Article : Etude de la fragmentation de l’ADN spermatique et

et de la s´egr´egation m´eiotique dans les gam`etes d’hommes

porteurs d’une mosa¨ıque gonosomique

R´esultats et dicussion

Les anomalies chromosomiques sont un des facteurs bien connus comme ´etant responsables de l’infertilit´e masculine. Les anomalies de nombre des chromosomes sexuels les plus fr´equentes chez l’homme sont XXY et XYY. Les hommes pr´esentant un caryotype XXY (syndrome de Klinefelter) ont souvent une perturbation de la spermatogen`ese qui provoque ensuite une oligozoospermie s´ev`ere ou une azoospermie (Morel et al. 2000). Au contraire, la majorit´e des hommes porteurs d’un caryotype XYY homog`ene ou en mosa¨ıque est fertile. Pourtant l’´etude de la s´egr´egation m´eiotique dans les spermatozo¨ıdes de ces hommes montre une augmentation mod´er´ee du taux des anomalies gonosomiques (Morel et al. 1999, Rives et al. 2003, 2005, Wong et al. 2008). Ainsi, ces hommes ont un risque ´elev´e de transmettre l’aneuplo¨ıdie `a leurs descendances. Les patients ayant un caryotype 45,X/46,XY peuvent ˆetre ph´enotypiquement masculin ou f´eminin. Les hommes porteurs d’une mosa¨ıque X0 sont souvent infertiles `a cause des param`etres spermatiques anormaux (Newberg et al. 1998, Alhalabi et al. 2013). Grˆace aux techniques d’AMP, ces hommes peuvent en b´en´eficier afin de devenir p`ere. L’analyse de leurs spermatozo¨ıdes permet d’´evaluer le risque de transmettre des anomalies de nombre des chromosomes sexuels `a l’embryon. Malheureusement, il n’existe dans la litt´erature que trois ´etudes men´ees sur l’analyse du taux d’aneuplo¨ıdie chez les hommes ayant la lign´ee cellulaire 45,X (Dale et al. 2002, Giltay et al. 2000, Newberg et al. 1998).

Dans notre ´etude, le taux des gam`etes aneuplo¨ıdes et le taux de la fragmentation de l’ADN spermatique ont ´et´e ´evalu´es sur trois hommes porteurs d’une mosa¨ıque gonosomique ayant la lign´ee 45,X. Le caryotype est 45,X/47,XYY ; 45,X/46,XY/47,XYY et 45,X/46,XY correspondant au patient P1, P2 et P3,

C. ETUDE DE LA FRAGMENTATION DE L’ADN SPERMATIQUE ET DE LA

S ´EGR ´EGATION M ´EIOTIQUE DANS LES GAM `ETES M ˆALES 95

respectivement. L’´equipement chromosomique a ´et´e analys´e par la technique FISH et la fragmentation de l’ADN spermatique a ´et´e ´evalu´ee par la technique TUNEL.

Parmi les spermatozo¨ıdes avec une aneuplo¨ıdie gonosomique, nous avons constat´e qu’environ 67% (P1) et 89% (P2) ´etaient disomiques XY. Ces spermatozo¨ıdes sont probablement issus des lign´ees germinales XYY. Toutefois, pour le patient P2, la non-disjonction des cellules germinales de la lign´ee XY peut ˆetre consid´er´ee. En effet, le trivalent XYY peut ˆetre la seule configuration qui pourrait ´echapper au point de contrˆole du stade pachyt`ene grˆace `a la saturation des sites d’appariement (Miklos. 1974). Les cellules qui r´eussirent `a ´echapper `a la d´egradation peuvent r´esulter en des spermatozo¨ıdes 24,XY et 24,YY (Wong et al. 2008). Pourtant, nous n’avons observ´e aucun spermatozo¨ıde 24,YY chez nos deux patients. De plus, Dale et al. ont d´etermin´e le taux d’aneuplo¨ıdie gonosomique chez un patient porteur d’un caryotype 45,X/47,XYY (15%/85%) ayant une oligoasth´enozoospermie. Ils ont trouv´e 0,09% des spermatozo¨ıdes 24,XY mais aucun spermatozo¨ıde 24,YY (Dale et al. 2002). Il est sugg´er´e que les spermatides YY n’ont pas pu passer le point de contrˆole et ont ´et´e ´elimin´es au cours de la spermatogen`ese (Milazzo et al. 2006). Cela pourrait expliquer l’oligozoospermie pr´esent´ee chez le patient 45,X/47,XYY.

