Surgical Oncology 1992 ; 1 : 145-150
Preservation of fertility following doxorubicin administration in the rat F . E . JOHNSON, G . J . LIEBSCHER, M . C . LAREGINA* AND K . C . TOLMAN Departments of Surgery and *Comparative Medicine, St Louis University Medical Center, St Louis, MO, USA
Several hundred thousand men receive chemotherapy each year; many are sterilized by this treatment . Testicular circulatory isolation (TCI), a regional drug exclusion approach to circumvent chemotherapy-related infertility, lessens doxorubicin-induced testicular injury in the rat . We evaluated the effect of TCI on doxorubicin-induced infertility in this study. Thirty-two eight-week-old male Sprague-Dawley rats were used . Eight rats received TCI for 45 min . Eight received doxorubicin (i .v . bolus) plus sham surgery . Eight received i .v . doxorubicin given immediately after institution of TCI . Eight controls received sham surgery alone . Mating studies began 2 months later . Six of the 8 males receiving TCI alone were fertile . In the doxorubicin-treated, sham-operated group, 0 of 7 animals were fertile . In the doxorubicin-treated group which also received TCI, 2 of 7 males were fertile . In the sham-operated group, all 8 rats were fertile . This is the first evidence that a regional drug exclusion technique can improve fertility in this model . Surgical Oncology 1992 ; 1 :145-150. Keywords : doxorubicin, drug toxicity, infertility, rat, regional drug delivery, xenobiotic.
INTRODUCTION
9,10-tetra hyd ro-6, 8,11-tri hyd roxy-8-(hydroxyacetyl)I-methoxy-5,12-naphthacenedione] and testicular injury in a rat model [2] ; we have also used the rat as a model for the regional drug exclusion technique, which is referred to as testicular circulatory isolation (TCI) . This model is simple, reproducible, and associated with good long-term health . Although the duration of TCI is highly correlated with testicular injury [3], Stern and co-workers have shown that 45 min of TCI has no long-term effects on testicular weight, sperm head count, or histology [4] . Lui et at have shown that TCI provides partial protection from doxorubicin-induced testicular toxicity [5] . However, since testicular function is only one component of fertility, and since testicular function is presumably the only one affected by TCI, we investigated TCI as a means to preserve fertility in the doxorubicin-treated rat model .
Drug-related testicular injury was appreciated early in the development of anti-cancer drugs . This was of little consequence at the time, since most drug regimens were ineffective and most patients died of their malignancy . Later, adjuvant chemotherapy regimens began to be evaluated, in which a fraction of the patients were expected to survive independent of any effect of chemotherapy . In these patients, drug-related infertility became a real clinical issue which has continued to the present time . Current drug treatment is often curative for cancers such as lymphomas, leukaemias, testicular cancer, and certain childhood cancers . In these patients retention of fertility is an important issue . We have approached this problem experimentally using an approach which involves temporary interruption of blood flow to the testicle during drug administration . Lui et al. have described the relationship between doxorubicin [(8 S-cis)-1o-[(3-amino2, 3, 6-tri-deoxy-alpha-L-lyxohexopyranosyl) oxy]-7, 8,
METHODS Animals
Correspondence : Dr Frank E . Johnson, Department of Surgery, St . Louis University Medical Center, 3635 Vista o Grand, PO Box 15250, St . Louis, MO 63110-0250, USA .
Sixty-eight eight-week-old male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing approxi145
1 46
F. E.
Johnson et al .
mately 225 g were used for this study . This is a fertile strain with a low incidence of spontaneous testicular abnormalities [6] . Three rats were housed per cage with free access to tap water and Purina rodent laboratory chow 5001 . One week was allowed for health screening and adaptation to the new environment prior to entering the study .
Drug Doxorubicin
(Adria Laboratory,
Wilmington,
Delaware) was dissolved in sterile 0 .15m NaCl immediately prior to administration, as recommended by the manufacturer.
