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From the Departments of * Pharmacology &
Therapeutics and Obstetrics & Gynecology,
McGill University, Montréal, Québec, Canada.
| Correspondence to: Bernard Robaire, Department of Pharmacology and Therapeutics, 3655 Promenade Sir-William-Osler, Montréal, Québec, Canada H3G 1Y6 (e-mail: Bernard.robaire{at}mcgill.ca). |
| Received for publication May 30, 2005; accepted for publication August 2, 2005. |
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Abstract |
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Key words: cis-Platinum, etoposide, bleomycin, testis, epididymis, progeny outcome, flagellar defect
After treatment for testicular cancer, patients experience a decrease in the number of spermatozoa produced and in their motility, as well as an increase in morphologically abnormal spermatozoa (Stephenson et al, 1995). Spermatogenesis recovers in most men after 5 years (Lampe et al, 1997). However, reports on the chromatin quality of the surviving spermatozoa are conflicting: one study found an increase in aneuploidy (De Mas et al, 2001), another found no change (Thomas et al, 2004), and yet another found a decrease in aneuploidy (Martin et al, 1997). The sperm chromatin structure assay revealed no increase in DNA fragmentation (Stahl et al, 2004); however, one case report described a man whose sole semen abnormality after treatment with BEP was an increase in DNA denaturation (Deane et al, 2004).
While studies of the effects of BEP on spermatogenesis in human testicular cancer patients provide valuable information, these data are confounded by the fact that the subjects have a diseased testis and have undergone orchidectomy. It has been shown that the semen quality of testicular cancer patients is already decreased at diagnosis (Petersen et al, 1999b; Jedrzejczak et al, 2004). The semen quality further decreases following orchidectomy, even before the initiation of chemotherapy (Petersen et al, 1999a). Furthermore, the patients in these studies have received various numbers of cycles of BEP, and the time from treatment to semen collection is variable. It is thus extremely difficult to conclude whether the effects on fertility are due to chemotherapy, orchidectomy, or the cancer itself.
The effects of BEP treatment on progeny outcome are difficult to examine in humans. It has been suggested that fertility in patients with testicular cancer was decreased by 30% after treatment (Huyghe et al, 2004). Several reports have suggested that there is no increase in congenital malformations among the progeny of testicular cancer patients who were treated with BEP (Senturia et al, 1985; Byrne et al, 1988). Because of small sample sizes, however, these reports do not have the power to detect an increase in relative risk of less than threefold to fivefold (Lahdetie et al, 1994). Animal studies indicate that paternal treatment with cis-platinum alone can affect progeny outcome: chronic cis-platinum treatment in the rat resulted in increased pre- and postimplantation loss, a change in sex-ratio of the offspring, as well as an increase in malformed and growth-retarded fetuses (Seethalakshmi et al, 1992). The effects of etoposide or bleomycin alone, or of the BEP combination, on progeny outcome have not been examined in an animal model. Furthermore, postnatal development of pups sired by male rats treated with any of the 3 drugs has yet to be examined.
We hypothesized that even in the absence of testicular cancer and orchidectomy, the chemotherapeutic regimen used to treat testicular cancer is deleterious to the production of spermatozoa, to their morphology and motility, and can affect fertility and progeny outcome. We tested this hypothesis in the rat model.
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Materials and Methods |
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Animals, Treatment, and Mating Protocol
Adult male (300–350 g) and virgin female (200–225 g)
Sprague-Dawley rats were purchased from Charles River Canada (St Constant,
Canada) and housed under controlled light conditions (14:10 hours light:dark)
in the Animal Resources Centre of McGill University. Animals were provided
with food and water ad libitum. All animal studies were conducted in
accordance with the principles and procedures outlined in the Guide to the
Care and Use of Experimental Animals prepared by the Canadian Council on
Animal Care (McGill Animal Research Centre protocol 4699).
