Human sperm chromosomes

Human Sperm Chromosomes Long-Term Effect of Cancer Treatment Anna Genesch, M. Rosa Caballin, Rosa Miro, Jordi Benet, Xavier Bonfill, and Josep Egozcue

ABSTRACT: The long-term cytogenetic effect of radio- or chemotherapy or both on male germ cells was evaluated by s t u d y of the chromosomal abnormalities in spermatozoa of four m e n treated for cancer 5-18 years earlier. The cytogenetic analysis of 422 s p e r m metaphases showed no differences in the aneuploidy rate. The incidence of structural chromosome aberrations was 14.0%, however, which is much higher than in controls. Thus, the high incidence of structurally aberrant spermatozoa observed in o u r long-term study indicates that antitumoral treatments

affect stem-cell spermatogonia and that aberrant cells can survive germinal selection and p r o d u c e abnormal spermatozoa.

INTRODUCTION Damage to the germinal epithelium and testis dysfunction are side effects of many cancer radio- and chemotherapies. Recovery of sperm production in treated men depends basically on total doses, on the developmental stage of the germ cells affected, on the individual response, and on the kind of cytotoxic drug used in chemotherapy [1]. So far, the cytogenetic effects of ionizing radiation and chemical drugs on germ cells have been evaluated through study of germ cell precursors or by extrapolation of data obtained from somatic cells. Genetic damage expressed as an increased frequency of reciprocal translocations has been observed in meiotic ceils from men treated with ionizing radiation [2] and mammals treated with chemical drugs [3]. However, until a few years ago, it was not possible to determine whether chromosome abnormalities induced in germ cell precursors were also present in spermatozoa (and thus susceptible of being transmitted to the offspring} or were completely eliminated during spermatogenesis. Introduction of the interspecific hamster-human fertilization technique [4] allowed direct visualization of human sperm chromosomes. With this technique, Martin et al. [5] observed an increased frequency of chromosomal abnormalities in spermatozoa from men analyzed shortly after radiotherapy. Whether those abnormalities decrease with time is not known, however. We present our results concerning the long-term cytogenetic effect of cancer therapies on the germ cells of four men treated for different types of cancer. The results of the analysis of one of the patients {patient 4) were published earlier [6]. From the Departament de Biologia Cel.lular i Fisiologia (A. G., R. M., J. B,, J. E.), the Departament de Biologia Animal, Biologia Vegetal i Ecologia. Universitat Aut6noma de Barcelona (R. C.), Servei d'Epidemiologia i Informaci6 Cliniques, Hospital de Sabadell, (X. B.).

Address reprint requests to: A. Genesca, M.D., Departament de Biologia Cel.lular i Fisiologia (Faeultat de Medicina), Universitat Aut6noma de Barcelona, E-08193 Bellaterra, Spain.

251 © 1990 Elsevier Scienc~ Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogene! 46:251-260 (1990) 0165-4608/90/$03.50

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A. Genesch et al.

MATERIALS AND METHODS Semen samples were obtained from four men treated with radio- or c h e m o t h e r a p y or both 5 - 1 8 years earlier for different types of cancer. Clinical data are shown in Table 1. H u m a n s p e r m chromosomes were obtained using the method described by Martin [7] and slightly modified by us. Motile h u m a n sperm were preincubated to allow capacitation for 5 - 6 hours in a modified Krebs-Ringer's solution, BWW m e d i u m [8] with 3.3% h u m a n serum albumin. Motile spermatozoa were obtained by a swim-up p r o c e d u r e [9]. After preincubation, h u m a n sperm were coincubated for 3 hours with zona-free Syrian hamster oocytes in BWW m e d i u m to allow interspecific fertilization. The oocytes were then washed free of sperm and cultured overnight in HAM's F-10 s u p p l e m e n t e d with bovine serum. After 12 hours the oocytes were transferred to F10 m e d i u m containing 0.4/zg/ml Colcemid for 4 to 7 hours. Finally, the oocytes were fixed by the method described by Tarkowski [10]. In addition, half of each semen sample was p r e i n c u b a t e d in TES-Tris yolk buffer for 2 days [11, 12] for performance of a second experiment with each sample.

Cytogenetic Analysis Cytogenetic analysis was performed after G-banding of chromosome c o m p l e m e n t s [13] as follows: H y p o h a p l o i d chromosome complements were verified by microscopic analysis of the region surrounding the egg. Nevertheless, since h y p o h a p l o i d complements could result from technical artifacts, we considered the a n e u p l o i d y rate as twice the h y p e r h a p l o i d y frequency. Structural aberrations were classified according to the International System of Human Cytogenetic Nomenclature [14]. We considered a deletion with its fragment present a chromosome break. Cells with both structural and numerical aberrations were i n c l u d e d in both groups.