Experimental design
Experiment 1 Thirty-two rats were randomly assigned to four treatment groups (n=8 per group) . Identifying ear notching was done after grouping . Animals were given general anaesthesia using methoxyflurane by nose cone plus intraperitoneal sodium pentobarbital (60 mg kg - ' body weight), with supplemental doses (15 mg kg -') given as needed . The left testicles of all rats were surgically exposed through a ventral scrotal incision and cooled using saline-soaked gauze on an ice-filled dish . Each animal received 1 ml of sterile 0.15 M NaCl subcutaneously after
Figure 1 . Testicular circulatory isolation : note vascular clamps in place . A Doppler probe is used to confirm cessation and return of testicular blood flow . Doxorubicin administration is by surgical exposure of the jugular vein in order to avoid extravasation .
induction . Rats in group 1 received left spermatic cord and gubernaculum clamping using vascular clamps for 45 min as described previously [4] (TCI : Fig . 1) . Rats in group 2 received doxorubicin (5 mg kg -1
body weight, i .v . bolus) plus sham surgery,
of birth . Pup length was measured with Vernier
which consisted of testicular exposure and cooling .
calipers accurate to 0 .1 mm . Pup weight was
Group 3 rats received a 45 min period of TCI plus i .v . doxorubicin, given immediately after application of
measured using an Ohaus electronic balance
vascular clamps to the gubernaculum and spermatic cord . In all doxorubicin-treated rats, drug adminis-
were noted, with microscopic evaluation reserved
accurate to 0 .01 g . Surface and visceral anomalies for grossly visible lesions .
tration was via the jugular vein exposed surgically .
We planned to perform several mating cycles for
Group 4 rats received sham surgery alone . All surgery was performed under sterile conditions .
all the males in this series, in order to evaluate fertil-
Post-operatively all rats were inspected daily ; those
tration . However, many of the drug-treated males
which expired prior to mating were not replaced .
died before the planned second mating cycle, which
On postoperative day 56, all surviving rats were caged with two 8-week-old female SpragueDawley rats for a period of 2 weeks (which exceeds the length of the oestrus cycle) . All females were then observed for 1 month and all resulting rat pups were counted, inspected, and necropsied on the day
ity at varying time points after doxorubicin adminis-
led us to perform a further experiment using a lower doxorubicin dose .
Experiment 2 A similar trial was then done on 36 rats using a lower dose of i .v. doxorubicin (2 .5 mg kg - ' body
Doxorubicin-induced infertility
147
weight) . Each of the groups in this experiment comprised 9 rats . In experiment 1, we observed the
length and weight (±SEM) were 51 .1±0 .2 mm and 6 .77 ± 0 .04 g, respectively . Neither were significantly
emergence of a pecking order in the female rats
different among groups (ANOVA) .
which appeared to prevent access of the male to the
A concurrent group of 7 untreated pairs had 5
second female. Thus, in experiment 2, only one
litters, giving a baseline indication of fertility for
female was caged with each male at post-operative
unmanipulated animals under the conditions of this
day 56 .
study . No gross surface or visceral abnormalities were found in any rat pup in either series ; thus no microscopic evaluation was done .
RESULTS
Experiment 1 DISCUSSION All doxorubicin-treated rats (groups 2 and 3) in this series failed to gain weight . One rat each from groups 2 and 3 expired prior to mating, confirming
It has been estimated that approximately 15,000 patients each year in the USA are curable with
that a toxic dose of doxorubicin had been used in
current chemotherapy, accounting for about 10% of
this work . In group 1, 6 of 8 males were fertile ; 11 of 16 females produced litters . In group 2, 0 of 7 males
all cured cancers per year [7) . Adjuvant therapy is used in several thousand additional patients annu-
were fertile ; 0 of 14 females produced litters . In
ally who are of reproductive age and are ultimately
group 3, 2 of 7 males were fertile ; 3 of 14 females
long-term disease-free survivors in unmaintained
produced litters . In group 4, 8 of 8 males were
remission from neuroblastomas, sarcomas, etc . In
fertile ; 11 of 16 females produced litters (Table 1) .
each of these groups, about half of the patients are
In this series, the overall mean litter size (± SEMI was 13±1 . Overall mean pup length and weight
male and aspects of quality of survival are emerg-
(±SEM) were 50 .0±0.2 mm and 6 .47±0.04 g,
ing ; prominent among these is the issue of fertility . Among chemotherapy-treated patients, permanent
respectively . We excluded group 2 from analysis
infertility is common [1, 8-12], related to agents
because all males were infertile and found no differ-
used (doxorubicin and alkylating agents such as pro-
ence in litter size, pup length, or pup weight among
carbazine are particularly potent germ-cell toxins),
groups 1, 3, and 4 (ANOVA) .
