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Absence of chromosomal instability in spermatozoa of men affected by testicular cancer

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Absence of chromosomal instability in spermatozoa of men affected by testicular cancer
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  Human Reproduction  vol.14 no.1 pp.247–251, 1999 Absence of chromosomal instability in spermatozoa of men affected by testicular cancer R.Alvarez 1,3 , L.Tusell 1 , A.Genesca` 1 , R.Miro´ 1 ,X.Garcia-del-Muro 2 and J.Egozcue 1 1 Departament de Biologia Cel.lular i Fisiologia, UniversitatAuto`noma de Barcelona and  2 Servei d’Oncologia Me`dica, HospitalDuran i Reinals, Spain 3 To whom correspondence should be addressed at: Unitat deBiologia, Departament de Biologia Cel.lular i Fisiologia, Facultatde Medicina, Universitat Auto`noma de Barcelona, E-08193Bellaterra, Spain Testicular germ cell cancer affects mainly young men. Itis the most frequent type of cancer in 20–35 year old men.Since cancer treatment using antineoplasic drugs andionizing radiation has a negative effect on the functionof the gonads, testicular cancer patients are offered theopportunity to cryopreserve their semen samples beforethe beginning of therapy. For this reason it would be of interest to know whether there is chromosome instabilityin their spermatozoa prior to any treatment. Using theinterspecific human–hamster fertilization system, we haveanalysed a total of 340 chromosome complements fromspermatozoa of control donors and 320 chromosome com-plements from testicular cancer patients. There were nosignificant differences in the frequencies of chromosomalaberrations between controls and cancer patients (9.7 and10.3% respectively;  P  0.4921). Our results indicate thatspermatozoa from untreated testicular cancer patients donotshowanincreased chromosomalinstabilityascomparedto control donors. Key words:  chromosome instability/human–hamster system/ sperm chromosomes/testicular cancer Introduction It is well documented that people affected by some kinds of cancer have an increased chromosomal instability, previous toany treatment (Hsu  et al ., 1983; Barrios  et al ., 1988, 1990,1991; Heim  et al ., 1989). In fact it has been suggested thatgenomic instability expressed as a higher baseline frequencyof chromosomal aberrations could be a driving force incarcinogenesis (Tlsty  et al ., 1995). Recent reports on chromo-somal instability in individuals with testicular germ celltumours have yielded inconclusive results. An increased incid-ence of chromosomal aberrations has been reported by vanden Berg-de Ruiter  et al . (1990), Delozier-Blanchet (1990)and Gundy  et al . (1990), while no chromosome instability hasbeen found by Vorechovsky and Zaloudik (1989), Osanto  et al .(1991) and Heimdal  et al . (1992). However, all these studieshave been carried out in somatic cells. There are no studies ©  European Society of Human Reproduction and Embryology  247about chromosomal instability in spermatozoa of patients withtesticular germ cell tumours.Testicular cancer is the most common tumour occurring inmen between the ages of 20 and 35 years. The incidence of testicular cancer varies significantly according to geographicarea and it is ~0.2% for Caucasian males (Bosl  et al ., 1997).Testicular cancer has become a curable illness with a  90% chance of survival after surgery, chemotherapy and/orradiotherapy (Kliesch  et al ., 1997). Antineoplastic drugs aswell as ionizing radiation can profoundly affect spermatogen-esis. The recovery of spermatogenesis after treatment dependson the treatment modalities, doses and individual susceptibility.Currently, it is not possible to predict accurately which of these men will regain spermatogenic function (Fossa  et al .,1983; Botchan  et al ., 1997). For this reason testicular cancerpatients are advised to cryopreserve semen samples before thebeginning of their treatment. However, if their spermatozoahad chromosomal instability, any future pregnancy achievedwith these semen samples should be accompanied by carefulprenatal care.These clinical implications make it important to ascertainwhether there is chromosomal instability in spermatozoa of men with testicular cancer. To investigate this question wehave compared the frequency of spontaneous chromosomalaberrations in spermatozoa of men affected by testicular cancer(after orchidectomy but before any further treatment) with thatfound in control donors. Materials and methods  Donors The subjects of this study were four patients aged 23–34 years withtesticular germ cell tumours treated with orchidectomy only (Table I).Semen samples from cancer patients were collected between 3 and26 months after orchidectomy. As control donors three healthy men(aged 28–45 years) with no history of exposure to mutagenic agentswere used. Cryopreservation of semen samples