RESULTS The mean penetration of hamster eggs in treated i n d i v i d u a l s was 48.9% whereas in control series it was 66.8% [p = 3.411 × 10-4). Our laboratory control series for penetration capacity is based on 13 individuals. All of them had been able to penetrate h u m a n eggs in an in vitro fertilization (IVF) program. Table 2 summarizes the frequency of chromosome aberrations found in the 422 spermatozoa analyzed from the four patients. Our control series for sperm chromosomes is based on 505 spermatozoa from three i n d i v i d u a l s analyzed in our laboratory [15]. All of t h e m were fertile. The percentage of spermatozoa with structural aberrations in treated i n d i v i d u a l s ranged from 12.4 to 14.9% (mean, 14%), which is statistically different from the percentage of structurally abnormal spermatozoa found in the control series (6.9%; p ~ 0.00060). The incidence of a n e u p l o i d y in sperm was taken as twice the percentage of h y p e r h a p l o i d y and ranged from 0 to 5.5%. There was no difference between the mean incidence of a n e u p l o i d y in treated i n d i v i d u a l s (3.3%) and in the control series (4.0%) (p - 0.7315). The frequency of structurally abnormal and aneuploid spermatozoa did not vary significantly among i n d i v i d u a l s (p = 0.9622 for structural abnormalities and p - 0.1494 for aneuploidy). The abnormal sperm c o m p l e m e n t s observed in treated i n d i v i d u a l s are shown in Table 3. Of the abnormal complements, 80.8% had structural aberrations and 19.2% were a n e u p l o i d (three had both kinds of abnormalities). Among structural aberrations,

Rhabdomyosarcoma Ewing sarcoma Wilms' tumor Wilms' tumor

1 2 3 4

13 × CYVADIC 12 x VAC RT: 2,200 rad RT: 4,000 rad 9 x D Act (2.3 mg)

Treatment 18 25 12 3

Age at treatment 23 30 23 21

Age at analysis

137 376

29 30 20 58

Eggs analyzed

48.9 66.8

100 40.0 25.0 36.2

Eggs penetrated (%)

Hamster egg penetration

Abbreviations: RT, radiotherapy; CYVADIC,cyclophosphamide plus Adriamycin plus vincristine plus DTIC; VAC, vincristine plus Adriamycin plus cyclophosphamide: D Act, u-actinomyein.

Mean Control

Diagnosis

C l i n i c a l d a t a a n d h a m s t e r egg p e n e t r a t i o n f r e q u e n c i e s

Case

Table 1

1 2 3 4 Total Control

21(14.6) 10(14.9) 17(13.9) 11(12.4) 59(14.0) 35(6.9)

Cells w i t h structural a b n o r m a l i t y , n(%)

results

9(6.3) 8(11.9) 7(5.7) 5(5.6) 29(6,8) 46(9.2)

H y p o h a p l o i d cells, n(%) 4(2.8) 1(1.5) 2(1.6) --7(1.7) 10(2.0)

H y p e r h a p l o i d ceils n(%)

5.5 3.0 3,3 -3.3 4.0

Aneuploid ceils (%}

19,4 17.9 15.6 12.4 16.6 10.9

Abnormal cells (%)~

*'Tolal percentage of abnormal t:eIls is not exactly the addilion of percentage of sperm with structural ~bnormalilies plus percentage of aneup|oid sperm because cc'tls with both kinds of abnormalities were included in both partial categories but were taken into accom~I only once in the ('olumn showing t)crc:entag(; af abnormal sperm,

144 67 122 89 422 505

Case

Sperm chromosome

Ceils a n a l y z e d (n)

Table 2

ba

Abnormal

Case 2 24,Y, + 1

Case 1 24,Y, + 22, + ace

Case 2

--

Case 3 23,XY,del(9)(p22),qr(6;11/ (q25.3;q24) 24,Y,+4

Case 3

Case 3 23,X,del(1)(q11) 23,X,del(2)(q33), + ace 22,Y,r(11),dic(4;20)(q35:p13), dic(14;18)(p12;q21), csb(2)(pl 3),fis(2) (pl 1ql 1),csb(6)(q23), + 2ace 23,X,5q 22,Y.dic{7;9)(q34;p24) 23,Y,8q+ 23,X,del(8){p21 ) 23,X,del(8)(q22) 23,Y,del(18)(q21 ), + ac:e 23,Y,+ 3ace 23,Y, + 2ac:e 23,Y, + 2ace 23,Y, + ace 23,X,ctb{1)(p34) 21.Y,--21, 22.dic(1;7)(p32;lH3) csb(5)(p11), + ace

