ARCHIVES OF ANDROLOGY 43:27–36 (1999) 27Copyright ã 1999 Taylor & Francis0148-5016/99 $12.00 + .00

SEX CHROMOSOME ANEUPLOIDIESIN SPERM OF 47,XYY MEN

F. MORELC. ROUXJ. L. BRESSON

Service de Cytogénétique–Immunocytologie–Biologie du Développementet de la Reproduction, Centre d’Etude et de Conservation des Oeufset du Sperme Humains Besançon Franche-Comté, Centre HospitalierUniversitaire Saint Jacques, et Institut d’Etude de Transfert des Gènes,Faculté de Médecine, Besançon, France

The sex chromosomal equipment in 26,675 sperm of 47,XYY males was analyzed. A total of 5.78% ofthe nuclei exhibited sex chromosome hyperhaploidy. Six studies have analyzed the sperm of 10 XYYpatients and, although these studies indicated some degree of elimination of the extra Y chromosomeduring spermatogenesis, a certain percentage of XYY germinal cells may also be able to achievemeiosis and produce sperm with gonosomal disomies. All these studies show an increased incidence ofgonosomal aneuploidies in sperm, but there are significant discrepancies concerning the extent of theseabnormalities. The global frequencies of sperm with an abnormal number of sex chromosomes rangedfrom 0.578 to 13.91%, depending on the patients. There are several explanations for these discrepan-cies: differences attributed to fluorescence in situ hybridization methodology, the use of dual or multicolorFISH, recruitment, interindividual variations, and intraindividual variations. This study reports an addi-tional series obtained from another XYY individual and compares and discusses the data on gonosomalhyperhaploidies in sperm of 47 XYY males using in situ hybridization analyses.

Keywords gonosomal aneuploidies, human sperm, in situ hybridization, XYY male

The 47,XYY sex chromosomal constitution is one of the most frequent gonosomal aneu-ploidies in the human species, comprising approximately 0.1% of all newborn males [13]. Overthe last 25 years, contradictory opinions have been reported on the behavior and the distributionof both Y chromosomes during meiosis and whether there is premeiotic elimination of the extraY chromosome by selective nondisjunction [1, 4, 14, 15, 24, 26]. Initially, these opinions werebased on results of meiotic cytogenetic studies [4, 15], on Y-specific stainings of testicular

We thank M. C. Clavequin and the Centre d’Etude et de Conservation des Oeufs et du Sperme humains, BesançonFranche-Comté. We are grateful to Professor Cooke of Western General Hospital of Edinburgh (United Kingdom) andProfessor Willard of Case Western Reserve University of Ohio (USA) for their pHY2.1 and pXBR probes. We thankP. Albert for proofreading the manuscript. This research was supported by the Association Régionale pour le Développementdes Etudes Biologiques en Génétique et Reproduction Humaines.

Address correspondence to Frédéric Morel, ESA CNRS 6025, Faculté de Médecine, Place Saint Jacques, 25030Besançon Cedex, France.

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28 F. Morel et al.

materials: histologic sections and dissociations [14, 24]. Some opinions were based on theanalysis of sperm complements by karyotyping using interspecific fertilization [1] and, recently,by analyzing synaptonemal complexes [26].

The opinions differ regarding the capacity of a 47,XYY germinal cell to complete a spermato-genetic cycle. Using a quinacrine fluorescent technique, Hultén and Pearson [14] found that 45%of primary spermatocytes contained two Y chromosomes associated as a YY bivalent, and anincreased frequency of YY bearing sperm as compared to control males. Luciani et al. [15], aftermeiotic studies in an XYY male, observed spermatocytes with X + Y + Y diakinetic univalentassociations. Chandley et al. [4] reported that an examination of chromosomes at meiosis, ontesticular biopsy with Q and C staining, showed apparently normal meiosis in two 47,XYY men.Benet and Martin [1] karyotyped 75 sperm from the ejaculated semen of a 47,XYY male andfound a normal incidence of X and Y bearing sperm. They observed no increase in the percent-age of sperm with sex chromosome abnormalities. Solari and Rey Valzacchi [26] studied anXYY male using synaptonemal complex analysis of microspread spermatocytes and by quan-tifying germ cells in semithin sections. The authors showed the prevalence of a YY synaptone-mal complex over XY synapsis in an XYY man with exclusive XYY spermatocytes.