Sinceithasbeendemonstratedthatcryopreservationof semensamplesdoes not affect the frequencies of sperm chromosome abnormalities(Chernos and Martin, 1989; Martin  et al ., 1991), semen samples werecryopreserved as follows: each aliquot was diluted in the same volumeof cryoprotectant medium consisting of 30% egg yolk, 14% glycerol,1.98% glucose, 1.72% sodium citrate and 2% glycine in aqueoussolution. Cryoprotectant medium was added sequentially drop bydrop, with constant mixing. Each aliquot was then placed in cryotubes.Cryotubes were placed in a special container (Nalgene Cryo 1°C;Nalgene, Rochester, NY, USA) filled with isopropyl alcohol. Thecontainer with the cryotubes was placed in a –80°C freezer for at   b  y g u e  s  t   onM a  y 6  ,2  0 1 4 h  t   t   p :  /   /  h  umr  e  p . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  R.Alvarez  et al . Table I.  Clinical data from testicular cancer patientsPatient Age (years) Tumour type Time (months)since orchidectomy1 34 Classical 3seminoma2 23 Embryonal 15carcinoma3 28 Mixed germ 26cell tumour b 4 25 Classical seminoma 1 a Time between orchidectomy and collection of semen samples. b Malignant teratoma, yolk sac tumour, embryonal carcinoma and seminoma. least 4 h. After this period, the cryotubes were transferred to a liquidnitrogen tank, until use. Sperm treatment Frozen semen samples were thawed in a 37°C incubator for 20 min.Thawed semen samples were washed twice with Biggers–Whitten–Whittingham (BWW) medium (Biggers  et al ., 1971) containing 0.3%human serum albumin (HSA) to eliminate seminal plasma. Motilespermatozoa were collected by a swim-up procedure (Andolz  et al .,1987) and centrifuged (600  g , 6 min). Since ionophore treatmentdoes not affect the frequencies of sperm chromosome abnormalities(Alvarez  et al ., 1996) the acrosome reaction of spermatozoa wasinduced by exposure to A23187 ionophore. Briefly, pelleted spermato-zoa were resuspended in 10  µ M calcium ionophore A23187 solutionin BWW 0.3% HSA for 10 min at 37°C, 5% CO 2 . The spermsuspension was then centrifuged to eliminate the ionophore, andresuspended in BWW medium with 3.3% HSA, adjusting the concen-tration to 1–2  10 5 motile spermatozoa/ml. The final suspension of ionophore-treated spermatozoa was kept in the incubator for 2 hbefore insemination.  Hamster superovulation and oocyte processing Adult Syrian hamster females were induced to superovulate byinjection of gonadotrophins. Oviducts were punctured in Petri dishescontaining BWW medium with 0.3% HSA to extract the cumulusmasses, which were transferred to a 0.1% hyaluronidase solution tofree the oocytes from cumulus cells. Finally, the oocytes were treatedwith a 0.1% trypsin solution to remove the zona pellucida. Gamete coincubation and egg culture Zona-free hamster eggs and human spermatozoa treated with calciumionophore A23187 were incubated in BWW medium 3.3% HSAdroplets under paraffin oil at 37°C in 5% CO 2  for 1–2 h. Duringcoincubation of gametes checks of penetration were carried out fromtime to time by pressing 5–10 oocytes under a coverslip placed overa slide on four wax spots and observed under a phase contrastmicroscope. As soon as the frequency of swollen sperm heads withtails per oocyte was 0.5 or higher, the other oocytes were washedand placed in F10 medium droplets. After 12 h of culture at 37°C in5%CO 2 ,theeggsweretransferredtoF-10mediumdropletscontaining0.4 mg/ml Colcemid for a further 4–6 h period. Chromosomal preparations and cytogenetic analysis The eggs were fixed by the method described by Tarkowski (1966).Human sperm metaphases were analysed after sequential uniformstaining–G banding. The classification of chromosome abnormalitieswasperformedaccordingtoISCN(1995).Chromosomeandchromatidgaps were not scored as abnormalities. 248 Statistical analysis To determine whether there was chromosomal instability inspermatozoa of men affected by testicular cancer, the frequencies of spermatozoa with chromosomal abnormalities from control and cancerpatients were compared using the two-tailed Fisher’s exact test. Results A total of 340 sperm chromosome complements from threecontrol donors and 320 sperm chromosome complements fromfour untreated cancer patients was analysed, showing 9.7 and10.3% of chromosome abnormalities respectively (Table II).There were no significant differences between both frequencies( P    0.4921). Furthermore, neither numerical nor structuralabnormalities were statistically different between both groups( P    0.1777,  P    0.3742 respectively). Total frequencies of chromosomal aberrations for control donors and cancer patientsfound in our study were in the range found by other authorsin control populations [from 6.7 to 15.2%: Rudak   et al . (1978)and Mikamo  et al . (1990) respectively].The incidence of chromosomal abnormalities in testicularcancer patients varied from 2.9 to 16.1%. Although a greatinter-individual