Males were randomly divided into 2 groups of 10 rats each. The rats from the control group were gavaged on days 1 through 5 of each week with 1 mL of 7:3 saline (Roche, Laval, Canada): DMSO (Fischer Scientific, Fair Lawn, NJ). On day 2 of each week, control rats were given 1 mL of saline by intraperitoneal injection. The animals from the drug-treated group were gavaged on days 1 through 5 of each week with 3.0 mg/kg cis-platinum (LKT Laboratories, St Paul, Minn) and 15.0 mg/kg etoposide (LKT Laboratories) dissolved in 7:3 saline:DMSO. On day 2 of each week, they were given an intraperitoneal injection of 1.5 mg/kg bleomycin (LKT Laboratories) dissolved in saline. This dose regimen was chosen based on the standard dose given to humans (Benedetto, 1999), adjusted for surface area according to the following formula: f x mg/kg = mg/m2, where f equals 6.0 for the rat (Bachmann et al, 1996). This dosing regimen differs from that used in humans in that humans are treated for 1 week per cycle of 3 weeks; each course of treatment generally consists of 2 or 4 cycles.
Males were mated after 6 weeks of treatment and at the completion of the 9-week treatment. On day 6 of the week (a non-treatment day), males were placed in a cage overnight with 2 naturally cycling females in proestrous, as determined by vaginal smears. The progeny of these females were used for the analysis of fetal development on gestation day 21. Four days later, on day 2 of the subsequent week, the males were mated again. The progeny of these females were used for analysis of postnatal growth and development. Females were checked for sperm by vaginal smear on the morning after mating. This was considered to be gestation day 0.
Tissue Collection and Histology
At the end of the 9-week treatment, males were anesthetized and the ventral
prostate, seminal vesicles, left testis, and left epididymis were removed and
weighed. The contralateral testis and epididymis were cleared with saline and
perfused with Bouin fluid as a fixative through the abdominal aorta. The
tissues were then excised, postfixed for an additional 24 hours in the same
fixative, dehydrated, and embedded in paraffin. To evaluate spermatogenesis,
5-µm testis sections were cut and stained with periodic acid–Schiff,
according to the manufacturer's instructions (Sigma). Sections were viewed
with a Leica AS LMD microscope (Leica, Wetzlar, Germany) equipped with a RS
Photometrics CoolSNAP fx digital camera (Roper Scientific, Tucson, Ariz).
The left epididymides were sectioned into caput-corpus and cauda regions. The caput-corpus epididymides were frozen in liquid nitrogen for the determination of spermatozoal counts; spermatozoa from the cauda epididymidis were used for motility and morphology analyses, as described below.
Spermatozoal Counts
The previously frozen caput-corpus epididymides were homogenized in 5 mL of
0.9% saline, 0.1% merthiolate, and 0.05% Triton X-100 (VWR International,
Mississauga, Canada), for 2 intervals of 15 seconds separated by a 30-second
interval. Heads of spermatozoa were counted using a hemocytometer to assess
the absolute number of sperm per caput-corpus epididymidis
(Robb et al, 1978).
Spermatozoal Motility
Spermatozoa from the cauda epididymides were used immediately for
computer-assisted sperm analysis (CASA), as previously described
(Slott et al, 1993), with the
exception that the medium used was as follows: Hanks balanced salt solution
(Gibco Invitrogen Co, Grand Island, NY), supplemented with 4.2 mg/mL HEPES,
0.35 mg/mL sodium bicarbonate, 2.0 mg/mL bovine serum albumin, 0.9 mg/mL
D-glucose, and 0.025 mg/mL soybean trypsin inhibitor, pH 7.3–7.4, at
37°C (Klinefelter et al,
1991). Briefly, the epididymis was trimmed free of fat, rinsed in
medium, clamped at the corpus-cauda junction, and severed at the corpus side
of the clamp. Several tubules of the distal cauda were pierced with a #11
scalpel, and the tissues were transferred to a Petri dish containing 10 mL of
medium, allowing spermatozoa to disperse into the medium. The tissues were
removed, and the spermatozoa were left to disperse for several minutes. An
aliquot of 10 µL of the spermatozoa-containing medium was transferred to a
prewarmed 80-µm–deep glass cannula for CASA analysis using the
HTM-IVOS system (Hamilton Thorne Research, Beverly, Mass) and version 12 of
the Toxicology software (analysis speed = 60 Hz; minimum track duration = 30
frames at 60 Hz; minimum average-path velocity [VAP] = 50; minimum
straightness [STR] = 80). For each animal, 4 slides, each with two
80-µm–deep chambers, were analyzed. At least 100 spermatozoa per
slide were analyzed. The following parameters were determined: percent of
motile spermatozoa, percent of progressively motile spermatozoa, curvilinear
velocity (VCL), straight line velocity (VSL), VAP, amplitude of lateral head
displacement (ALH), beat cross frequency (BCF), linearity (LIN = VSL/VCL
x 100), and straightness (STR = VSL/VAP x 100).
