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From the Departments of * Pharmacology and
Therapeutics and Obstetrics and Gynecology,
McGill University, Montreal, Canada
| Correspondence to: Dr Bernard Robaire, Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6 (e-mail: Bernard.robaire{at}mcgill.ca). |
| Received for publication September 11, 2007; accepted for publication February 18, 2008. |
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Abstract |
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Key words: Cancer therapeutics, testis
Spermatogenesis is often impaired in testicular cancer patients prior to chemotherapy as a result of the cancer itself (Agarwal, 2005). In addition, BEP chemotherapy has substantial detrimental effects on spermatogenic function; most patients are rendered temporarily azoospermic or oligozoospermic, depending on the dose and duration of treatment (Petersen et al, 1994). Normal spermatogenesis recovers in about 50% of the patients after 2 years and in the large majority (80%) 5 years after the completion of chemotherapy (Lampe et al, 1997; Howell and Shalet, 2005; Magelssen et al, 2006). However, in some patients, sperm production does not reinitiate and permanent infertility ensues. Thus, fertility is a major concern for testicular cancer patients undergoing BEP chemotherapy.
In animal models, previous studies have shown that either acute or chronic administration of the chemotherapeutic drugs cisplatin or etoposide induces adverse effects on various male reproductive parameters shortly after exposure. For instance, subchronic administration of cisplatin over a 9-week period resulted in a decrease in reproductive organ weights, including testes and epididymides, decreased sperm motility, and increased preimplantation and postimplantation loss; malformed and growth-retarded fetuses were observed among the progeny sired by cisplatin-treated males (Seethalakshmi et al, 1992). Acute exposure to cisplatin resulted in decreased reproductive organ weights, as well as increased preimplantation loss (Huang et al, 1990; Kinkead et al, 1992). However, no studies have investigated the potential persistence of these effects, particularly with respect to progeny outcome, after completion of treatment. Likewise, chronic etoposide administration induced drastic alterations in spermatogenesis at high doses, but the impact of such treatment on progeny outcome remains unknown (Kadota et al, 1989; Kawaguchi et al, 2000). In addition, these studies all consisted of exposure to a single drug. However, as mentioned above, the current BEP regimen relies on the synergistic effects of 3 different anticancer drugs over at least a 9-week period. Therefore, to accurately mimic the impact of this regimen, bleomycin, etoposide, and cisplatin have to be given chronically and in combination.
To date, chemotherapy for testicular cancer has not been associated with increased birth defects or abnormal progeny, but the number of subjects in these studies has been relatively small (Hartmann et al, 1999). The relative risk for abnormal progeny among the offspring of testicular cancer survivors remains poorly defined and requires further study. Furthermore, it is very difficult to predict the long-term effects of the BEP regimen on fertility and progeny outcome in patients prior to the initiation of chemotherapy as the condition of each patient may vary at the time of diagnosis and with the treatment modalities of the disease (Gandini et al, 2006). Owing to this lack of information, animal studies may provide a better understanding of how BEP affects fertility and progeny outcome in the short- and long-term. We have reported previously that the combination of bleomycin, etoposide, and cisplatin, when chronically administered for 9 consecutive weeks to adult male rats, resulted in a drastic reduction in spermatozoal count and decreased progeny survival but, interestingly, did not compromise fertility (Bieber et al, 2006). The purpose of this study was to investigate, using the rat as a model, the long-term effects of the subchronic combined-administration of bleomycin, etoposide, and cisplatin, mimicking the clinical chemotherapeutic exposure in humans, on male reproductive functions, with emphasis on fertility and progeny outcome.