Pour le patient P3 45,X/46,XY, le taux d’aneuplo¨ıdies gonosomiques et de diplo¨ıdies est plus ´elev´e par rapport au groupe t´emoin mais la diff´erence n’est pas statistiquement significative. La fr´equence des gam`etes aneuplo¨ıdes chez notre patient est plus faible par rapport `a celle montr´ee dans la litt´erature (Newberg et al. 1998, Giltay et al. 2000). Les spermatozo¨ıdes 24,XY et 24,YY peuvent d´eriver des cellules germinales 46,XY avec une non-disjonction pendant la m´eiose I (Templado et al. 2013). Cette perturbation peut ˆetre expliqu´ee par le fait que la mosa¨ıque pourrait provoquer un environnement testiculaire anormal et des alt´erations biochimiques.

Les fausses couches spontan´ees `a r´ep´etition et la diminution du taux de naissances sont li´es `a une augmentation du taux de fragmentation de l’ADN spermatique (Carell et al. 2003, Frydman et al. 2008). Dans cette ´etude, le taux de fragmentation de l’ADN spermatique chez nos trois patients est l´eg`erement plus ´elev´e en comparaison avec le groupe t´emoin mais la diff´erence n’est pas significative. En fait, un taux ´elev´e de la fragmentation de l’ADN spermatique a ´et´e identifi´e chez des hommes porteurs d’une anomalie de structure (Perrin et al. 2009) ou chez des hommes infertiles ayant un caryotype normal mais des param`etres spermatiques anormaux (Brahem et al. 2012, Tang et al. 2010).

La comparaison de nos r´esultats avec ceux de la litt´erature s’av`ere difficile `a cause du taux diff´erent de cellules anormales pour chaque patient. De plus, la proportion des lign´ees cellulaires anormales peut varier d’un tissu `a l’autre chez un mˆeme patient. Les patients P2 et P3 ont un spermogramme normal et ont eu des enfants naturellement tandis que le patient P1 a une oligoasth´enot´eratozoospermie s´ev`ere. Malheureusement nous n’avons pas pu ´etudier la mosa¨ıque dans le tissu testiculaire chez nos trois patients. En conclusion, nous avons trouv´e une proportion l´eg`erement plus ´elev´ee des aneuplo¨ıdies des chromosomes sexuels et autosomiques ainsi qu’un taux de fragmentation de l’ADN spermatique chez les trois patients porteurs d’une mosa¨ıque gonosomique. Pourtant, cette augmentation est rencontr´ee souvent chez les hommes infertiles ayant un caryotype normal. Les hommes porteurs d’une mosa¨ıque gonosomique qui n’ont pas pu ˆetre p`ere naturellement, peuvent b´en´eficier d’une prise en charge en AMP. Un diagnostic pr´enatal pourra ˆetre envisag´e pour ces couples.