In situ hybridization makes it possible to directly obtain the constitution in sex chromo-somes of ejaculate sperm and therefore sheds new light on this question. Over the last 4 years,6 studies have been published on the segregation of gonosomes during the spermatogenesisof XYY males by analyzing the gonosomal constitution of ejaculate spermatozoa using in situhybridization [3, 5, 12, 16, 17, 20].

This article reports the results of an additional series obtained from another XYY individualand compares and discusses the data on gonosomal hyperhaploidies in spermatozoa of 47,XYYmales available up to now from in situ hybridization analyses.

MATERIALS AND METHODS

This study concerns one sample of frozen semen of 47,XYY male sperm provided by theSpermothèque de la Fédération Française de Centre d’Etude et de Conservation des Oeufs etdu Sperme humains (CECOS) with values for conventional semen parameters falling withinnormal ranges (Table 1). The man had already had children and was volunteering to become asperm donor, but the cytogenetic analysis of blood peripheric lymphocytes showed a 47,XYYkaryotype.

Preparation and decondensation of sperm nuclei, preparation of the molecular DNA probes,fluorescence in situ hybridization, criteria for hybridized nuclei selection, and analysis were

Table 1. Materials and methods used in this study

Autosomal Scored PatientSperm Chromosome dual FISH Decondensation spz (XY referred

Age parameters Discovery (probes) (probes) treatment dual FISH) to as No.

40 Normala Sperm X-Y 8–18 DTeT,b 26,675 11donor (pXBR-pHY2.1) (pJM128-L1.84) papain

aAccording to WHO standards [28].bDTeT, Dithioerythritol.

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Sex Chromosome Aneuploidies in Sperm 29

performed as described by Mercier et al. [20]. After thawing, sample was washed and 250 µLof sperm suspension in phosphate-buffered saline at a concentration of 25 × 106 spermatozoa/mL was dropped onto a slide and fixed in Carnoy solution (methanol:acetic acid, 3:1). Nucleardecondensation was performed in a solution of 0.2 M Tris HCl, pH 8.6, containing 1.25%papain, 0.16% dithioerythritol, and 0.5% dimethylsulfoxide. The slides were incubated over-night with a mixture of digoxigenin-labeled Yq heterochromatin specific probe (pHY2.1) [6]and biotinylated X specific a satellite probe (pXBR) [27].

After posthybridization washings, the hybrids were detected by aminomethylcoumarin ace-tic acid (AMCA)-labeled avidin D (Vector Laboratories, Biosys, Compiègne, France), followedby a mixture of biotinylated anti-avidin D and fluorescein isothiocyanate (FITC)-labeled anti-digoxigenin (Boehringer Mannheim, Meylan, France) and finally AMCA-avidin D. The slideswere mounted using a mixture of glycerol, antifade reagent (Vector Laboratories) and propidiumiodide (PI; Sigma Aldrich, Saint Quentin Fallavier, France). Two-color FISH with the probesL1.84 and pJM128, both provided by the American Type Culture Collection (ATCC, Rockville,MD), was performed on one slide of the 47,XYY male semen sample to identify the frequencyof diploid cells. The slides were analyzed using a Zeiss Axiophot fluorescent microscope and26,675 sperm nuclei were counted using strict selection criteria (Table 1).

RESULTS

A total of 26,675 sperm nuclei were analyzed by dual FISH. Hybridization efficiency was95.15%. The mean was 41.58% for spermatozoa exhibiting one X chromosome and 47.79%for spermatozoa exhibiting one Y chromosome. There was an excess of Y-bearing cells with aratio X:Y of 0.86:1. In this sample, total frequency of cells with numerical errors for sexchromosome was 5.78% (Table 2). The frequency of disomic sperm was 1% for the X chromo-some and 1.64% for the Y chromosome. The proportion of XY bearing sperm was 3.01%.Other patterns of chromosomal aneuploidies had 0.12% XYY nuclei and 0.01% XXY nuclei.