and dose-related [13] . Animal data support these clinical observations [2, 14-17] ; some agents have
Experiment 2
only temporary effects 118] while others have per-
In the rats receiving low-dose doxorubicin, there were no deaths or apparent morbidity . All animals gained weight normally . Eight of 9 rat pairs in group
manent toxicities [2] . Inadequate recovery from cytotoxic therapy-related infertility is apparently a function of stem cell killing [19) . Although data are
1 were fertile . In group 2, 8 of 9 rat pairs were
limited, the pattern of sexual behaviour in chemo-
fertile . Seven of 9 rat pairs in group 3, and 6 of 9 rat
therapy-treated patients who are cured of their
pairs in group 4, produced litters .
cancer approaches pre-morbid levels [20, 21], and infertility appears to severely degrade the quality of
The overall mean litter size (±SEM) for all four groups in this series was 12±1, which was not significantly different among groups (ANOVA) . Mean pup
Table 1 . Fertility among experimental groups
survival . This topic has recently been reviewed [20-22], though major informational gaps exist in
Group
1 (TCI*)
2 (DOX*+ SHAM*)
3 (DOX+ +TCI)
4 (SHAM)
DOX dose, mg kg - ' Fertile/total
2 .5 5 .0 8/9 6/8
2 .5 5 .0 8/9 0/7f
2 .5 5 .0 7/9 2/7'
2 .5 6/9
*TCI indicates testicular circulatory isolation ; DOX, doxorubicin ; SHAM, sham
surgery. ?Before mating, 1/8 died of DOX toxicity .
5 .0 8/8
1 48
F. E. Johnson et al,
the literature . Limited data obtained via planned biopsies (mainly in leukaemics) shows that severe disruption of histology is very common [23, 24] and parallels data derived from measurement of testicular size [25], fertility [12], or sperm counts [26] . Two arguments have been made against efforts to preserve fertility in such patients . First is the possibility of transmitting unfavourable gene pools into such patient's offspring . However, family studies of cancer patients and children of chemotherapy-treated cancer patients have generally not demonstrated an increased incidence of cancer or other disorders (27-29] . Second is the gonadal dysfunction which is known to exist in some male cancer patients even before the onset of treatment [22, 30] . More recent evidence suggests that this may be related to the presence of cancer rather than an intrinsic testicular abnormality and is often reversible with successful cancer therapy [22] . The fever of Hodgkin's disease, for example, appears to account for many of the seminal fluid abnormalities of this disease . We feel these considerations should not stand in the way of efforts to minimize gonadal toxicity of anti-cancer agents . Approaches to this problem have been numerous but rather passive : sperm banking, reliance on spontaneous recovery from testicular injury, manipulation of drug choice/dose/schedule, etc . Serious difficulties have been encountered with each of these approaches . Sperm banking is generally seen as a major solution, but the available literature (which is rather sparse) suggests that this is severely flawed [22] . The approach tested in the current work is one of few active interventions which have been described . Lui et al. [5] have demonstrated significant protection of the rat testicle from doxorubicin-related drug toxicity using TCI during bolus doxorubicin administration . Regional drug-delivery approaches have been useful in other organs to limit drug toxicities . For over 30 years, isolation-perfusion of the extremities has been an accepted technique which permits very high drug levels to be delivered to a region of the body involved with cancer . Innovative approaches which regionalize drug delivery to lung, liver, brain, and other organs have been reported . The testicular anatomy is certainly favourable for a similar approach . A potential difficulty of this regional drug exclusion technique would be that testicular metastases