96 ORIGINAL ARTICLE

A study of aneuploidy and DNA fragmentation in spermatozoa of three

men with sex chromosome mosaicism including a 45,X cell line

MINH HUONG NGUYEN 1,2 , FREDERIC MOREL 1,2,3 , LOUIS BUJAN 4,5 ,

PASCALE MAY-PANLOUP 6 , MARC DE BRAEKELEER 1,2,3 & AURORE PERRIN 1,2,3

1 Laboratoire d ’ Histologie, Embryologie et Cytog é n é tique, Facult é de M é decine et des Sciences de la Sant é , Universit é de Bretagne Occidentale, Brest, France, 2 Institut National de la Sant é et de la Recherche M é dicale (INSERM) U1078, Brest, France, 3 Service de Cytog é n é tique et Biologie de la Reproduction, H ô pital Morvan, CHRU Brest, Brest, France, 4 Research Group on Human Fertility EA3694, Universit é Toulouse III Paul Sabatier, Toulouse, France, 5 CECOS Midi-Pyr é n é es and GERMETHEQUE biobank, Groupe d ’ activit é de m é decine de la reproduction, CHU Paule de Viguier, Toulouse, France, and 6 Laboratoire de biologie de la reproduction, CHU Angers, Angers, France

Abstract

Meiotic segregation of mosaic males with a 45,X cell line has been little examined. In this study, we evaluated the risk of aneuploid gametes using l uorescence in situ hybridization (FISH) and DNA fragmentation in ejaculated spermatozoa of three men with sex chromosome mosaicism including a 45,X cell line. Triple- and dual-color FISH were performed. Sperm DNA fragmentation was detected using the TUNEL assay. A signii cantly increased frequency of XY disomic spermatozoa was observed for patients (P)1 and P2. A signii cant increase in diploidy and autosomal aneuploidy was found in P2 and P3, respectively. The rate of DNA fragmentation was not different from that observed in a control group. Data from the literature are scarce (only 3 cases reported), making comparison of the present data difi cult, especially as the frequencies of the cell lines comprising the mosaicism differed between patients. Furthermore, the proportion of the different cell lines can differ from one tissue to another in the same patient. Whether the relative levels of the several cell lines present in the mosaicism can inl uence the rate of aneuploid spermatozoa remains unknown.

Keywords: Sex chromosome mosaicism , meiotic segregation , DNA fragmentation , aneuploidy

Introduction

In industrialized countries, infertility affects about 15 – 20% of couples. The causes of infertility are variable, being of male origin in one-third of the couples, female in another third, and mixed in the remaining third. Many factors, including chromosomal abnormalities, are known to inl uence spermatogenesis and be respon-sible for male infertility. The presence of a chromosomal abnormality (structural or numerical) can interfere with normal spermatogenesis resulting in oligo and/or terato and/or asthenozoospermia, even azoospermia.

In humans, the most common chromosomal abnor-mality is aneuploidy, sex chromosome aneuploidy being more frequent than autosomal aneuploidy in neonates (Templado et al., 2013). The most frequent numerical sex chromosome anomalies in men are XXY and XYY.

Other types of numerical sex chromosome anomalies are rare. They include males with a XYY chromosomal constitution and those with a 45,X/46,XY karyotype.

The majority of mosaic and non-mosaic 47,XYY males are fertile. However, studies performed in spermatozoa using l uorescence in situ hybridization (FISH) in XYY males revealed a moderate increased rate of gonosomal abnormalities (Morel et al., 1999; Rives et al., 2003, 2005; Wong et al., 2008) and those with this condition might be at a higher risk of passing on a chromosomal aneuploidy to their offspring.

Patients with a 45,X/46,XY karyotype can be phenotypically male or female. Males with X0 mosa-icism are often infertile due to abnormal sperm param-eters (Alhalabi et al., 2013; Newberg et al., 1998). As these males could benei t from assisted reproductive Human Fertility, 2015; 18(2): 96–99

© 2014 The British Fertility Society

ISSN 1464-7273 print/ISSN 1742-8149 online DOI: 10.3109/14647273.2014.988663

Correspondence: Pr. Marc De Braekeleer, Laboratoire de Cytog é n é tique, H ô pital Morvan, B â timent 5bis, CHRU Brest, 2, avenue Foch, F-29609 Brest cedex, France. Tel: ⫹ 33 (0)2 98 22 36 94. Fax: ⫹ 33 (0)2 98 22 39 61. E-mail: marc.debraekeleer@univ-brest.fr

(Received 4 April 2014 ; revised 19 June 2014 ; accepted 3 July 2014 )

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technologies (ARTs), the analysis of spermatozoa is required to assess the risk of transmitting numerical sex chromosome abnormalities to the embryo. However, data on aneuploidy in the literature are rare and con-cern only three mosaic males with a 45,X cell line (Dale et al., 2002; Giltay et al., 2000; Newberg et al., 1998).