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Materials and Methods |
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Animal Groups and Treatment Regimen
Animals were divided randomly into 3 different treatment groups (n = 15 per
group): a control group (0x), and 2 BEP-treated groups (0.33x and
0.5x). Dose selection was based on the human clinical regimen. In the
clinic, patients undergoing adjuvant BEP chemotherapy commonly receive a daily
dose of 20 mg/m2 cisplatin and 100 mg/m2 etoposide on
days 1–5 and a weekly dose of 30 mg bleomycin. These standard doses for
each drug were converted to rat doses by adjusting for body weight/surface
area ratio (Bachmann et al,
1996) and represent the 1x dosage. In a 1x dosage,
animals would receive 15 mg/kg body weight (bw) of etoposide, 3 mg/kg bw of
cisplatin, and 1.5 mg/kg bw of bleomycin. In the 0.33x BEP group,
animals received one-third of the 1x dose, or 5 mg/kg etoposide, 1 mg/kg
cisplatin, and 0.5 mg/kg bleomycin. Accordingly, the 0.5x BEP animals
were given 7.5 mg/kg etoposide, 1.5 mg/kg cisplatin, and 0.75 mg/kg
bleomycin.
Rats in the BEP groups were treated with 3 cycles of bleomycin, etoposide, and cisplatin for a total of 9 weeks. In order to closely mimic the time-schedule of the human clinical regimen, etoposide and cisplatin were administered on days 1–5 and bleomycin on days 2, 9, and 16 of each 21-day cycle. Cisplatin and bleomycin, in 0.9% saline, were given by intraperitoneal (IP) injection. Etoposide was dissolved in a 3:7 (vol/vol) mixture of dimethylsulfoxide (DMSO)-saline and administered by gavage. Age-matched rats in the control group were treated in the same manner and received equivalent volumes of saline and DMSO. All drug solutions were prepared fresh daily before administration. For the recovery experiment, the treatment period was followed by a recovery period of 3 cycles (9 weeks) before the rats were euthanized.
The 0.33x BEP dose was the maximum tolerated dose consistent with good survival during the recovery phase. Only 1 animal died in this treatment group before the end of the recovery period. The 0.5x BEP dose resulted in high mortality, with 7 out of 17 rats dying during the treatment; data from this group at the end of treatment are provided only to emphasize potential systemic and germ cell toxicity of this subchronic BEP treatment.
Tissue Collection and Preparation
At the time of euthanasia, animals were anesthetized with an IP injection
of a mixture of ketamine hydrochloride (50 mg/kg), acepromazine (1 mg/kg),
xylazine hydrochloride (5 mg/kg) and 0.9% saline. Prior to perfusion, blood
was collected under anesthesia; the serum was then separated and stored at
–80°C. The left testis and epididymis were ligated, dissected out,
weighed, and rapidly frozen in liquid nitrogen and stored at –80°C
until further use. The contralateral testis and epididymis were first cleared
with 0.9% saline and then perfused through the abdominal aorta with Bouin
fixative for 10 minutes. Following perfusion, the testis and epididymis were
excised and postfixed for an additional 24 hours in the same fixative. Tissues
were dehydrated and then embedded in paraffin
(Serre and Robaire, 1999).
Histology
For histological analysis, 5 µm-thick sections were cut on a microtome,
mounted on microscope glass slides (Superfrost Plus microscope glass slides;
Fisher Scientific Inc, Montreal, Canada) and dried overnight at 37°C.
Tissue sections were deparaffinized with xylene and rehydrated through a
graded series of ethanol changes; staining of sections with periodic
acid-Schiff (PAS) was carried out using the Sigma PAS-kit 395B-1KT, according
to the manufacturer's instructions. Sections were examined and photographed by
light microscopy.
Terminal Deoxynucleotidyl Transferase–Mediated Nick-End Labeling Assay
Testicular germ cell apoptosis was assessed by terminal deoxynucleotidyl
transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) assay using
ApopTag Peroxidase In Situ Apoptosis Detection kit (S7100; Chemicon
International Inc, Temecula, California), according to the manufacturer's
instructions. Following TUNEL staining, sections were counterstained with
Harris Hematoxylin and mounted under glass coverslips with Permount (Fisher).
The sections were examined and scored under a light microscope (Leica DM LB2
microscope; Leica, Wetzlar, Germany) equipped with an RS Photometrics CoolSNAP
fx digital camera (Roper Scientific, Tucson, Arizona). TUNEL-positive germ
cells were quantified by counting the number of TUNEL-positive cells in each
round cross-section of the seminiferous tubules. Sections from 5 animals per
group were analyzed, and apoptotic germ cells were counted in at least 200
seminiferous tubules per testis section. As negative controls, a section from
each animal was processed as described above, but terminal transferase was
omitted from the TdT labeling buffer.
