testicular tubules and result in low spermcounts in 47,XYY males (Table I). Naturally, the ques-tion arises as to whether 47,XYY men with low spermcounts have more gonosomally disomic sperm. Unfor-tunately, the data available in the literature did notprovide a clue to this question (Table II). It is possiblethat there is either no relationship between spermcounts and the proportion of gonosomally aneuploidsperm or that this relationship has been obscured bythe differences among studies caused by the factorsdiscussed above.

The frequency of diploid sperm was not significantlydifferent in the 47,XYY patient, when compared to themean of 10 control donors (Table IV). Total frequenciesof diploid sperm in our 47,XYY man were 0.37% afterthree-color FISH with probes for chromosome X, Y and1, and 0.38% after two-color FISH with probes for chro-mosome 13 and 21 (Table IV). A similar frequency oftotal diploid sperm (0.30%) was reported in a 47,XYYmale by Blanco et al. [1997] using three-color FISH,with no statistically significant difference from the con-current controls. In two other reports, frequencies ofdiploid sperm in 47,XYY patients (0.11–0.15%), as-sessed using two-color FISH with probes for chromo-some 8 and 18, were lower, but not significantly differ-ent from those of the concurrent controls (0.07%)[Mercier et al., 1996; Morel et al., 1999].

Four children with autosomal aneuploidy whose fa-ther had a 47,XYY karyotype have been reported so farand all of them had trisomy 21 [Hauschka et al., 1962;Stoll et al., 1979; Grass et al., 1984]. In the first twocases, the parental origin of the extra chromosome 21was not determined [Hauschka et al., 1962; Stoll et al.,1979]. In the other two, an examination of chromosomepolymorphisms indicated that the supernumerarychromosome 21 came from the mothers [Grass et al.,1984; Stoll et al., 1979]. Our two-color FISH analysis ofsperm from two 47,XYY patients [Martin et al., 1999;the present communication](Table III), do not show asignificant increase in the frequency of disomy 21. Withthe single exception of a patient who had an elevatedfrequency of disomy 13 [Martin et al., 1999], no signifi-cant increase was seen in the frequencies of disomicsperm for all six autosomes assessed in nine 47,XYYmen, as compared to concurrent controls (Table II). Be-cause chromosome 13 was studied in two patients (38and 28 years old, respectively) in this laboratory usingthe same scoring criteria and similar experimental con-ditions, the significant elevation of disomy 13 in the 38years old patient cannot be explained by experimental

Meiotic Chromosome Segregation in XYY Males 45

differences. Increased age, also, is an unlikely explana-tion because an increased frequency of disomic spermhas been found only for sex chromosomes [Griffin et al.,1995; Martin et al., 1995; Robbins et al., 1995], andonly for YY sperm in this laboratory [Kinakin et al.,1997], leaving inter-individual variability as the mostlikely source of the discrepancy. Based on our presentstudy and the literature, we can conclude that if aninter-chromosomal effect exists during meiosis in47,XYY males, it might affect only specific chromo-somes in a minority of men.

ACKNOWLEDGMENTS

We are grateful to Drs. Juzhen Luo, Guixiang Zhou,Jingsheng Wu and Huaiping Zhu for collecting semensamples. Special thanks to Evelyn Ko for her experttechnical assistance and to Tina Hoang for proofread-ing the manuscript. This research was supported by agrant from the Medical Research Council of Canada toRHM.

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46 Shi and Martin