or second primary testicular cancers [31) would receive decreased treatment . However, such metastases are distinctly uncommon [32], and' second primary testis cancers occur in only about 1-2% [33] or less [34] of testicular cancer patients . An obvious exception would be involvement of the testicle with leukaemia, which reportedly occurs in 5-15% of patients (35] . However, secondary treatments can reliably eradicate testicular relapses . In childhood lymphoma patients, testicular involvement is not seen in early-stage disease and is only seen in 3% of patients overall . Most instances are unilateral, and salvageable [36] . High-dose chemotherapy and marrow autotransplantation will probably be applied increasingly in the future and carries the risk of even greater testicular injury . TCI may prove useful in these patients . The use of other testicular toxins (such as radiopharmaceuticals) [37] represents another area of hazard which might be suitable for the application of TCI . We are aware that hormonal manipulation of susceptibility of spermatogenic epithelium to toxic agents has been reported - using LHRH [38] and testosterone [39], for example . These approaches have fallen short of their theoretical promise, just as there has been incomplete testicular protection with TCI as reported here . These approaches are not mutually exclusive, suggesting that combined hormonal manipulation and TCI at the time of drug delivery may be valuable . By diminishing the metabolic demand of spermatogenic cells, such medical therapy may not only partially protect against cytotoxicity of the chemotherapy agents, but may also protect against the ischemic injury of TCI . Doxorubicin, an anthracycline, is a common component of many multi-agent treatment regimens for various cancers afflicting young men . Certain pharmacological aspects of doxorubicin metabolism suggest that mechanical testicular circulatory isolation may be worthwhile . When doxorubicin is administered as a single i .v . bolus, its plasma concentration decays as a double exponential function of time for the first 48 h . While the plasma T 1/2 alpha is estimated to be 20 min in the rat [40], in man this is only 4-7 min [40, 41] . This suggests that short periods of testicular circulatory isolation may be adequate to avoid doxorubicin-induced toxicity . We assayed fertility on day 56±2 for several
Doxorubicin-induced infertility reasons . The spermatogenic cycle in rats is normally 48 days [42] ; it may be longer after exposure to a cytotoxic insult such as ionizing irradiation
[43] .
Fifty-six days should provide enough time for surviving stem cells to progress through the pathway of maturation and produce a new generation of spermatozoa . This time span is also short enough to
149
7. DeVita VT, Chapter 13 in DeVita VT, Hellman S, Rosenberg SA (ads), Cancer. Principles and Practice of Oncology (2nd Ed .) Philadelphia : Lippincott, 1985 . 8. Kreuser ED, Harsch U, Hetzel WD, et al. Gonadal toxicity in patients with testicular cancer after chemotherapy. EurJCancer Clin Onco11986 ; 22 : 2899. Sorio R, Tirelli U, Zagonel V, Monfardini S . Sexual function among adult patients with malignant lympho-
minimize regeneration of surviving stem cells .
mas undergoing combination chemotherapy .
Spermatogenic cells which were already differen-
ASCO 1987 ; 6 : 204 .
tiated at the time of doxorubicin administration
Proc
10 . Roeser HP, Stocks AE, Smith AJ . Testicular damage
should have left the testis by this time . We thus felt
due to cytotoxic drugs and recovery after cessation of
that day 56 would provide a good point to assess
therapy . Aust NZJ Med 1978 ; 8 : 250 . 11 . Schilsky RL, Lewis BJ, Sherins RJ, Young AC . Gonadal
the value of TCI in fertility preservation . This study emphasizes the difficulty of single-dose
dysfunction in patients receiving chemotherapy for
doxorubicin administration for establishment of a
cancer . Ann lnt Med 1980 ; 93 : 109 . 12 . Charak BS, Gupta R, Mandrekar P, et al. Testicular
testicular injury, since a dose of 5 mg kg - ' body weight produced approximately 10% animal mortality by day 56, whereas 50% dose reduction abolished the effect on fertility . Nonetheless, TCI appeared to provide partial protection from doxorubicin-induced infertility in our first experiment. We find this particularly exciting since, prior to this work, we had carried out several pilot mating studies of rats treated with doxorubicin at 5-6 mg
dysfunction
after
cyclophosphamide
vincristine-
procarbazine-prednisolone chemotherapy for advanced Hodgkin's disease . Cancer 1990 ; 65 : 1903-6 . 13 . Austin GJS, Pektasicles D, Bagshawe KD, et al. Fertility after chemotherapy for male and female germ cell tumours . lntjAndrol 1987 ; 10 : 389 . 14 . Horstman MG, Meadows GG, Yost GS . Separate mechanisms for procarbazine spermatotoxicity and anticancer activity . Cancer Res 1987 ; 47 : 1547 .
kg - ' and observed universal infertility (unpublished) .