Relating to the evaluation of diploid cell number using 8 and 18 chromosome probes, thehybridization onto one sperm smear of the 47,XYY male sperm sample showed that 95.25% ofnuclei exhibited a signal of hybridization. Among the 10,081 scored nuclei, 15 nuclei (0.15%)showed four spots, which corresponded to two 8 signals and two 18 signals; 26 nuclei (0.26%)showed two 8 spots and one 18 spot, and 20 nuclei (0.20%) exhibited one 8 spot and two 18spots. The frequency of double disomic nuclei for both autosomes could be estimated at 0.00052%(0.26 × 0.20%). Thus the frequency of diploid nuclei were identical to the frequency of nucleiwith two 8 and two 18 signals (0.15%).

Table 2. Presumed karyotype of gametes in the 47,XYY patient studied

TotalPresumed karyotype (%) gonosomal

abnormalities Patient23,X 23,Y 24,XY 24,XX 24,YY 47,XYY 47,XXY 0a (%) No.

41.58 47.79 3.01 1 1.64 0.12 0.01 4.85 5.78 11

aNonfluorescent: Nonhybridization or nullisomy.

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DISCUSSION

The comparison of this study and the others shows that although the hypothesis concerningthe nearly complete elimination of the extra Y chromosome during spermatogenesis seems tobe established, there are significant discrepancies about the estimations of the global frequen-cies of cells exhibiting an abnormal number of sex chromosomes.

Materials and Methods Concerning Gametes in 47,XYY Men

Tables 1 and 3 summarize the clinical, biological, and methodological data concerning the11 hybridohistochemical analyses of the gonosomal equipment of ejaculate spermatozoa from47,XYY men. These men, 27–42 years of age, were selected for different indications: fertilepatients and voluntary sperm donors, patients consulting for infertility, and infertile patientsreferred for intracytoplasmic spermatozoa injection (ICSI). In all cases, standard cytogeneticanalysis of lymphocyte chromosomes revealed a 47,XYY homogeneous karyotype (patients 1–5, 8–11); otherwise, the data was not documented (patients 6, 7). The semen characteristicswere assessed, according to WHO standards [28] as normal for patients 4, 6, 7, 11, moderatelyaltered for patients 1, 2, 5, and very perturbed for patients 3, 8, 9, 10 (Tables 1 and 3).

In 7 patients, the study was carried out using at least dual gonosomal hybridization (one Xand one Y probe). In these 7 cases, a standard dual hybridization procedure either with afluorescent revelation (patients 1, 4, 8, 9, 11) or an enzymatic revelation with a diaminobenzidineor a peroxidase-tetramethylbenzidine reaction (patients 2, 3) was performed. In the four othercases, the authors used triple FISH (one X, one Y, and one autosomal probe) with a fluorescentrevelation (patients 5, 6, 7, 10). In all cases, sperm nuclei were partially decondensed, butusing different solutions and different decondensation times (Table 3).

In all the studies, cells were scored by applying strict selection criteria, and certain spermnuclei were not included in the scoring: those with overlapping sperm heads, nuclei that werenonswollen or over swollen [20], or overlapped spermatozoa or sperm heads without a well-defined boundary were not evaluated [3]. All sperm nuclei with split or superimposed signalswere classified as ambiguous [5]. The number of analyzed sperm for each sample was verydifferent (from 410 sperm for patient 9 to 100,000 for patient 4). In the case of severe oligo-zoospermia or oligoasthenoteratozoospermia, only a limited number of sperm nuclei could beanalyzed (patients 5, 8, 9).

The estimation of global frequencies of sperm with an abnormal number of sex chromo-somes ranged from 0.578% (patient 6) to 13.91% (patient 4), depending on the patients, withthe disomy frequencies evaluated for XY (0.24 to 8.91%), XX (0 to 2.7%) and YY (0.08 to4.43%) (Table 4).

