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

R. Alvarez1,3, L. Tusell1, A. Genescà1, R. Miró1, X. Garcia-del-Muro2 and J. Egozcue1

1 Departament de Biologia Cel.lular i Fisiologia, Universitat Autònoma de Barcelona and 2 Servei d'Oncologia Mèdica, Hospital Duran i Reinals, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Testicular germ cell cancer affects mainly young men. It is the most frequent type of cancer in 20–35 year old men. Since cancer treatment using antineoplasic drugs and ionizing radiation has a negative effect on the function of the gonads, testicular cancer patients are offered the opportunity to cryopreserve their semen samples before the beginning of therapy. For this reason it would be of interest to know whether there is chromosome instability in their spermatozoa prior to any treatment. Using the interspecific human–hamster fertilization system, we have analysed a total of 340 chromosome complements from spermatozoa of control donors and 320 chromosome complements from testicular cancer patients. There were no significant differences in the frequencies of chromosomal aberrations between controls and cancer patients (9.7 and 10.3% respectively; P = 0.4921). Our results indicate that spermatozoa from untreated testicular cancer patients do not show an increased chromosomal instability as compared to control donors.

Key words: chromosome instability/human–hamster system/sperm chromosomes/testicular cancer


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It is well documented that people affected by some kinds of cancer have an increased chromosomal instability, previous to any treatment (Hsu et al., 1983; Barrios et al., 1988Go, 1990Go, 1991Go; Heim et al., 1989Go). In fact it has been suggested that genomic instability expressed as a higher baseline frequency of chromosomal aberrations could be a driving force in carcinogenesis (Tlsty et al., 1995Go). Recent reports on chromosomal instability in individuals with testicular germ cell tumours have yielded inconclusive results. An increased incidence of chromosomal aberrations has been reported by van den Berg-de Ruiter et al. (1990), Delozier-Blanchet (1990) and Gundy et al. (1990), while no chromosome instability has been found by Vorechovsky and Zaloudik (1989), Osanto et al. (1991) and Heimdal et al. (1992). However, all these studies have been carried out in somatic cells. There are no studies about chromosomal instability in spermatozoa of patients with testicular germ cell tumours.

Testicular cancer is the most common tumour occurring in men between the ages of 20 and 35 years. The incidence of testicular cancer varies significantly according to geographic area and it is ~0.2% for Caucasian males (Bosl et al., 1997Go).

Testicular cancer has become a curable illness with a >90% chance of survival after surgery, chemotherapy and/or radiotherapy (Kliesch et al., 1997Go). Antineoplastic drugs as well as ionizing radiation can profoundly affect spermatogenesis. The recovery of spermatogenesis after treatment depends on 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., 1983Go; Botchan et al., 1997Go). For this reason testicular cancer patients are advised to cryopreserve semen samples before the beginning of their treatment. However, if their spermatozoa had chromosomal instability, any future pregnancy achieved with these semen samples should be accompanied by careful prenatal care.

These clinical implications make it important to ascertain whether there is chromosomal instability in spermatozoa of men with testicular cancer. To investigate this question we have compared the frequency of spontaneous chromosomal aberrations in spermatozoa of men affected by testicular cancer (after orchidectomy but before any further treatment) with that found in control donors.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Donors
The subjects of this study were four patients aged 23–34 years with testicular germ cell tumours treated with orchidectomy only (Table IGo). Semen samples from cancer patients were collected between 3 and 26 months after orchidectomy. As control donors three healthy men (aged 28–45 years) with no history of exposure to mutagenic agents were used.


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Table I. Clinical data from testicular cancer patients
 
Cryopreservation of semen samples
Since it has been demonstrated that cryopreservation of semen samples does not affect the frequencies of sperm chromosome abnormalities (Chernos and Martin, 1989Go; Martin et al., 1991Go), semen samples were cryopreserved as follows: each aliquot was diluted in the same volume of cryoprotectant medium consisting of 30% egg yolk, 14% glycerol, 1.98% glucose, 1.72% sodium citrate and 2% glycine in aqueous solution. Cryoprotectant medium was added sequentially drop by drop, 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. The container with the cryotubes was placed in a –80°C freezer for at least 4 h. After this period, the cryotubes were transferred to a liquid nitrogen 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., 1971Go) containing 0.3% human serum albumin (HSA) to eliminate seminal plasma. Motile spermatozoa were collected by a swim-up procedure (Andolz et al., 1987Go) and centrifuged (600 g, 6 min). Since ionophore treatment does not affect the frequencies of sperm chromosome abnormalities (Alvarez et al., 1996Go) the acrosome reaction of spermatozoa was induced by exposure to A23187 ionophore. Briefly, pelleted spermatozoa were resuspended in 10 µM calcium ionophore A23187 solution in BWW 0.3% HSA for 10 min at 37°C, 5% CO2. The sperm suspension was then centrifuged to eliminate the ionophore, and resuspended in BWW medium with 3.3% HSA, adjusting the concentration to 1–2x105 motile spermatozoa/ml. The final suspension of ionophore-treated spermatozoa was kept in the incubator for 2 h before insemination.