Spermatid/Spermatozoal Head Counts
A weighed portion of the decapsulated left testis was homogenized in 5 ml
of 0.9% NaCl, 0.1% thimerosal, and 0.5% Triton X-100 with a Polytron (Brinkman
Instruments, Westbury, New York) twice for 15 seconds, separated by a
30-second period, as previously described
(Robb et al, 1978). Spermatid
heads in an aliquot from each testis homogenate were counted with a
hemocytometer.
Measurement of Serum Testosterone Levels
Serum testosterone concentrations were measured using a testosterone
enzyme-linked immunosorbent assay (ELISA) kit (catalog no 55R-RE52151; IBL
Immunobiological Laboratories, Hamburg, Germany) following the manufacturer's
instructions.
Analysis of Progeny Outcome Parameters
After the completion of the 9-week BEP treatment, each male from the
0.33x BEP-treated and control groups (n = 10–15) was caged
overnight with 2 selected female rats in proestrus. To assess fertility and
progeny outcome parameters during the recovery phase, the BEP-treated rats
were mated 3, 6, and 9 weeks after the cessation of treatment. The following
morning, vaginal smears were examined for the presence of spermatozoa to
determine whether copulation had occurred; this day was defined as gestation
day 0. All confirmed-pregnant females were euthanized on gestation day 20 by
CO2 asphyxiation and then cesarean-sectioned. The ovaries were
removed, and the numbers of corpora lutea (representing the numbers of
ovulated oocytes) were counted. The uteri were dissected and inspected for
implantation and resorption sites, and the numbers of implantation and
resorption sites were recorded.
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Statistical Analysis
All data were analyzed using SigmaStat (SPSS Inc, Chicago, Illinois).
Values are presented as means ± SEM. Differences between groups were
examined for statistical significance using a Student's t test when
only 2 groups were compared or 1-way analysis of variance (ANOVA). If ANOVA
indicated P values of .05 or below, a Bonferroni t test was
performed to determine the significance of difference between all groups.
Nonparametric Mann-Whitney tests were performed when data were not distributed
normally. P values of .05 or below were regarded as significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Spermatid Head Counts
In the 9 week BEP-treated groups, there was a dose-dependent decrease in
the spermatid/spermatozoa head counts that was significant only in the
0.5x group compared to control
(Figure 3G). Treatment with
0.33x BEP followed by the 9-week recovery period resulted in a 20.6%
decrease in spermatid/spermatozoa head count that was not statistically
significant (P = .07; Figure
3G).
Effects of BEP Treatment on Serum Testosterone Concentrations
Treatment with BEP for 9 weeks did not alter serum testosterone
concentrations in the 0.33x dosage group
(Figure 4). Although serum
testosterone concentrations were reduced in the 0.5x BEP group by nearly
50%, the difference was not statistically significant (P = .10),
presumably due to the wide fluctuations in serum testosterone known to occur
in the rat (Robaire and Bayly,
1989). In addition, serum testosterone concentrations were not
different in the 0.33x BEP group compared to the control group following
the recovery phase.
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Effects of BEP Exposure on Germ Cell Apoptosis
As demonstrated above, BEP treatment is clearly associated with abnormal
spermatogenesis and a reduction in germ cell content in the seminiferous
epithelium in a dose-dependent manner. Therefore, we investigated whether
testes from BEP-treated rats had an increased number of apoptotic germ cells
when compared with testes from control animals, using the TUNEL assay. Under
normal physiologic conditions, apoptosis occurs spontaneously in testicular
germ cells, affecting principally spermatogonia and spermatocytes. As
expected, testis cross-sections from control rats had few TUNEL-positive cells
(Figure 4A) and were
characterized by a low number of overall TUNEL-positive tubules
(Figure 4E). Whereas exposure
to 0.33x BEP had no significant effect on the testis weight, an increase
in both the number of TUNEL-positive tubules and the number of TUNEL-positive
cells per tubule was observed compared to the control after 9 weeks of
treatment (Figure 4B, E, and
F). In addition, the number of TUNEL-positive tubules in the
0.5x BEP group was increased significantly compared with the control
group. However, there was no significant increase in the number of
TUNEL-positive cells per TUNEL-positive tubules; this may indicate that the
dramatic depletion of germ cells observed in those testis sections resulted
from massive apoptosis (Figure 4C, E, and
F). No significant differences in either TUNEL parameter were
detected between 0.33x BEP-exposed and control rats after the 9-week
recovery period (Figure 4E and
F). Therefore, the increase in apoptotic germ cells in the
seminiferous epithelium of 0.33x BEP-treated rats was not
persistent.