15 . DaCunha MF, Meistrich ML, Nader S . Absence of testi-
It is also noteworthy that TCI alone did not diminish
cular protection by a gonadotropin-releasing hormone analogue against cyclophosphamide-induced testicular
fertility . These studies are encouraging enough that we have begun preliminary trials using a similar clamping
approach,
not requiring
surgery, to
regionalize drug delivery in humans .
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18 . Vawda Al, Davies AG . Effects of cisplatin on the
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mouse testis. Acta Endocrinol 1996 ; 112 : 436 . stem cell survival and testis function after cytotoxic therapy . Br J Cancer 1986 ; 53 : 89 . 20. Devlen J, Maguire P, Phillips P, et al. Psychological problems associated with diagnosis and treatment of lymphomas I . Retrospective study. Br Med J 1987 ; 295 : 953 .
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KC, Johnson FE . Long-term outcome following testicu-
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25. Sames MA, Rautonen J . Small testicles with impaired
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production of sperm in adult male survivors of child-
lymphoma involving the testis : clinical features and
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Radiat
Res
Preservation of fertility following doxorubicin administration in the rat F . E . JOHNSON, G . J . LIEBSCHER, M . C . LAREGINA* AND K . C . TOLMAN Departments of Surgery and *Comparative Medicine, St Louis University Medical Center, St Louis, MO, USA
Several hundred thousand men receive chemotherapy each year; many are sterilized by this treatment . Testicular circulatory isolation (TCI), a regional drug exclusion approach to circumvent chemotherapy-related infertility, lessens doxorubicin-induced testicular injury in the rat . We evaluated the effect of TCI on doxorubicin-induced infertility in this study. Thirty-two eight-week-old male Sprague-Dawley rats were used . Eight rats received TCI for 45 min . Eight received doxorubicin (i .v . bolus) plus sham surgery . Eight received i .v . doxorubicin given immediately after institution of TCI . Eight controls received sham surgery alone . Mating studies began 2 months later . Six of the 8 males receiving TCI alone were fertile . In the doxorubicin-treated, sham-operated group, 0 of 7 animals were fertile . In the doxorubicin-treated group which also received TCI, 2 of 7 males were fertile . In the sham-operated group, all 8 rats were fertile . This is the first evidence that a regional drug exclusion technique can improve fertility in this model . Surgical Oncology 1992 ; 1 :145-150. Keywords : doxorubicin, drug toxicity, infertility, rat, regional drug delivery, xenobiotic.
INTRODUCTION
9,10-tetra hyd ro-6, 8,11-tri hyd roxy-8-(hydroxyacetyl)I-methoxy-5,12-naphthacenedione] and testicular injury in a rat model [2] ; we have also used the rat as a model for the regional drug exclusion technique, which is referred to as testicular circulatory isolation (TCI) . This model is simple, reproducible, and associated with good long-term health . Although the duration of TCI is highly correlated with testicular injury [3], Stern and co-workers have shown that 45 min of TCI has no long-term effects on testicular weight, sperm head count, or histology [4] . Lui et at have shown that TCI provides partial protection from doxorubicin-induced testicular toxicity [5] . However, since testicular function is only one component of fertility, and since testicular function is presumably the only one affected by TCI, we investigated TCI as a means to preserve fertility in the doxorubicin-treated rat model .
Drug-related testicular injury was appreciated early in the development of anti-cancer drugs . This was of little consequence at the time, since most drug regimens were ineffective and most patients died of their malignancy . Later, adjuvant chemotherapy regimens began to be evaluated, in which a fraction of the patients were expected to survive independent of any effect of chemotherapy . In these patients, drug-related infertility became a real clinical issue which has continued to the present time . Current drug treatment is often curative for cancers such as lymphomas, leukaemias, testicular cancer, and certain childhood cancers . In these patients retention of fertility is an important issue . We have approached this problem experimentally using an approach which involves temporary interruption of blood flow to the testicle during drug administration . Lui et al. have described the relationship between doxorubicin [(8 S-cis)-1o-[(3-amino2, 3, 6-tri-deoxy-alpha-L-lyxohexopyranosyl) oxy]-7, 8,
METHODS Animals
Correspondence : Dr Frank E . Johnson, Department of Surgery, St . Louis University Medical Center, 3635 Vista o Grand, PO Box 15250, St . Louis, MO 63110-0250, USA .