In this study, we evaluated the risk of aneuploid gam-etes and DNA fragmentation in ejaculated spermatozoa from three men with sex chromosome mosaicism, and in a 45,X cell line.

Materials and methods Patients and sperm characteristics

Patient P1 was referred to a medical geneticist when he was 15 years old for asymmetric lower limbs, short stature, and obesity. Cytogenetic analysis on peripheral blood lymphocytes revealed a 45,X constitution in 16% of the metaphases and a 47,XYY constitution in the remaining 84%. The same mosaicism was found in i -broblasts. At the age of 17 years, because of concern of a possible infertility, a semen analysis was performed. It revealed a severe oligoasthenoteratozoospermia with 2 million spermatozoa/mL in a volume of 0.5 mL and 15% motility. Sperm cryopreservation was done.

Patient P2, a phenotypically normal male who had previously fathered a daughter, volunteered as a semen donor. Cytogenetic analysis on peripheral blood lym-phocytes revealed three different cell lines: 45,X in 20% of the metaphases, 46,XY in 70%, and 47,XYY in the remaining 10%. Sperm count was 225 million/mL in a volume of 3.5 mL, with 45% normal motility and 24% normal morphology.

Patient P3, a phenotypically normal male who had previously fathered 3 daughters, volunteered to become a semen donor. Cytogenetic analysis on peripheral blood lymphocytes showed a 45,X/46,XY karyotype (67%/33%). Sperm analysis showed teratozoospermia with 53 million spermatozoa/mL in a volume of 6.3 mL, with 35% motility and 9% normal morphology.

Four fertile men with normal karyotype and normal sperm parameters served as control subjects for the aneu-ploidy study and twenty-four for the DNA fragmentation analysis. All participants gave their informed consent. Sperm preparation

Sperm were collected by masturbation after 3 – 7 days of sexual abstinence. Each sample was washed i rst in phosphate-buffered saline (PBS) and then centrifuged. The pellets were i xed on a slide with Carnoy ’ s solution (methanol:acetic acid; 3:1, vol:vol). Sperm nuclei were decondensed in 1 M NaOH solution and then washed in 2xsaline sodium citrate (SSC).

Probes

Triple-color FISH using alpha-satellite centromeric probes specii c for chromosomes X (DXZ1, spectrum

green, Abbott, Rungis, France), Y (LSI SRY, spectrum orange, Abbott), and 8 (CEP8, spectrum aqua, Abbott) for patient P1, or 18 (CEP18, spectrum aqua, Abbott) for patients P2 and P3 was performed. In this experi-ment, centromeric probes for chromosome 8 or 18 were used as a quality control of hybridization and to distinguish diploidy from disomies. Dual-color FISH using 13q14/21q22 (spectrum green/spectrum orange, respectively, Kreatech, Strasbourg, France) was also performed. Because of cross-hybridization between centromeres of acrocentric chromosomes, locus-specii c identii er (LSI) probes need to be used to give the rate of disomies 13 and 21.

FISH procedure

Sperm slides were immersed in a jar of 2xSSC/0.4% NP40 solution and dehydrated by passage through an ethanol series of increasing concentration (70 ° /90 ° /98 ° ). Denaturation was performed both on sperm nuclei and probes for 1 minute at 70 ° C. Hybridization was carried out overnight at 37 ° C. After incubation, the slides were washed in 0.4xSSC/0.3% NP40 for 45 seconds at 72 ° C then in 2xSSC/0.1% NP40 at room temperature for 30 s. Slides were dehydrated in ethanol series, air-dried in dark, counterstained with 4 ¢ , 6-diamino-2-phenylindole (DAPI), and observed using a Zeiss Axioplan micro-scope (Zeiss, Le Pecq, France).