Hamster superovulation and oocyte processing
Adult Syrian hamster females were induced to superovulate by injection of gonadotrophins. Oviducts were punctured in Petri dishes containing BWW medium with 0.3% HSA to extract the cumulus masses, which were transferred to a 0.1% hyaluronidase solution to free the oocytes from cumulus cells. Finally, the oocytes were treated with a 0.1% trypsin solution to remove the zona pellucida.

Gamete coincubation and egg culture
Zona-free hamster eggs and human spermatozoa treated with calcium ionophore A23187 were incubated in BWW medium 3.3% HSA droplets under paraffin oil at 37°C in 5% CO2 for 1–2 h. During coincubation of gametes checks of penetration were carried out from time to time by pressing 5–10 oocytes under a coverslip placed over a slide on four wax spots and observed under a phase contrast microscope. As soon as the frequency of swollen sperm heads with tails per oocyte was 0.5 or higher, the other oocytes were washed and placed in F10 medium droplets. After 12 h of culture at 37°C in 5% CO2, the eggs were transferred to F-10 medium droplets containing 0.4 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 uniform staining–G banding. The classification of chromosome abnormalities was performed according to ISCN (1995). Chromosome and chromatid gaps were not scored as abnormalities.

Statistical analysis
To determine whether there was chromosomal instability in spermatozoa of men affected by testicular cancer, the frequencies of spermatozoa with chromosomal abnormalities from control and cancer patients were compared using the two-tailed Fisher's exact test.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A total of 340 sperm chromosome complements from three control donors and 320 sperm chromosome complements from four untreated cancer patients was analysed, showing 9.7 and 10.3% of chromosome abnormalities respectively (Table IIGo). There were no significant differences between both frequencies (P = 0.4921). Furthermore, neither numerical nor structural abnormalities were statistically different between both groups (P = 0.1777, P = 0.3742 respectively). Total frequencies of chromosomal aberrations for control donors and cancer patients found in our study were in the range found by other authors in control populations [from 6.7 to 15.2%: Rudak et al. (1978) and Mikamo et al. (1990) respectively].


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Table II. Spermatozoa with chromosomal abnormalities in control donors and testicular cancer patients
 
The incidence of chromosomal abnormalities in testicular cancer patients varied from 2.9 to 16.1%. Although a great inter-individual variability was found, these differences were not statistically significant (P > 0.05, two-tailed Fisher's exact test). Furthermore, none of the testicular cancer patients showed a statistically significant difference in chromosomal abnormalities when compared to controls.

Types and frequencies of structural abnormalities in control donors as well as in cancer patients are shown in Table IIIGo. Most structural abnormalities were chromosome breaks and acentric fragments (Figure 1Go) in both controls and cancer patients. There were no statistical differences in the frequencies of the different types of structural chromosome abnormalities between both groups.


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Table III. Spontaneous structural chromosome abnormalities found in spermatozoa of control donors and testicular cancer patients
 


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Figure 1. G-banded human sperm-derived chromosome complement from a patient affected with testicular cancer showing a structural chromosome abnormality (chsb 9q12). Arrowheads indicate the two fragments of chromosome 9.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have analysed the incidence of chromosome aberrations in human sperm-derived chromosome complements from untreated patients with testicular cancer. The possibility of an increased risk of sperm chromosomal abnormalities is important because some studies have demonstrated that there is no selection of spermatozoa based on chromosomal contents (Epstein and Travis, 1979Go; Martin, 1989Go).

Statistical analyses indicate that the baseline values of aberrant metaphases were not different between cancer patients and control donors (10.3 and 9.7% respectively). Similar results have been reported by Martin et al. (1997). These authors found that the frequency of chromosomal aberrations in testicular cancer patients previous to any treatment was 10.2%, a value that was not statistically different from their control donors. All these results together suggest that testicular cancer patients do not have an increased risk of transmission of chromosomal aberrations to their offspring compared to controls. However, it must be noted that these results are based on only a few testicular cancer patients, and a high inter-individual variability was found, raising the possibility that a larger sample might reveal statistically significant differences.