Reversibility of the Effects of Subchronic BEP Treatment on Progeny Outcome
The effects of paternal subchronic BEP exposure on progeny outcome at the
end of treatment, and after 3, 6, or 9 weeks of recovery, were assessed by
examining preimplantation and postimplantation loss, litter size, sex ratio,
and fetal weights. BEP treatment (0.33x BEP group) resulted in a 5-fold
increase in preimplantation loss compared to control
(Figure 5A, P <
.001); preimplantation loss remained elevated in litters sired by BEP-treated
males up to 9 weeks posttreatment. A 3-fold increase in postimplantation loss
was observed in the BEP group relative to control after 9 weeks of treatment
(Figure 5B; P <
.05). Interestingly, unlike preimplantation loss, which remained persistently
elevated, the incidence of postimplantation loss did not differ from the
control in the BEP-treated group during the recovery period. As a consequence
of the increases in preimplantation and postimplantation loss, a significant
reduction in litter size was observed in the 0.33x BEP group compared
with the controls (Figure 5C).
The number of pups per litter remained low in the BEP-exposed group after the
3-week recovery period but did not differ from controls in the 6-week and
9-week recovery groups. Sex ratios were not altered by paternal exposure to
BEP (Figure 5D). The mean
weights of male and female fetuses were not affected in any treatment group,
nor did exposure to BEP induce an increase in the incidence of external
malformations in the fetuses (data not shown).
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Discussion |
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Impairment of spermatogenesis is one of the earliest signs of the adverse effects of BEP chemotherapy on testicular function as sperm counts of patients decrease following the initiation of chemotherapy (Gandini et al, 2006). In the present study, we showed that exposure to 0.33x low-dose BEP treatment caused the degeneration of germ cells, the formation of multinucleated giant cells, the shedding of immature germ cells within the lumen, and Sertoli cell vacuolization. The 0.5x BEP treatment resulted in more drastic effects on the seminiferous epithelium, with extensive germ cell depletion and tubule atrophy after 9 weeks of treatment.
In the mammalian testis, spontaneous germ cell death occurs to eliminate damaged germ cells from the seminiferous epithelium, thereby maintaining cellular homeostasis of the epithelium. In the adult rat testis, various studies have shown that drug-induced germ cell death is mediated by apoptosis (Cai et al, 1997; Blanco-Rodriguez et al, 1998; Sjöblom et al, 1998). In this study, by in situ analysis of TUNEL-positive cells, we demonstrate that the germ cell degeneration observed in adult rats exposed to BEP treatment is mediated by apoptosis. At low-dose 0.33x BEP exposure, a marked increase in apoptotic germ cells was observed at the end of the 9-week treatment; the rate of apoptosis may increase gradually with BEP exposure. In contrast, with exposure to an increased dose, 0.5x BEP, testes with the most extensive degeneration and atrophic tubules showed a relatively low number of apoptotic germ cells, presumably as a result of extensive germ cell depletion. Apoptosis is a relatively rapid mechanism of cell death; therefore, it is possible that the high dose 0.5x BEP treatment induced a more drastic increase in germ cell death in the early days of treatment, resulting in the severe reduction of germ cell content and tubule atrophy observed at the end of dosing. In addition, BEP-induced apoptosis was more pronounced in spermatogonia and spermatocytes, suggesting that BEP treatment may target actively dividing germ cells.