Sperm DNA fragmentation analysis using TUNEL assay Sperm DNA fragmentation was detected by the ApopTag Red Kit (MP Biomedicals, Illkirch, France) according to the manufacturer ’ s recommendations. The procedure has been described elsewhere (Perrin et al., 2009). Briel y, after immersion in a jar containing a 2xSSC/0.4% NP40 solution and dehydration, the slides were covered by equilibration buffer for 30 seconds. Then a mix of reaction buffer and terminal deoxyribo-nucleotidyl transferase was added to the slides allowing DNA elongation. They were incubated in a dark moist chamber at 37 ° C for 1 h. After having stopped the en-zyme reaction, the slides were washed twice in PBS, and the DNA elongation was revealed by incubating the cells with anti-digoxigenin antibody coupled to rhod-amine for 30 min in a dark moist chamber at 37 ° C. The slides were washed twice in PBS and air-dried. They were counterstained with DAPI and observed using a Zeiss Axioplan microscope. The spermatozoa with frag-mented DNA had partial or total red-colored nuclei, and those with intact DNA were blue. A total of 500 sperm heads were analyzed for each patient and only spermatozoa with a l agellum were counted.

Statistical analysis

Chi-square tests were performed to compare the fre-quency of each hybridization pattern obtained between patients and controls. A value of p ⬍ 0.05 was consid-ered to be signii cant.

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98 M. H. Nguyen et al.

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Results

Analysis of aneuploidy

A total of 27069 and 24409 sperm nuclei were scored in triple FISH and dual FISH, respectively (Table I).

The incidences of gonosomal and autosomal aneu-ploidy for patient P1were 0.45% and 0.58%, respectively; these values were not signii cantly increased compared with those for controls. Nevertheless, a highly signii -cant number of XY disomic and diploid spermatozoa were observed (0.3% vs 0.08% and 0.74% vs 0.3%).

For patient P2, the rate of gonosomal aneuploidy was higher, although not statistically signii cantly ( p ⫽ 0.06), than in the controls. This increase was due to a high frequency of XY disomic spermatozoa ( p ⬍ 0.001). A signii cant increase in autosomal aneuploidy, due to a high rate of disomy 21 spermatozoa, was also observed. For patient P3, no signii cant difference was observed in the rate of gonosomal or autosomal aneuploidy, nor diploidy compared with controls.

Analysis of sperm DNA fragmentation

The rate of spermatozoa with fragmented DNA was 1.6%, 2.7%, and 2.6% for patients P1, P2, and P3, respectively. These proportions were not statisti-cally different from that observed in a control group (1.2% ⫾ 0.95).

Discussion

In this study, the rates of sex chromosome aneuploidy and DNA fragmentation were investigated in spermato-zoa of three men with rare gonosomal mosaicism. There are several previous reports on the rate of aneuploidy in spermatozoa from homogeneous 47,XYY or mosaic 46,XY/47,XYY males (Blanco et al., 2001; Giltay et al., 2000; Morel et al., 1999; Rives et al., 2003, 2005; Wong et al., 2008), but only three studies have analyzed the frequency of aneuploidy in mosaic X/XY or X/XYY

patients (Dale et al., 2002; Giltay et al., 2000; Newberg et al., 1998).

About 67% and 89% of spermatozoa presenting a gonosomal aneuploidy in patients P1 and P2, respec-tively, were XY disomics. These spermatozoa were prob-ably initiated in XYY germ cells, although, in patient 2, non-disjunction of XY germ cells cannot be excluded. The XYY trivalent was suggested to be the sole coni gu-ration that could escape the pachytene checkpoint, due to saturation of pairing sites (Miklos, 1974), therefore resulting in 24,XY or 24,YY sperm (Wong et al., 2008). However, no 24,YY spermatozoon was found in our two patients. Dale et al. (2002) determined the rate of sex chromosome aneuploidy in a 45,X/47,XYY (15%/85%) patient presenting with primary infertility and oligoas-thenozoospermia. Using triple FISH, they found 0.09% of sperm nuclei to be 24,XY, but none to be 24,YY.