Sixty-eight eight-week-old male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing approxi145
1 46
F. E.
Johnson et al .
mately 225 g were used for this study . This is a fertile strain with a low incidence of spontaneous testicular abnormalities [6] . Three rats were housed per cage with free access to tap water and Purina rodent laboratory chow 5001 . One week was allowed for health screening and adaptation to the new environment prior to entering the study .
Drug Doxorubicin
(Adria Laboratory,
Wilmington,
Delaware) was dissolved in sterile 0 .15m NaCl immediately prior to administration, as recommended by the manufacturer.
Experimental design
Experiment 1 Thirty-two rats were randomly assigned to four treatment groups (n=8 per group) . Identifying ear notching was done after grouping . Animals were given general anaesthesia using methoxyflurane by nose cone plus intraperitoneal sodium pentobarbital (60 mg kg - ' body weight), with supplemental doses (15 mg kg -') given as needed . The left testicles of all rats were surgically exposed through a ventral scrotal incision and cooled using saline-soaked gauze on an ice-filled dish . Each animal received 1 ml of sterile 0.15 M NaCl subcutaneously after
Figure 1 . Testicular circulatory isolation : note vascular clamps in place . A Doppler probe is used to confirm cessation and return of testicular blood flow . Doxorubicin administration is by surgical exposure of the jugular vein in order to avoid extravasation .
induction . Rats in group 1 received left spermatic cord and gubernaculum clamping using vascular clamps for 45 min as described previously [4] (TCI : Fig . 1) . Rats in group 2 received doxorubicin (5 mg kg -1
body weight, i .v . bolus) plus sham surgery,
of birth . Pup length was measured with Vernier
which consisted of testicular exposure and cooling .
calipers accurate to 0 .1 mm . Pup weight was
Group 3 rats received a 45 min period of TCI plus i .v . doxorubicin, given immediately after application of
measured using an Ohaus electronic balance
vascular clamps to the gubernaculum and spermatic cord . In all doxorubicin-treated rats, drug adminis-
were noted, with microscopic evaluation reserved
accurate to 0 .01 g . Surface and visceral anomalies for grossly visible lesions .
tration was via the jugular vein exposed surgically .
We planned to perform several mating cycles for
Group 4 rats received sham surgery alone . All surgery was performed under sterile conditions .
all the males in this series, in order to evaluate fertil-
Post-operatively all rats were inspected daily ; those
tration . However, many of the drug-treated males
which expired prior to mating were not replaced .
died before the planned second mating cycle, which
On postoperative day 56, all surviving rats were caged with two 8-week-old female SpragueDawley rats for a period of 2 weeks (which exceeds the length of the oestrus cycle) . All females were then observed for 1 month and all resulting rat pups were counted, inspected, and necropsied on the day
ity at varying time points after doxorubicin adminis-
led us to perform a further experiment using a lower doxorubicin dose .
Experiment 2 A similar trial was then done on 36 rats using a lower dose of i .v. doxorubicin (2 .5 mg kg - ' body
Doxorubicin-induced infertility
147
weight) . Each of the groups in this experiment comprised 9 rats . In experiment 1, we observed the
length and weight (±SEM) were 51 .1±0 .2 mm and 6 .77 ± 0 .04 g, respectively . Neither were significantly
emergence of a pecking order in the female rats
different among groups (ANOVA) .
which appeared to prevent access of the male to the
A concurrent group of 7 untreated pairs had 5
second female. Thus, in experiment 2, only one
litters, giving a baseline indication of fertility for
female was caged with each male at post-operative
unmanipulated animals under the conditions of this
day 56 .
study . No gross surface or visceral abnormalities were found in any rat pup in either series ; thus no microscopic evaluation was done .
