1 Human Fertility Research Group and CECOS Midi-Pyrénées, Centre de Stérilité Masculine, Service d'Uro-Andrologie, Hôpital La Grave and 2 Service de Génétique Médicale, Hôpital Purpan, Toulouse, France
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Abstract |
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Key words: chemotherapy/FISH /PEB side effects/sperm aneuploidy/testicular cancer
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Introduction |
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The incidence of congenital malformation does not appear to be increased in offspring of male cancer patients following chemotherapy. However, published reports are either case studies or have small sample sizes which are not able to detect less than a 3- to 5-fold increase in relative risks (Meistrich, 1993). Moreover, few studies have assessed the genetic consequences of chemotherapy on spermatozoa. Rather, they addressed different chemotherapy regimens and their results are conflicting, showing the presence (Jenderny and Röhrborn, 1987
; Genesca et al., 1990a
,b
; Brandriff et al., 1994
; Monteil et al., 1997
; Robbins et al., 1997
; Martin et al., 1999
) or absence (Jenderny et al., 1992
; Martin et al., 1995
, 1997a
, Martin et al., b
) of chromosomal alterations. In the case of testicular cancer chemotherapy, only one group has studied the effects of the PEB regimen on sperm chromosomes. In four patients who provided sperm samples between 2 and 13 years after chemotherapy, neither structural (Martin et al., 1997b
) nor numerical (Martin et al., 1997a
) abnormalities were found. On the other hand, one patient investigated soon after the beginning of 4 cycles of PEB regimen (at 59 and 366 days), showed a significant increase in sperm chromosomal abnormalities (Martin et al., 1999
).
Using fluorescence in-situ hybridization (FISH) of chromosome-specific DNA probes, the aim of this study was to compare spermatozoa aneuploidy in patients recently submitted to adjuvant PEB chemotherapy, with a control group.
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Materials and methods |
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The PEB chemotherapeutic regimen (based on body surface area) was 120 mg/m2 etoposide and 20 mg/m2 cisplatin from day 1 to day 5, and 30 mg bleomycin on days 2 and 9. This regimen was repeated two, three or four times at monthly intervals according to the tumoral grade.
Sample preparation
After thawing, semen samples were washed twice in a buffer solution (0.1 mol/l TrisHCl, pH 8.0) and, following the final centrifugation at 400 g, were resuspended in 0.52.0 ml buffer. One drop of the suspension was smeared onto a clean glass microscope slide and allowed to air-dry. Slides were either stored in a slide box and kept at 20°C or used immediately in preparation for FISH.
The slides were dehydrated through 70, 85 and 100% ethanol for 2 min each. After air-drying, the sperm decondensation was performed using a published method (Wyrobek et al., 1990), with minor modifications. Briefly, slides were incubated at room temperature for ~8 min (range: 530 min) in 10 mmol/l dithiothreitol (DTT; Sigma-Aldrich, Saint Quentin Fallavier, France) in 0.1 mol/l TrisHCl, pH 8.0, and for 12 min (range: 545 min) in 10 mmol/l lithium 3,5-diiodosalicylic acid (LIS; Sigma-Aldrich) and 1 mmol/l DTT in 0.1 mol/l TrisHCl, pH 8.0. The slides were then briefly rinsed in 0.1 mol/l TrisHCl, pH 8.0, allowed to air-dry, and then fixed with methanol:acetic acid (3:1) for a minimum of 20 min and air-dried.
FISH
Directly-labelled alpha satellite centromere-specific probes (Vysis, Downers Grove, IL, USA) were used for this FISH assay: (i) Spectrum Green CEP 7 alpha, Spectrum Orange CEP 16 alpha in a two-colour hybridization; and (ii) Spectrum Green CEP X alpha, Spectrum Orange CEP Y alpha, Spectrum Green CEP 18 alpha and Spectrum Orange CEP 18 alpha in a three-colour hybridization. The DNA was denatured in 70% formamide for 5 min at 74°C, dehydrated through an increasing concentration (70, 85 and 100%) of cold ethanol, air-dried and placed for 2 min on a hot plate with a surface temperature of 42°C. The labelled probes were added to hybridization buffer according to the manufacturer's instructions. The hybridization solution was dropped onto the sperm preparation, and a coverslip (22x22 mm) was added and sealed with rubber cement. Hybridizations were conducted overnight in a dark moist box, at 42°C. The slides were then washed in 0.4x standard saline citrate (SSC), pH 7.0, at 74°C for 2 min, then for 1 min in 0.1% Nonidet P-40 (NP-40; Sigma-Aldrich)/2xSSC) at room temperature and air-dried. 4,6-Diamidino-2-phenylindole (DAPI) counterstain (12 µl) was applied to each slide, and a coverslip added and sealed with rubber cement.
Scoring and analysis
The slides were examined using a Zeiss Axioplan 2 epifluoresence microscope equipped with a triple band pass filter for DAPI, fluorescein isothiocyanate (FITC) and tetramethyl-rhodamine isothiocyanate (Rhodamine). The Spectrum Green CEP X alpha, CEP 7 alpha appeared green, the Spectrum Orange CEP Y alpha, CEP 16 alpha appeared red, and the Spectrum Green CEP 18 alpha/Spectrum Orange CEP 18 alpha appeared yellow. The chromosome 18 probe was used as an internal control to distinguish between sex chromosome disomy and diploidy.