Spermatozoal Morphology
The cauda epididymides were minced into 7 mL of phosphate-buffered saline
(PBS) (Roche). The suspension was filtered, washed several times with PBS,
fixed for 1 hour in 1% glutaraldehyde (Mecalab LTD, Montreal, Canada) in PBS,
and washed again with PBS. The resulting pellet was suspended in 1% agarose
(Gibco Invitrogen) and embedded for electron microscope analysis as follows.
The samples were washed 2 times in 0.1 M sodium cacodylate buffer containing
3% sucrose, pH 7.4; postfixed in 1% osmium tetroxide and 1.5% potassium
ferrocyanide; and embedded in epoxy resin. Spermatozoal ultrastructure was
analyzed on the electron microscope (Philips 410 Electron Microscope,
Eindhoven, The Netherlands). At least 100 midpiece cross-sections and 100
principal-piece cross-sections per sample were photographed. The number of
midpiece cross-sections and principal-piece cross-sections with any
abnormality, as well as the number with a cytoplasmic droplet, were
recorded.
Analysis of Pregnancy Outcome
On gestation day 21, sperm-positive females were sacrificed by
CO2 asphyxiation and laparotomized. The ovaries were removed, and
the numbers of corpora lutea were recorded. The uteri were removed and
examined for any abnormalities, including resorption moles. The numbers of
implantation sites in the uteri were recorded. The extent of preimplantation
loss per litter was calculated as follows: (number of corpora lutea - number
of implantation sites)/(number of corpora lutea). The number of
postimplantation losses was calculated as follows: (number of implantation
sites - number of live fetuses)/(number of implantation sites). The fetuses
were removed successively, blotted, weighed, examined for external
malformations, sacrificed by hypothermia, and immersion-fixed in Bouin
fixative. After fixation, the fetuses were dissected and the major organs
examined for internal malformations. The sex-ratio was calculated as the ratio
of the number of male fetuses to the total litter size.
Postnatal Growth and Development
Pregnant females were placed alone in a cage on gestation day 20. From day
21 onward they were monitored regularly for signs of labor. Labor was
considered complete when all pups were cleaned and nursing and when the
vaginal area was free of blood. Upon completion of labor, pups were removed
from the cage, weighed, sexed, examined for malformations, labeled by
toeclipping, and returned to the mother. The pups were weighed biweekly until
weaning, after which time they were weighed once per week until postnatal day
62. Pups were weaned on postnatal day 21, at which time 4 males and 4 females
from each litter were placed in separate cages, and the remaining pups were
sacrificed. The pups were monitored daily for any changes, and the days of eye
opening, vaginal opening, and preputial separation were recorded. Female pups
were sacrificed by CO2 asphyxiation at 10 weeks of age, and the
spleen, kidneys, and ovaries were weighed. Male pups were sacrificed at 13
weeks, and the spleen, kidneys, left testis, left epididymis, seminal
vesicles, and ventral prostate were weighed.
Statistical Analysis
The number of postnatal deaths was analyzed using the Fischer's exact test.