The results of Han et al. [12] concerning the different types of disomic, diploid, hyperdip-loid, and tetraploid cells were 1, 3.35, 0.05, and 0.25%, respectively, but the ploidy levels wereestimated by measuring the size of each cell nucleus against a microscope eyepiece graticule:cells that were considered disomic had a nucleus of normal size (diameter or length 6–8 µm)with two distinct spots, while those containing two spots in a large nucleus (diameter or length9–20 µm ) were considered diploid. The few cells with three distinct spots and a large nucleus

30 F. Morel et al.

Results Concerning Chromosomes of Gametes in 47,XYY Men

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Tab

le 3

.R

ecap

itul

atio

n of

dif

fere

nt m

ater

ials

and

met

hods

con

cern

ing

the

stud

ies

of g

amet

es i

n 47

,XY

Y m

en

Aut

osom

alP

atie

ntC

hrom

osom

edu

al F

ISH

Dec

onde

nsat

ion

Sco

red

num

ber

Ref

eren

ces

Age

Spe

rm p

aram

eter

sD

isco

very

(pro

bes)

(pro

bes)

trea

tmen

tsp

z

1H

an e

t al

. [1

2]42

Hyp

ospe

rmia

and

Con

sult

atio

n X

(T

RX

)E

DT

A,a

2,00

6sl

ight

ter

atos

perm

iafo

r in

fert

ility

Y (

HR

Y)

DT

Ta

2M

arti

ni e

t al

. [1

6]29

Oli

goas

then

ospe

rmia

ICS

I ap

poin

tmen

t X

(pB

amX

5)D

TT

3,30

0 Y

(D

YZ

1)3

33S

ever

e ol

igos

perm

iaIC

SI

appo

intm

ent

3,50

0

4M

erci

er e

t al

. [2

0]37

Nor

mal

bS

perm

don

or X

(pX

BR

)

8–1

8D

TeT

,a10

0,00

0 Y

(pH

Y2.

1)(p

JM12

8–L

1.84

)pa

pain

5B

lanc

o et

al.

[3]

?O

ligo

asth

enos

perm

iaC

onsu

ltat

ion

X (

Vys

is)

DT

T1,

974

for

infe

rtil

ity Y

(V

ysis

) 1

8 (V

ysis

)

6C

hevr

et e

t al

. [5

]32

Nor

mal

bS

perm

don

or X

(pX

BR

2)D

TT

24,3

15 Y

(pH

Y2.

1)

737

Nor

mal

bV

asec

tom

y 1

(pU

C1.

77)

10,8

27

8M

enni

cke

et a

l. [1

7]33

Oli

goas

then

oter

ato-

Infe

rtili

ty X

, pY

3.4c

DT

T,

LIS

a1,

000

sper

mia

934

X,

pY3.

441

0

1027

X,

pY3.

4, 1

89,

099

a ED

TA

, et

hyle

ne d

iam

ine

tetr

aace

tic

acid

; D

TT

, di

thio

thre

itol

; D

TeT

, di

thio

eryt

hrit

ol;

LIS

, li

thiu

m s

alt.

b Acc

ordi

ng t

o W

HO

sta

ndar

ds [

28].

c Cen

trom

etri

c al

pha

sate

llit

e pr

obes

wer

e pu

rcha

sed

from

Onc

or.

31

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32

Tab

le 4

.R

ecap

itul

atio

n of

dif

fere

nt r

esul

ts c

once

rnin

g th

e ch

rom

osom

al e

quip

men

t an

d th

e pr

esum

ed k

aryo

type

of

gam

etes

in

47,X

YY

men

(hom

ogen

izat

ion

of r

esul

ts)

Pre

sum

ed k

aryo

type

(%

)

Tot

algo

noso

mal

Pat

ient

23,

23,

24,

24,

24,

25,

46,

46,

46,

47,

47,

92,

92,

24X

/Yab

norm

alit

ies

No.