Since germ cell tumours include two subtypes of seminoma, three subtypes of teratoma, embryonal carcinoma, choriocarcinoma and yolk sack tumours (Bosl et al., 1997Go), it is possible that chromosomal instability could vary within tumour type, explaining the great inter-individual variability observed and indicating that a higher number of patients should be analysed before making a conclusion.

The exact aetiology of testicular cancer is not yet completely understood. In some cases germ cell tumours are believed to arise from meiotic spermatocytes which have undergone crossing-over and in which DNA repair is unsuccesful (Bosl et al., 1997Go). These spermatocytes should undergo apoptosis. However, the excess in copy number and expression of the 12p gene and additional mutational genetic events involving cell cycle control prevent apoptosis. These transformed cells are XY, have a 2C or a higher chromosome number, have the potential for widespread gene loss through genomic instability, and an increased 12p copy number and overexpression of a gene with oncogenic potential (Bosl et al., 1997Go).

Due to the nature and origin of testicular cancer, the possible risk of transmissible genetic changes that cannot be measured using classical cytogenetic techniques, or of genetically determined disease in the offspring, cannot be ignored when using assisted fertilization techniques with fresh or frozen spermatozoa from these patients. However, the reported data of in-vitro fertilization centres and data obtained from offspring of oncological patients (including testicular cancer patients) indicate no increased risk of malformations (Dodds et al., 1993Go; Meschede et al., 1995Go). Nevertheless, the offspring of testicular cancer patients should be included in the follow-up of these patients, because the increased risk of developing a testicular cancer in their sons should be taken into consideration (Kliesh et al., 1997).

Previous studies carried out in our laboratory (Genescà et al., 1990Go) showed that the incidence of numerical and structural chromosome abnormalities, in spermatozoa from patients treated for testicular cancer with antineoplastic drugs 2–7 years after cancer treatment, was significantly greater than in controls. Furthermore, the incidence of chromosomal aberrations was also significantly greater compared to another series of men treated with antitumoural agents for non-testicular diseases a long time before the sperm analysis (Genescà et al., 1990Go). Therefore, this increased level of chromosomal abnormalities in sperm of testicular cancer patients, compared to those treated for other kinds of cancer, must be due to the effects of antineoplastic drugs, and not to an increased chromosomal instability in testicular cancer patients. All these results taken together strongly recommend that cancer patients have their semen cryopreserved, when possible, prior to the initiation of chemo- or radiotherapy.

Studies of chromosomal instability in somatic cells (lymphocytes and fibroblasts) from testicular cancer patients have also yielded contradictory results. An increased incidence of chromosomal aberrations has been reported by van den Berg-de Ruiter et al. (1990), Delozier-Blanchet (1990) and Gundy et al. (1990). On the other hand, no chromosome instability has been reported by Vorechovsky and Zaloudik (1989), Osanto et al. (1991) and Heimdal et al. (1992). All these reports are characterized by the presence of a great inter-individual variability in the levels of chromosomal abnormalities in testicular cancer patients. As pointed by Heimdal et al. (1992), due to the high inter-individual variability, it is probable that studies of a limited number of patients and controls should give conflicting results.

Tzancheva and Konitowski (1997) showed, in a preliminary study of a group of 15 untreated cancer patients, that although the rate of chromosomal aberrations was similar in both cancer and control donors, the lymphocytes of cancer patients display an increased susceptibility to treatment with bleomycin and caffeine, suggesting a latent chromosomal instability. The occurrence of multiple primary neoplasms in patients with testicular germ cell cancer treated with radiotherapy supports the existence of a latent chromosomal instability (Dieckmann et al., 1994Go).

Thus, the failure of the present and other studies performed in human lymphocytes to demonstrate increased chromosome breakage in patients with testicular cancer, does not rule out a role for chromosome instability in the susceptibility to this disease, because chromosome instability could exist in a latent form.

In summary, as yet, an uneventful pregnancy and a normal future development of the new-born cannot be guaranteed. In such cases, providing patients with current knowledge concerning the issue of testicular malignancy and paternity, and assisting them in taking appropriate decisions is, therefore, highly recommended. Once a pregnancy has been achieved it is mandatory to provide close prenatal care and postnatal follow-up.


    Acknowledgments
 
This work has been performed with the financial support from the Consejo de Seguridad Nuclear. We also acknowledge the financial support given by CIRIT to the Cell Biology Unit (CRQ 93–2025) and (1995 SGR-0478).


    Notes
 
3 To whom correspondence should be addressed at: Unitat de Biologia, Departament de Biologia Cel.lular i Fisiologia, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on December 3, 1997; accepted on October 14, 1998.