All other data were analyzed using the Student's t test, with
Bonferonni correction as appropriate. Data are presented as the mean plus or
minus standard error of the mean (SEM). The level of significance was
considered P < .05.
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Results |
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Testis Histology
We examined the histopathology of the testis of rats exposed to BEP for 9
weeks. Testis cross-sections of BEP-treated rats were characterized by severe
atrophic and germ cell–depleted seminiferous tubules compared to
controls, reflecting the decrease in testis weight
(Figure 3A and B). Loss
(sloughing) of immature germ cells into the lumen, giant multinucleated cell
formation, along with extensive vacuolization of seminiferous tubules and
Sertoli cell–only tubules were observed among the sections of treated
rats (Figure 3C through F).
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We observed an increase in the percent of spermatozoa with morphological abnormalities in the midpiece of the flagella after BEP treatment (Figure 6). The midpiece of a control spermatozoon consists of 9 microtubule doublet pairs surrounding 1 central doublet. Each of the 9 peripheral doublet pairs is associated with 1 outer dense fiber. Surrounding the outer dense fibers is the mitochondrial sheath (Figure 7A). In the control group, the only abnormalities seen were an abnormal spatial arrangement of outer dense fibers and the presence of 2 or more flagellar sections within 1 membrane. Some of the abnormalities observed in the midpiece cross-sections of spermatozoa from BEP-treated rats (Figure 7) were the same as the 2 abnormalities seen in the control group. Interestingly, additional abnormalities, not observed in the control group, were found in spermatozoa from the BEP-treated animals. These included an absence of the mitochondrial sheath, an abnormal number of outer dense fibers (either too many or too few), hemilateral absence of the axoneme and the outer dense fibers, a malformed mitochondrial sheath, the presence of some normal outer dense fibers and some that appear small and malformed, as well as sections that were malformed and had a combination of the abnormalities listed above, in addition to excess cytoplasm that contained outer dense fibers and debris (Figure 7). There was no increase in the percent of sperm with a cytoplasmic droplet in the BEP-treated group (data not shown).
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In addition to defects in the midpiece, an increase in the percent of spermatozoa with abnormalities in the principal piece of the flagella was observed (Figure 6). The principal piece of a spermatozoon from a control rat (Figure 8A) consists of the same 9 plus 2 arrangement of microtubules as the midpiece; however, outer dense fibers 3 and 8 are replaced by the 2 longitudinal columns of the fibrous sheath. Surrounding the outer dense fibers are the circumferential ribs. In this region of the flagella, the abnormalities seen in the BEP group were also seen in the control group, but at significantly higher incidences compared to the control group (Figure 6); these included a hemilateral absence of the outer dense fibers, with or without an intact axoneme, and a missing outer dense fiber (Figure 8).
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Fertility and Progeny Outcome
In pregnancies sired by males exposed to BEP for 6 or 9 weeks, all
sperm-positive females became pregnant; there were no changes in pre- or
postimplantation loss, litter size, or sex-ratio
(Table). In addition, there
were no effects on fetal weights. There were no external malformations in
either of the control groups or in the 6- or 9-week BEP-exposed groups. No
internal malformations were observed in any of the major organs, such as the
liver, kidneys, lungs, or heart.
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The second group of females that were mated to males after 6 and 9 weeks of BEP treatment were allowed to proceed through gestation to delivery. All the females mated to males that were treated for 6 weeks with BEP were pregnant and delivered live pups between gestation days 21 and 22. The average number of pups per litter that survived past day 1 was 14.9 ± 0.65 in the 6-week BEP group and 13.1 ± 1.1 in the control group. Additionally, the growth curves for the pups sired by 6-week BEP-treated males did not differ from the pups sired by control animals (data not shown). Developmental markers (dates of eye opening, vaginal opening, and preputial separation) did not differ between control and BEP-treated groups.