variability was found, these differences werenot statistically significant ( P  0.05, two-tailed Fisher’s exacttest). Furthermore, none of the testicular cancer patientsshowed a statistically significant difference in chromosomalabnormalities when compared to controls.Types and frequencies of structural abnormalities in controldonors as well as in cancer patients are shown in Table III.Most structural abnormalities were chromosome breaks andacentric fragments (Figure 1) in both controls and cancerpatients. There were no statistical differences in the frequenciesof the different types of structural chromosome abnormalitiesbetween both groups. Discussion We have analysed the incidence of chromosome aberrationsin human sperm-derived chromosome complements fromuntreated patients with testicular cancer. The possibility of anincreased risk of sperm chromosomal abnormalities is import-ant because some studies have demonstrated that there is noselection of spermatozoa based on chromosomal contents(Epstein and Travis, 1979; Martin, 1989).Statistical analyses indicate that the baseline values of aberrant metaphases were not different between cancer patientsand control donors (10.3 and 9.7% respectively). Similarresults have been reported by Martin  et al . (1997). Theseauthors found that the frequency of chromosomal aberrationsin testicular cancer patients previous to any treatment was10.2%, a value that was not statistically different from theircontrol donors. All these results together suggest that testicularcancer patients do not have an increased risk of transmissionof chromosomal aberrations to their offspring compared tocontrols. However, it must be noted that these results are basedon only a few testicular cancer patients, and a high inter-individual variability was found, raising the possibility that alarger sample might reveal statistically significant differences.   b  y g u e  s  t   onM a  y 6  ,2  0 1 4 h  t   t   p :  /   /  h  umr  e  p . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  Sperm chromosome analysis in testicular cancer patients Figure 1.  G-banded human sperm-derived chromosome complement from a patient affected with testicular cancer showing a structuralchromosome abnormality (chsb 9q12). Arrowheads indicate the two fragments of chromosome 9. Table II.  Spermatozoa with chromosomal abnormalities in control donors and testicular cancer patients n  Spermatozoa with Spermatozoa Total (%)numerical with structuralabnormalities a (%) abnormalities (%)ControlsC1 98 2  2 (4.1) 6 (6.1) 9 (9.2) b C2 76 1  2 (2.6) 6 (7.9) 8 (10.5)C3 166 3  2 (3.6) 10 (6.0) 16 (9.6)Total 340 6  2 (3.5) 22 (6.5) 33 (9.7)PatientsP1 94 – 11 (11.7) 11 (11.7)P2 87 2  2 (4.6) 10 (11.5) 14 (16.1)P3 34 – 1 (2.9) 1 (2.9)P4 105 – 7 (6.7) 7 (6.7)Total 320 2  2 (1.3) 29 (9.1) 33 (10.3) n  number of sperm chromosome complements analysed. a Aneuploidy was conservatively calculated as twice the frequency of hyperhaploidy. b A cell showed both a numerical and a structural abnormality. Table III.  Spontaneous structural chromosome abnormalities found in spermatozoa of control donors and testicular cancer patientsBreaks Exchanges n  chsb fis chtb Subtotal t radials other Subtotal Totalace cht-ace (%) (%) (%)del cht-delControls 340 16 1 1 18 (5.3) 1 2 2 5 (1.5) 23 (6.8)Cancer patients 320 22 1 2 25 (7.8) 1 5 2 8 (2.5) 33 (10.3) n  number of sperm chromosome complements analysed; chsb  chromosome breaks; ace  acentric fragments; del  terminal deletions; fis  fision;chtb  chromatid break; cht-ace  chromatid acentric fragment; cht-del  chromatid deletion; t  translocation; ‘other’ includes marker chromosomes andrings. Since germ cell tumours include two subtypes of seminoma,three subtypes of teratoma, embryonal carcinoma, chorio-carcinoma and yolk sack tumours (Bosl  et al ., 1997), it ispossible that chromosomal instability could vary within tumourtype, explaining the great inter-individual variability observedand indicating that a higher number of patients should beanalysed before making a conclusion.The exact aetiology of testicular cancer is not yet completelyunderstood. In some cases germ cell tumours are believedto arise from meiotic spermatocytes which have undergonecrossing-over and in which DNA repair is unsuccesful (Bosl et al ., 1997). These spermatocytes should undergo apoptosis.However, the excess in copy number and expression of the24912p gene and additional mutational genetic events involvingcell cycle control prevent apoptosis. These transformed cellsare XY, have a 2C or a higher chromosome number, have thepotential for widespread gene loss through genomic instability,and an increased 12p copy number and overexpression of agene with oncogenic potential (Bosl  et al ., 1997).Due to the nature and srcin of testicular cancer, the possiblerisk of transmissible genetic changes that cannot be measuredusing classical cytogenetic techniques, or of genetically deter-mined disease in the offspring, cannot be ignored whenusing assisted fertilization techniques with fresh or frozenspermatozoa from these patients. However, the reported dataof in-vitro fertilization centres and data obtained from offspring   b  y g u e  s  t   onM a  y 6  ,2  0 1 4 h  t   t   p :  /   /  h  umr  e  p . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om  R.Alvarez  et al . of oncological patients (including testicular cancer patients)indicate no increased risk of malformations (Dodds  et al .,1993; Meschede  et al ., 1995). Nevertheless, the offspring of testicular cancer patients should be included in the follow-upof these patients, because the increased risk of developing atesticular cancer in their sons should be taken into consideration(Kliesh  et al ., 1997).Previous studies carried out in our laboratory (Genesca` et al ., 1990) showed that the incidence of numerical andstructural chromosome abnormalities, in spermatozoa frompatients treated for testicular cancer with antineoplastic drugs2–7 years after cancer treatment, was significantly greaterthan in controls. Furthermore, the incidence of chromosomalaberrations was also significantly greater compared to anotherseriesofmentreatedwithantitumouralagentsfornon-testiculardiseases a long time before the sperm analysis (Genesca` et al ., 1990). Therefore, this increased level of chromosomalabnormalities in sperm of testicular cancer patients, comparedto those treated for other kinds of cancer, must be due tothe effects of antineoplastic drugs, and not to an increasedchromosomal instability in testicular cancer patients. All theseresults taken together strongly recommend that cancer patientshave their semen cryopreserved, when possible, prior to theinitiation of chemo- or radiotherapy.Studies of chromosomal instability in somatic cells (lympho-cytes and fibroblasts) from testicular cancer patients havealso yielded contradictory results. An increased incidence of chromosomal aberrations has been reported by van denBerg-de Ruiter  et al . (1990), Delozier-Blanchet (1990) andGundy  et al . (1990). On the other hand, no chromosomeinstability has been reported by Vorechovsky and Zaloudik (1989), Osanto  et al . (1991) and Heimdal  et al . (1992). Allthese reports are characterized by the presence of a great inter-individual variability in the levels of chromosomal abnormalit-ies in testicular cancer patients. As pointed by Heimdal  et al .(1992), due to the high inter-individual variability, it is probablethat studies of a limited number of patients and controls shouldgive conflicting results.Tzancheva and Konitowski (1997) showed, in a preliminarystudy of a group of 15 untreated cancer patients, that althoughthe rate of chromosomal aberrations was similar in both cancerand control donors, the lymphocytes of cancer patients displayan increased susceptibility to treatment with bleomycin andcaffeine, suggesting a latent chromosomal instability. Theoccurrence of multiple primary neoplasms in patients withtesticular germ cell cancer treated with radiotherapy supportsthe existence of a latent chromosomal instability (Dieckmann et al ., 1994).Thus, the failure of the present and other studies performedin human lymphocytes to demonstrate increased chromosomebreakage in patients with testicular cancer, does not rule outa role for chromosome instability in the susceptibility to thisdisease, because chromosome instability could exist in alatent form.In summary, as yet, an uneventful pregnancy and a normalfuture development of the new-born cannot be guaranteed.In such cases, providing patients with current knowledgeconcerning the issue of testicular malignancy and paternity,250and assisting them in taking appropriate decisions is, therefore,highly recommended. Once a pregnancy has been achieved itis mandatory to provide close prenatal care and postnatalfollow-up. Acknowledgements This work has been performed with the financial support from theConsejo de Seguridad Nuclear. We also acknowledge the financialsupport given by CIRIT to the Cell Biology Unit (CRQ 93–2025)and (1995 SGR-0478). References Alvarez, R., Tusell, L., Genesca`, A.  et al . (1996) Chromosome aberrations inhuman spermatozoa treated with Ca 2  ionophore A23187.  Mol .  Hum .  Reprod  .,  2 , 481–484.Andolz, P., Bielsa, M.A., Genesca`, A.  et al . (1987) Improvement of spermquality in abnormal samples using a modified swim-up procedure.  Hum .  Reprod  .,  2 , 99–101.Barrios, L., Caballı´n, M.R., Miro´, R.  et al . (1988) Chromosome abnormalitiesin peripheral blood lymphocytes from untreated Hodgkin’s patients.  Hum . Genet  .,  78 , 320–324.Barrios, L., Miro´, R., Caballı´n, M.R.  et al . (1990) Chromosome instability inBlader carcinoma patients.  Cancer Genet  .  Cytogenet  .,  49 , 107–111.Barrios, L., Caballı´n, M.R., Miro´, R.  et al . 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