Ref

eren

ceX

YX

YX

XY

YX

YY

XY

XX

YY

XY

YX

XY

XX

YY

XY

YY

+ a

uto

0a(%

)

1H

an e

t46

.444

.60.

250.

30.

40.

052.

70.

30.

350.

050.

20.

054.

354.

65al

. [1

2]b

2M

arti

ni e

t43

.537

.52.

32

0.8

0.8

0.1

8.2

6al

. [1

6]c

345

.435

5.4

2.7

2.3

0.6

0.1

3.8

11.1

4M

erci

er e

t35

.57

45.6

98.

910.

324.

430.

050.

24.

8213

.91

al.

[20]

5B

lanc

o et

50.3

45.3

80.

30.

151.

010.

3d0.

22.

361.

76al

. [3

]

6C

hevr

et e

t43

.11

55.2

0.24

0.02

0.08

0.17

0.02

0.04

0.00

80.

240.

88e

0.57

8al

. [5

]7

42.7

656

.21

0.52

0.19

0.11

0.02

0.18

0.84

8M

enni

cke

50.5

47.5

0.8

0.7

0.5

2et

al.

[17]

951

.745

.11

1.2

13.

2

1052

.15

40.7

63.

110.

311.

020.

050.

020.

010.

142.

4f

4.52

a Non

fluo

resc

ent:

Non

hybr

idiz

atio

n or

nul

liso

my.

b Est

imat

ion

of p

loid

y ra

te a

ccor

ding

to

nucl

ear

diam

eter

.c C

lass

ific

atio

n ac

cord

ing

to c

rite

ria

of M

erci

er e

t al

. [2

0] f

or h

omog

eniz

atio

n of

res

ults

.d P

erce

ntag

e of

dip

loid

y.e A

mbi

guou

s.f A

mbi

guou

s (0

.38%

) an

d 22

,–X

/Y (

2.02

).

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(9–20 µm) were presumed to be hyperdiploid, and cells with four distinct spots and a verylarge nuclear diameter were scored as tetraploid. Both our research and that of Martini et al.[16] reported a high incidence of aneuploidy: 6% (patient 2), 11.1% (patient 3), 13.91% (pa-tient 4), and 5.78% (patient 11) with a majority of XY disomies (Tables 2 and 4). Mennicke etal. [17] evaluated this incidence at 2 and 3.2% in dual FISH (patients 8, 9) and at 4.52%(patient 10) in triple FISH with probes to chromosomes 18, X and Y. Likewise, Blanco et al.[3] and Chevret et al. [5] used a triple probe FISH procedure with probes to chromosomes 18,X and Y (patient 5) or probes to chromosomes 1, X and Y (patients 6, 7). The gonosomaldisomy levels were estimated at only 1.46% for patient 5 by Blanco et al. [3] and 0.34 and0.71% for patients 6 and 7 by Chevret et al. [5]. Blanco et al. [3] calculated the diploidspermatogenetic cells (spermatogonia or early spermatocytes) to be 0.3%, and Chevret et al.[5] found 0.23 and 0.13%. For patient 6, two tetraploid sperm cells (0.008%) were also ob-served by Chevret et al. [5].

The ratio of X-bearing to Y-bearing sperm was not significantly different from the expected1:1 [3, 12, 16]. Mennicke et al. [17] found that the sex ratio seemed to show a higher propor-tion of 23,X sperm. On the other hand, both Chevret et al. [5] and our team observed an excessof Y-bearing sperm with an X:Y ratio of 0.78:1 (patients 4, 6), 0.76:1 (patient 7), and 0.87:1(patient 11).

The results of these six research teams showed an increased incidence of gonosomal abnor-malities in the sperm of 47,XYY men in comparison to those estimated in a reference popula-tions [7, 11, 18, 22]. It would appear that the chromosomal risk might be greater for theoffspring of XYY men than for those in the general population. Nevertheless, in 47,XYY men,the number of sperm nuclei with a gonosomal abnormality remains small, which correspondsto the well-known hypothesis that premeiotic or meiotic repair exists [1, 3, 4, 12, 20, 24]. Thisrepair process eliminates most of the extra Y chromosomes or cells with an extra Y chromo-some. Estop et al. [9] studied the meiotic segregation of 24 sperm obtained from a 47,XXYmale, and their results support the hypothesis that XXY cells can complete meiosis but that amale with a 47,XXY karyotype with no evidence of mosaicism may produce sperm withnormal sex chromosomes. The collected data here show significant discrepancies concerningthe percentage of XYY germ cells capable of achieving meiosis and producing sperm withabnormal gonosomal equipment (from 0.578 to 13.91% of aneuploid spermatozoa).