All of the females mated to males exposed to BEP for 9 weeks were also
sperm positive. However, unlike the females mated to males after 6 weeks of
BEP treatment, only 1 of 10 sperm-positive matings resulted in a litter of a
normal size (Figure 9). Of the
others, we observed one mother eat all of her pups while they were still
alive, one had 1 pup that died within 1 day, 3 had litters of 5 or less, and 4
others that were sperm positive were never observed with any pups. It is
noteworthy that the 1 normal-sized litter in the 9-week group was not sired by
the 1 male that had normal sperm motility. Interestingly, only 2 of the 10
sperm-positive females mated to BEP-exposed males went into labor on gestation
day 22 (P 
.002, 
2 analysis), compared with 7 out
of 7 females mated to control males. Of the other 8 females mated to
BEP-treated males, parturition occurred on gestation day 23 in 4, and for the
remaining 4 females, there was no observable evidence of surviving offspring;
in contrast, all females mated to control animals went into parturition on day
22 or earlier. Of the surviving pups sired by BEP-treated males, no
abnormalities were observed in postnatal growth
(Figure 10), developmental
markers (data not shown), or organ weights (data not shown).
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Discussion |
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In the current study, male rats were treated continuously for 9 weeks with BEP, and the reproductive organs and sperm quality were analyzed upon the completion of treatment. The BEP-treated animals experienced a substantial decrease in the weights of both the testes and epididymides. Reduction of the testis size (weight) along with disrupted spermatogenesis are well-known side effects of cisplatin-based chemotherapy (Lampe et al, 1997; Howell and Shalet, 2001). We observed that exposure to chronic BEP chemotherapy resulted in marked alterations of seminiferous tubule histology, with severely impaired spermatogenesis. At the testicular level, 9-week chronic BEP chemotherapy induced germ cell depletion and tubule atrophy. Abnormalities in the spermatogenesis process may account for the resulting low spermatozoal count observed in that group. We found that 71% of the BEP-treated rats had a spermatozoal count that was decreased by more than 90% when compared to controls. Relatively few studies have examined the impact of BEP treatment on the number of spermatozoa in men immediately after the completion of chemotherapy. The spermatozoal concentration in men who have undergone BEP gradually improves after completion of treatment (Tomomasa et al, 2002), and thus it is difficult to compare the response in humans with the results of the current study.
In a study by Stephenson et al (1995), 57% of human patients treated with BEP had less than 50% motile spermatozoa. In the rat, motility of less than 70% is considered abnormal (Seed et al, 1996). In the current study, 86% of drug-treated rats had spermatozoal motility below 70%. In addition to the percent motility, the VAP, VSL, VCL, BCF, and ALH values were significantly altered in the BEP-treated rats. Of these parameters, VSL, VCL, and BCF have been shown to be correlated with fertility (Toth et al, 1991). This may explain why fertility remains a problem even in patients who are not oligospermic (Hansen et al, 1991).
No studies to date have examined the morphology of spermatozoa from human testicular cancer patients at the electron microscope level. We observed an increase in the incidence of morphologically abnormal spermatozoa, as well as a larger variety of abnormalities, in BEP-treated rats. Interestingly, several of the midpiece abnormalities we observed uniquely in BEP-treated rats have been reported after other drug treatments, such as cyclosporine A (Masuda et al, 2003) or triptolide, a diterpene triepoxide isolated from a Chinese plant (Huynh et al, 2000). These abnormalities, however, are extremely rare in control rats (Syntin and Robaire, 2001). The flagellum is formed during spermiogenesis in the testis, indicating that the increase in abnormalities in BEP-treated rats probably reflects a defect in spermiogenesis, rather than epididymal maturation. This defect may be at the gene expression level and may occur as early as during the pachytene spermatocyte phase of spermatogenesis, as this is when the first genes required for flagellar formation are expressed (Horowitz et al, 2005). Nevertheless, a number of the flagellar defects found in selenium-deficient rats were detected only after the spermatozoa left the caput epididymidis (Olson et al, 2004), indicating that the defects we observed may also originate during spermatozoal maturation in the epididymis. In murine models, defects in the flagella of spermatozoa are frequently associated with infertility (Huttner et al, 1993; Olson et al, 2005).