During spermatogenesis, primary spermatocytes un-dergo meiosis to form haploid spermatids. In our studied patients, a high proportion of these cells with gonosomal aneuploidy would therefore have been expected. Our data and those from Dale et al. (2002) strongly suggest that sex chromosome aneuploidy in spermatids causes these cells to be selected for destruction (Odorisio et al., 1998). The data also suggest that YY spermatids could have been eliminated during spermiogenesis (Milazzo et al., 2006). This might explain why patients with 45,X/47,XYY presented with oligozoospermia.

Mosaic 45,X/46,XY individuals are often infertile and present with a spectrum of phenotypes ranging from almost normal male to Turner syndrome female (Newberg et al., 1998). The rates of gonosomal aneu-ploidy and dianeu-ploidy in spermatozoa of patient P3 were higher, although not statistically signii cantly, than those of the control group. Spermatozoa with a 24,XY or 24,YY constitution could have originated from non-disjunction in the 46,XY germ cell line during meiosis I (Templado et al., 2013). Meiotic segregation using triple FISH was reported in two males presenting with primary infertility and oligoasthenoteratozoospermia.

Table I. Aneuploidy levels in sperm assessed by triple and dual FISH.

Patient 1 Patient 2 Patient 3 Controls

Karyotype 45,X(16%)/47,XYY(84%) 45,X(20%)/46,XY(70%)/47,XYY(10%) 45,X(67%)/46,XY(33%) 46,XY

Three-color FISH

No. of sperm scored 1348 2204 2643 20874

Total sex aneuploidy (%) 0.45 0.46 0.42 0.24

XY disomy (%) 0.3 * 0.41 * * * 0.19 0.08

XX disomy (%) 0.15 0.05 0.15 0.09

YY disomy (%) 0 0 0.08 0.07

Two-color FISH

No. of sperm scored 1216 2124 2333 18736

Total aneuploidy 0.58 _{0.94 * * *} 0.25 0.29 Disomy 13 0.33 0.28 0.04 0.14 Disomy 21 0.25 _{0.66 * * *} 0.21 0.15 Diploidy 0.74 * * 0.53 0.42 0.34 * p ⬍ 0.05 ( versus controls). * * p ⬍ 0.01 ( versus controls). * * * p ⬍ 0.001 ( versus controls).

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Newberg et al. (1998) found signii cantly higher fre-quencies of gonosomal (1.92% vs 0.70%) and chromo-some 18 (0.89% vs 0.28%) disomy sperm from a male with a 45,X(10%)/46,XY(90%) karyotype compared with those observed from a fertile control. Giltay et al. (2000) found a slightly increased rate of gonosomal aneuploidy in a male with a 45,X(39%)/46,XY(61%) karyotype compared with those observed in three nor-mospermic donors (2.2% vs 0.8 – 1.9%).

The rate of sperm DNA fragmentation was not dif-ferent between patients with a rare sex chromosome mosaicism and the control group. In fact, an elevated proportion of gametes with fragmented DNA had pre-viously been found in males carrying a structural chro-mosomal abnormality (Perrin et al., 2009) and in infer-tile men with a normal karyotype and abnormal sperm parameters (Brahem et al., 2012; Tang et al., 2010). It should be noted that two men in the present series had already fathered naturally.

The comparison of our results with those from the literature is difi cult because the frequencies of the cell lines composing the mosaicism differed between patients. Furthermore, the proportion of the different cell lines can be different from one tissue to another in the same patient. A limitation of the study is the lack of testicular tissue analysis, which would permit the true rate of mosaicism in germinal tissue to be identii ed dei nitively but to obtain such tissue would have been ethically unacceptable in these situations. Whether the