Following are several explanations for these different views.

Methodological Differences in Using Interphasicin Situ Hybridization and/or Interpreting the Results

This hypothesis is rather unlikely and would probably not cause such large variations; andcurrent estimations of disomy frequencies in control populations are approximately the same[7, 11, 19, 21].

Using Dual or Multi-FISH

If we assume that, in these patients with a 47,XYY homogeneous karyotype, the achievedspermatogenesis is due to germinal cells remaining XYY, then dual FISH with X and Y probesprovides an adequate and direct distinction between a disomic spermatozoa and an XYY dip-loid cell of the germinal line. On the other hand, if we assume that spermatogenesis in an XYYmale is particularly due to disomic cells (or those that became disomic), then XY dual FISH

Sex Chromosome Aneuploidies in Sperm 33Sy

st B

iol R

epro

d M

ed D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y U

nive

rsita

ets-

und

Lan

desb

iblio

thek

Due

ssel

dorf

on

12/1

7/13

For

pers

onal

use

onl

y.

34 F. Morel et al.

does not allow us to directly distinguish nuclei with one signal each for X and Y like XYdisomic sperm (24,XY) from a diploid cell (46,XY). Nevertheless, recent studies [2, 8, 25]have shown that there is no significant difference in diploid rate estimations, on the sameejaculate, whether using dual FISH with two autosomal probes (patients 4 and 11) or tripleFISH (X probe + Y probe + autosomal probe). Thus, using dual or multi-FISH would notexplain the observed differences either.

Interindividual Variations

This possibility corresponds to the various differences of opinion and results whatever thetechnique, described in the literature. Gabriel-Robez et al. [10], clearly explained this phenom-enon in studying the synaptic behavior of sex chromosomes in two XYY men. In one patient,spermatocytes had lost the extra Y chromosome before the meiotic prophase and it is generallythought that the extra Y is lost during spermatogenesis. This male with a normal sperm countcould be fertile. In the second patient, the second Y was not lost and a variety of synapticconfigurations at early pachytene were found: XY bivalent, XYY trivalent, XY bivalent with aY univalent, X univalent with YY synapsis; this man had severe oligoasthenoteratozoospermia.Similarly, Solari and Rey Valzacchi [26] studied an infertile XYY man, using a synaptonemalcomplex analysis of microspread spermatocytes and by quantifying germ cells in semithinsections. All spermatocytes micrographed had an XYY constitution, and the biopsy presenteda homogeneous arrest of spermatogenesis at the spermatocyte/young spermatid stages. Interindividualvariations for all usual sperm parameters in the fertile male have already been demonstrated.

Intraindividual Variations

This hypothesis, which would consider fluctuations in the spermatogenetic process devel-opment of the XYY patient, has not yet been documented. Yet, intraindividual variations con-cerning other sperm parameters are known [23].

CONCLUSION

It seems likely that the extra Y chromosome could be found in a mature gamete populationmore or less large in some XYY individuals than in others. Depending on the reliability of thespermogram, the assessment of the individual risk of gonosomal abnormalities for the off-spring could be very different.

REFERENCES

1. Benet J, Martin RH (1988): Sperm chromosome complements in a 47,XYY man. Hum Genet 78:313–315.

2. Bernardini L, Martini E, Geraedts JPM, Hopman AHN, Lanteri S, Conte N, Capitanio GL (1997):Comparison of gonosomal aneuploidy in spermatozoa of normal fertile men and those with severemale factor detected by in situ hybridization. Mol Hum Reprod 3:431–438.

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