To the best of our knowledge, this is the first study to assess the impact in the rat model of concurrent administration of bleomycin, etoposide, and cis-platinum. Each of these drugs, however, has previously been given individually to rats (Seethalakshmi et al, 1992; Russell et al, 2000, 2004). Of the 3 drugs, cis-platinum has been reported to affect the numbers of spermatozoa and their motility (Seethalakshmi et al, 1992). After 9 weeks of daily intraperitoneal treatment with 0.5 mg/kg cis-platinum, a decrease in sperm count of approximately 60% and a decrease in sperm motility of approximately 50% were reported in rats. The effect on motility is much greater than we observed in our study; however, the effect on sperm count is much smaller. The greater effect on motility with cis-platinum alone may be a result of the route of administration, although it is interesting to note that the percent of sperm that were motile in the control rats in that study (56%) is well below what is considered normal (Seed et al, 1996). However, the greater effect on numbers of spermatozoa in our study is likely to be due to an additive effect of the 3 drugs and underscores the value of elucidating the impact of the combination regimen in the rat model.
As a result of the effects of BEP treatment on sperm count, motility, and morphology, we anticipated a decrease in fertility; a reduction in sperm production of more than 90% has been demonstrated to impair fertility in the rat (Robaire et al, 1984). Furthermore, changes in motility and morphology are likely to impair fertility. Interestingly, paternal exposure to cis-platinum alone has been reported to increase the incidence of fetal malformations and deaths (Seethalakshmi et al, 1992). Surprisingly, after either 6 or 9 weeks of paternal BEP treatment, there was no adverse impact on fetal development, as assessed on gestation day 21; fetal morphology was normal, as were the numbers of live fetuses per litter. First, the sperm number required to maintain fertility in BEP-treated rats was less than 10% compared to controls. Second, even though a significant proportion of the remaining 10% have decreased motility and flagellar defects, their function was not affected sufficiently to alter progeny outcome. These results are in stark contrast to experiments with cyclophosphamide, another anticancer drug. After paternal treatment with this drug, at doses that did not alter male reproductive organ weights or sperm counts, there were increases in pre- and postimplantation loss and congenital malformations in the progeny (Trasler et al, 1985).
When the females mated to males treated with BEP for 9 weeks were allowed to deliver, a significant proportion of their pups did not survive past postnatal day 1. We observed that of 10 litters, only 1 was of a normal size. There was no decrease in litter size nor was there an increase in fetal deaths when pregnancy was terminated on gestation day 21, indicating that it is likely that many of the sperm-positive mothers that were allowed to proceed through gestation may have delivered full litters of live pups, which subsequently died and were eaten or were eaten alive. No obvious lethal defects were observed among the fetuses examined on gestation day 21. We speculate that delayed parturition had an adverse impact on postnatal survival in the 4 litters in which this occurred. Prenatal maternal exposures to dexamethasone, diethylstilbestrol, ethylene glycol dimethyl ether, 2-methoxyethanol, or Aroclor 1254 have been reported to delay parturition in rats (White et al, 1983; Chatterjee et al, 1993; Marty and Loch-Caruso, 1998; Lee et al, 2003). Delayed parturition is frequently associated with an increase in perinatal mortality (Rands et al, 1982; Leonhardt et al, 1991; Zimmerman et al, 1991). Alternatively, the pups may have had a fatal functional deficit that we did not observe. To the best of our knowledge, this is the first study to show an effect of paternal chemical exposure on the timing of parturition and postnatal survival of progeny.
These results clearly show that BEP treatment has a deleterious impact on the quality of spermatozoa in the male rat, resulting in a decrease in spermatozoal numbers and motility, an increase in morphologically abnormal spermatozoa, and harmful effects to the postnatal development of the progeny. These data indicate that the chemotherapeutic agents, rather than cancer or orchidectomy, may affect the quality of spermatozoa in patients who have been treated for testicular cancer.
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Acknowledgments |
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Footnotes |
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Both authors contributed equally to this work. 
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