in treated individuals

23,Y,del(3)(q23).del(6](q22.3), + ace 23,X,del(4)(ql 1 ),del{16)(p12) 23,X,inv(7){p15p22) 23,Y,del(8)(q22.3) 23,Y, + ace 23,Y,tr(2;13){q23;q14),del{13) (q34) 23,X,ctb(7)(p211,csb(14}[q22) 23.Y,ctb{3)(p14),csb(9)(q22), + ace 2 1 , X , - 6, 16,+ ace 20,Y, 7, 8 , - 1 2 , - 1 3 , d e 1 ( 6 ) (q13),del(13)(q12), 14q + , + mar, + ace

complements

H y p e r h a p l o i d plus structurally abnormal c o m p l e m e n t s

Case 1 25,X, + 7,+ 17 24,X,+ 16 24,Y, + 20

Hyperhaploid complements

23,X,del(1 )(p33), + ace 24,Y,fis(2)(pl l q l l ) 22,Y,qr(2;10)(ql 3;q22) 22,X,t(5;14)(p15.1 ;q31.2J 23,Y,inv(6)(p21.1 p23), inv(21)(ql 1.2q22.3) 23,Y,inv(6)(p25q22) 23,Y,csb(6)(q16.2) 22,Y,t(8;9)(q22;q31) 22,X,qr(9;12)(p13;ql 3) 22,Y, - 13, + f(13)(q22q34) 23,X,inv(16)(pllql 2] 23,X,del(17)(q21 ), + ace 23,Y,inv(18](q11.2q21.3) 24,Y,fis(19)(pllq11) 23,X,del(20)(pl 1) 23,Y,inv(21)(qll.2q22.3) 23,X,csb(6)(p 1 l q l 1) 22,X,tr(1 ;7)(ql 2;p22) 22,X, - 4,del(16)(p121 22,Y, 9 , + 2 a c e

Case 1

Case 2

sperm chromosome

Structurally abnormal c o m p l e m e n t s

Table 3

Case 4

Case 4

+ ace

--

Case 4 23,Y,del{1){q11) 24,X,fis(1)(p11q11) 24.Y,fis(1)(p1 l q l l ) 24,Y,fis(1 )(pl l q l 1) 25,Y,fis(1)(pl lq11) fis(9)(pllq11) 23,Y,del(5)(q131 23,Y,del(9)(p21), + ace 23.Y,qr(1 ;9)(p36;q22) 23,Y,qr{9;19) 23,Y, + ace 23,Y,tr(3;D),csb(20)(q11.2),

ba ¢71

256

A. Genesca et al.

21

f 9

°i

Figure 1

C h r o m o s o m e c o m p l e m e n t of a s p e r m a t o z o o n earring an u n i d e n t i f i e d acentric fragm e n t . T h e k a r y o t y p e is 23,Y, + ace.

deletions and acentric fragments (Fig. 1) were the most frequent, although chromosome breaks, translocations, inversions, marker chromosomes, fissions, tri- and quadriradials, rings, and dicentric chromosomes were also found in spermatozoa from men treated for cancer. A few cells had multiple chromosome aberrations (Fig. 2). DISCUSSION

Sperm p r o d u c t i o n is i m p a i r e d by ionizing radiation therapies [16, 17]. However, the fertility of an i n d i v i d u a l d e p e n d s not only on the absolute number of spermatozoa but also on the proportion of spermatozoa able to fertilize [18]. Martin et al. [19] observed that both sperm concentration and functional capabilities (assessed by the ability of s p e r m to penetrate hamster eggs) remain poor 2 to 3 years after radiotherapy. In our series, one patient (not reported in this article) treated with radio- and chemotherapy 2 years earlier had a penetration capacity of hamster oocytes of almost 0% 2 years after treatment and one patient (unpublished observations) had a penetration score of 0% 8 years after treatment. Our results reflect a long-term impaired functional capability of spermatozoa frmn men treated with physical and chemical mutagens. Cytogenetic studies in spermatozoa of men exposed to radio- or c h e m o t h e r a p y or both are still scarce [5, 6, 20, 2~, 22] (Table 4). In general, all these studies show a clear increase in structurally abnormal sperm complements as compared with their own control series. In all of them, the frequency of structurally abnormal spermatozoa was also higher than the frequency of a n e u p l o i d sperm. The incidence of a n e u p l o i d y in treated individuals, when considered as twice the frequency of h y p e r h a p l o i d y , is only statistically different from their control series in the studies of Martin et al. [5] and Brandriff et al. [201 {p = 0.0078 and p = 0.00005, respectively). The results obtained so far are still too scarce to allow valid conclusions regarding the effect of cancer treatments on induction of a n e u p l o i d y in spermatozoa. The results of our long-term study show that many years after treatment there is