It is well documented that testicular cancer patients experience impaired spermatogenesis shortly after the initiation of BEP chemotherapy (Oliver, 1996). However, it is not clear whether drug-induced apoptosis is solely responsible for the loss of germ cells and subsequent decreased production of spermatozoa. Interestingly, the number of apoptotic germ cells did not appear to be markedly increased in the testis cross-sections examined following the 9-week recovery period, demonstrating that the apoptotic germ cell death is either transitory or reversible. Taken together, the apparently normal testis histology and the absence of enhanced apoptosis in the majority of BEP-treated male rats after the recovery period suggest that, at least histologically and numerically, apparently spermatogenesis recovers effectively after BEP treatment. These results are in agreement with previous animal experiments where single drug exposures were used (Zhang et al, 2001; Seaman et al, 2003; Stumpp et al, 2004).
The action of BEP chemotherapeutics on seminiferous epithelial germ cells is the consequence of the combination of 3 different anticancer agents with 3 separate modes of action. Cisplatin alone is known to have adverse effects on testis histopathology and progeny outcome when chronically administered to rats. Cisplatin functions by damaging DNA through the formation of chemically stable DNA adducts. Cisplatin-induced DNA adducts accumulate in the testis following repeated dosing (Poirier et al, 1992); this persistence of DNA adducts in rat testes may suggest that germ cells are less efficient in eliminating adducts, thus leading to prolonged adverse effects of the drug in these cells. A dose-dependent accumulation of DNA adducts in rat spermatozoa was demonstrated following acute cisplatin exposure but, interestingly, without inducing adverse developmental effects (Hooser et al, 2000). Therefore, it is possible that cisplatin-induced DNA adducts may have accumulated in germ cells during spermatogenesis with the cisplatin-based BEP treatment; these cisplatin-induced DNA adducts may result in DNA strand break formation in spermatozoal DNA during the treatment period. Unrepaired DNA damage in spermatozoa after chemotherapy is a known cause of mutations or chromosomal aberrations, contributing to progeny outcome defects. Thus, we cannot exclude that the genetic integrity of spermatozoa exposed to BEP may have been compromised.
In the current study, we addressed whether the BEP treatment for testicular cancer had a prolonged negative impact on fertility and the outcome of progeny sired after paternal exposure. We showed that BEP treatment for 9 weeks caused a significant increase in the incidence of both preimplantation and postimplantation loss when rats were mated with untreated females at the end of the treatment. Interestingly, in the reversal study, BEP treatment resulted in an increase in preimplantation loss that persisted up to 9 weeks after completion of treatment. This preimplantation loss may represent unfertilized, ovulated oocytes or the death of early embryos prior to implantation. According to the timing of spermatogenesis in rats, the abnormal progeny parameters observed at the end of the 9-week BEP treatment may represent the effects of BEP treatment on spermatozoa that were first exposed as spermatogonia and throughout their complete spermatogenic development. The persistence of preimplantation loss at 3 and 6 weeks after completion of BEP treatment may in turn reflect the consequences of BEP treatment on germ cells that were first exposed as spermatogonia and up to the premeiotic differentiation but not during the postmeiotic steps or spermiogenesis, as the treatment had stopped by that time. Following 9 weeks of recovery, preimplantation loss remained elevated; the spermatozoa responsible for fertilizing these embryos were exposed to BEP treatment during their spermatogonial differentiation stages.
Understanding the long-term effects of BEP exposure is essential for testicular cancer patients, particularly for those seeking to have children after chemotherapy; the potential risk for abnormal progeny after paternal BEP exposure requires further studies. Based on our results, it is clear that BEP treatment has prolonged adverse effects on progeny outcome in rats. Our observations, if translated to the human clinical situation, emphasize that spermatozoa produced during BEP chemotherapy exposure may increase risk for abnormal progeny outcome.
In conclusion, we demonstrate that paternal exposure to subchronic BEP induces transient defects on spermatogenesis and exerts prolonged effects on preimplantation loss in progeny sired more than 1 spermatogenic cycle after the termination of paternal exposure to BEP.
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Acknowledgments |
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Footnotes |
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indicates a significant
difference (P < .05) between the values for the 0.33x and
0.5x BEP groups.