To avoid scoring bias, the same stringent scoring criteria were used by one examiner (P.D.M.) on both groups. Sperm nuclei were scored as having two signals when they displayed two signals of equal size and intensity, separated by at least the diameter of one signal (Martin and Rademaker, 1995) and well positioned within the sperm head. Overlapping spermatozoa or sperm heads without a well-defined boundary were not evaluated. Nullisomies of one of the tested chromosomes were not directly scored and were considered as equivalent to the incidence of disomies (Downie et al., 1997
; Egozcue et al., 1997
). A total of 10 000 consecutive sperm nuclei were scored from each combination of probes.
The data were analysed by the 2 test. A P-value < 0.05 was considered significant.
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Results |
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Discussion |
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The current study, using 7, 16, 18, X and Y -specific chromosome probes demonstrated a significant increase in sperm aneuploidy (disomy 16, 18, XY and diploidy) in treated patients compared with control men.
To date, only one other group has reported a study on sperm damage after PEB chemotherapy in patients treated for testicular cancer. This group (Martin et al., 1997a) studied disomy for chromosomes 1, 12, X, Y, XY and diploidy by FISH in four testicular cancer patients both before and 2 to 13 years after treatment with PEB. These authors did not find any increased risk of numerical chromosomal abnormalities. Sperm karyotyping was used to confirm this result, and the possibility of an increased risk of structural abnormalities in the same patients was excluded (Martin et al., 1997b
). However, in a patient submitted to four PEB cycles, the study of sperm cell chromosomes by FISH before, during (59 days) and after (366 days) chemotherapy showed an increased aneuploidy, which is in agreement with the current results. In fact, a significant increase of the XY disomy and of diploid sperm cells was noticed (Martin et al., 1999
). The current results led to a similar observation showing a significant increase in the frequency of aneuploidy during the first 18 months following initiation of the PEB chemotherapy.
Among the drugs used, etoposide appears to be a good candidate for aneuploid sperm induction as it interacts with topoisomerase II during anaphase I and II of meiosis. Topoisomerase II activity is required for removing regions of DNA catenation between (sister and non-sister) chromatids. This decatenation enables segregation of homologous chromosomes and sister chromatids (Murray and Szostak 1985; Holm et al., 1989
). Etoposide inhibits topoisomerase II activity by forming a DNAtopoisomerase IIdrug ternary complex, and induces a non-disjunction of recombinant chromosomes (Rose et al., 1990
) and of sister chromatids (Downes et al., 1991
). Experiments in vivo showed that etoposide treatment results in an increased risk of aneuploid germ cells, in mouse oocytes (Mailhes et al., 1994
) and also in spermatocytes (Kallio and Lahdetie, 1996
). Abnormal chromosome segregation has been shown with bleomycin in yeast (Sora et al., 1983
; Käfer, 1990
) and in Drosophila melanogaster (Traut, 1980
), but the main effect of the drug is the induction of DNA double-strand breaks resulting in structural chromosome abnormalities, as shown in hamster primary embryonic cells (Xiao and Natarajan, 1999
) and in human lymphocytes (Ellard et al., 1995
). No increased risk of aneuploidy was shown with cisplatin (cis-diamminodichloroplatinum II) in either male or female germ cells of D. melanogaster (Brodberg et al., 1983
) or in murine metaphase II oocytes (Higdon et al., 1992
).
The impact of the drugs used is further demonstrated by discrepancies between various chemotherapeutic regimens used in different cancers (Table III). In two studies (Jenderny and Röhrborn, 1987
; Genesca et al., 1990a
), interspecific fertilization of hamster eggs was used to show a significant increase in the incidence of stable structural chromosome abnormalities (deletions, translocations, inversions). This was seen in the spermatozoa of two patients treated for non seminoma testicular cancer by PVB (cis-platinum diaminedichloride, vinblastine, bleomycin) or PVB + PEB, 2 and 5 years after treatment, and also four additional patients, treated 5 to 18 years earlier by chemotherapy and/or radiotherapy for sarcoma or Wilms' tumour (Genesca et al., 1990b
). Conversely, in the study of a PVB-treated testicular cancer patient (Jenderny et al., 1992
), no increase in chromosome structural abnormalities was found. Indeed, the earliest studies are controversial with regard to numerical chromosome aberrations. Some reports claimed an increase in numeric chromosome anomalies (Jenderny and Röhrborn, 1987
; Genesca et al., 1990a
; Brandriff et al., 1994
), while others did not (Genesca et al., 1990b
; Jenderny et al., 1992
). All of these studies used the interspecific hamster test, and it is possible that the low number of sperm cells analysed might have biased these results.
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Whatever the cause and duration of sperm aneuploidy, the consequences could be spontaneous abortion, stillbirth or birth defects in chromosomally abnormal children (Hassold et al., 1996). In fact, under experimental conditions, no selection of chromosomally abnormal spermatozoa occurs during fertilization, and numerous studies (Genesca et al., 1990a
,b
; Brandriff et al., 1994
; Marchetti et al., 1999
) have shown (by using several methods) that aneuploid spermatozoa are as competent as normal spermatozoa for fertilizing mouse oocytes.
It is believed that patients must be informed of these facts, so that genetic counselling can be offered. With regard to PEB chemotherapy, while the hypothesis of a transient chromosomal side effect appears possible, more studies on sperm chromosomes are required before and during the follow-up of the treatment. Definitive conclusions will help practitioners in counselling about the necessity and duration of contraception after chemotherapy treatment, while a chromosomal examination could be proposed in cases of pregnancy of these patients' partners, soon after the end of chemotherapy.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 22, 2000; accepted on February 26, 2001.