1 2 4

[20] [21] Present study

Rhabdonlyosarconla, Ewing sarcoma. Wihns' tumor

Seminoma

lymphoma, teratoma, medulloblastoma, Hodgkin

S e nl i n o m a ,

Tumor

RT RT/CT RT/CT

RT

Treatment

69 41 422

149

3 too/3 yr

1 yr 1 - 2 yr 5 - 1 8 vr

No. of s p e r m analyzed

Time after treatmeut

Abbreviations: RT. radio|herapy: CT. chemotherapy. Results reclassified wilh the incidence of aneuploidy considered as twice the rate of hyperhaploidy.

9

[ 51

No. of men

Sperm chromosomes in cancer-treated individuals.

Reference

Table 4

14.0

17.1

23.0

5.6 2.4 3.3

6.7

(o/o)~'

Uo) 7.4

S p e r m with numerical abnormalities

S p e r m with structural abnormalities

",,1

258

A. Genesc~ et al.

O Figure 2 Chromosome complement and karyotype of an spermatozoon with multiple aberrations. The karyotype is 22,Y, - 21, - 22, dic(1;7)(p32;p13),csb(5)(p11), + ace.

still a clear increase in the frequency of chromosome abnormalities in spermatozoa from m e n treated for cancer as compared with control series. This high incidence of aberrant spermatozoa in m e n exposed to antitumoral therapies indicates that m a n y of the abnormalities were i n d u c e d by the treatment in stem-cell spermatogonia. With regard to genetic risk, the damage i n d u c e d in stem-cell spermatogonia will give rise to production of abnormal germ cells during the i n d i v i d u a l ' s entire reproductive lifespan, because stem-cell spermatogonia constitute a self-renewing cell population. Our results do not agree with the conclusion of Adler [23] who considered that chromosome aberrations i n d u c e d by chemical mutagens on spermatogonia do not survive to selection. Some data indicate the existence of a strong selection against

S p e r m C h r o m o s o m e s i n M e n T r e a t e d for C a n c e r

259

a b e r r a n t cells i n d u c e d b y p h y s i c a l a n d c h e m i c a l m u t a g e n s b e t w e e n t h e stages of s p e r m a t o g o n i a a n d s p e r m a t o c y t e [24, 25] a n d p o s s i b l y also f r o m t h e s p e r m a t o c y t e stage o n [26]. O u r r e s u l t s i n d i c a t e , h o w e v e r t h a t m a n y of t h e a b e r r a n t cells m a y survive germinal selection and produce abnormal spermatozoa.

The authors thank Cristina Preciado supported by a grant from CIRIT, Generalitat de Catalunya, for expert technical assistance. This research was supported by Project No. 1476/82 from CAICYT, Ministerio de Educacion y Ciencia.

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seminoma patient treated with radiation. In: Book of Abstracts of the International Conference on Reproduction and Human Cancer, Bethesda, p. 100. 21. Jenderny J, Rohrborn G (1987): Chromosome analysis of h u m a n sperm. I. First results with a modified method. Hum Genet 76:385-388. 22. Genesc~ A, Mir6 R, Caballin MR, Benet J, Bonfill X, Egozcue J. (1987]: Sperm chromosome studies in three treated cancer patients. Book of Abstracts of the International Conference on Reproduction and Human Cancer, Bethesda, p. 99. 23. Adler ID (1982): Mouse spermatogonia and spermatocyte sensitivity to chemical mutagens. Cytogenet Cell Genet 33:95-100. 24. Rathenberg R, Schwegler H, Miska W (1976): Comparative investigations on cytogenetic effects of x-irradiation on the germinal epithelium of male mice and chinese hamster. Hum Genet 34:171. 25. Brewen JG, Preston RJ (1978): Analysis of chromosome aberrations in mammalian germ cells. In: Chemical Mutagens, Principles and Methods for Their Detection, Vol. 5, A Hollaender, FJ de Serres, eds, Plenum Press, New York, p. 127. 26. Ford CE, Searle AG, Evans EP, West BJ (1969): Differential transmission of translocations induced in spermatogonia of mice by irradiation. Cytogenetics 8:447-470.