RESULTS
Experiment 1 DISCUSSION All doxorubicin-treated rats (groups 2 and 3) in this series failed to gain weight . One rat each from groups 2 and 3 expired prior to mating, confirming
It has been estimated that approximately 15,000 patients each year in the USA are curable with
that a toxic dose of doxorubicin had been used in
current chemotherapy, accounting for about 10% of
this work . In group 1, 6 of 8 males were fertile ; 11 of 16 females produced litters . In group 2, 0 of 7 males
all cured cancers per year [7) . Adjuvant therapy is used in several thousand additional patients annu-
were fertile ; 0 of 14 females produced litters . In
ally who are of reproductive age and are ultimately
group 3, 2 of 7 males were fertile ; 3 of 14 females
long-term disease-free survivors in unmaintained
produced litters . In group 4, 8 of 8 males were
remission from neuroblastomas, sarcomas, etc . In
fertile ; 11 of 16 females produced litters (Table 1) .
each of these groups, about half of the patients are
In this series, the overall mean litter size (± SEMI was 13±1 . Overall mean pup length and weight
male and aspects of quality of survival are emerg-
(±SEM) were 50 .0±0.2 mm and 6 .47±0.04 g,
ing ; prominent among these is the issue of fertility . Among chemotherapy-treated patients, permanent
respectively . We excluded group 2 from analysis
infertility is common [1, 8-12], related to agents
because all males were infertile and found no differ-
used (doxorubicin and alkylating agents such as pro-
ence in litter size, pup length, or pup weight among
carbazine are particularly potent germ-cell toxins),
groups 1, 3, and 4 (ANOVA) .
and dose-related [13] . Animal data support these clinical observations [2, 14-17] ; some agents have
Experiment 2
only temporary effects 118] while others have per-
In the rats receiving low-dose doxorubicin, there were no deaths or apparent morbidity . All animals gained weight normally . Eight of 9 rat pairs in group
manent toxicities [2] . Inadequate recovery from cytotoxic therapy-related infertility is apparently a function of stem cell killing [19) . Although data are
1 were fertile . In group 2, 8 of 9 rat pairs were
limited, the pattern of sexual behaviour in chemo-
fertile . Seven of 9 rat pairs in group 3, and 6 of 9 rat
therapy-treated patients who are cured of their
pairs in group 4, produced litters .
cancer approaches pre-morbid levels [20, 21], and infertility appears to severely degrade the quality of
The overall mean litter size (±SEM) for all four groups in this series was 12±1, which was not significantly different among groups (ANOVA) . Mean pup
Table 1 . Fertility among experimental groups
survival . This topic has recently been reviewed [20-22], though major informational gaps exist in
Group
1 (TCI*)
2 (DOX*+ SHAM*)
3 (DOX+ +TCI)
4 (SHAM)
DOX dose, mg kg - ' Fertile/total
2 .5 5 .0 8/9 6/8
2 .5 5 .0 8/9 0/7f
2 .5 5 .0 7/9 2/7'
2 .5 6/9
*TCI indicates testicular circulatory isolation ; DOX, doxorubicin ; SHAM, sham
surgery. ?Before mating, 1/8 died of DOX toxicity .
5 .0 8/8
1 48
F. E. Johnson et al,
the literature . Limited data obtained via planned biopsies (mainly in leukaemics) shows that severe disruption of histology is very common [23, 24] and parallels data derived from measurement of testicular size [25], fertility [12], or sperm counts [26] . Two arguments have been made against efforts to preserve fertility in such patients . First is the possibility of transmitting unfavourable gene pools into such patient's offspring . However, family studies of cancer patients and children of chemotherapy-treated cancer patients have generally not demonstrated an increased incidence of cancer or other disorders (27-29] . Second is the gonadal dysfunction which is known to exist in some male cancer patients even before the onset of treatment [22, 30] . More recent evidence suggests that this may be related to the presence of cancer rather than an intrinsic testicular abnormality and is often reversible with successful cancer therapy [22] . The fever of Hodgkin's disease, for example, appears to account for many of the seminal fluid abnormalities of this disease . We feel these considerations should not stand in the way of efforts to minimize gonadal toxicity of anti-cancer agents . Approaches to this problem have been numerous but rather passive : sperm banking, reliance on spontaneous recovery from testicular injury, manipulation of drug choice/dose/schedule, etc . Serious difficulties have been encountered with each of these approaches . Sperm banking is generally seen as a major solution, but the available literature (which is rather sparse) suggests that this is severely flawed [22] . The approach tested in the current work is one of few active interventions which have been described . Lui et al. [5] have demonstrated significant protection of the rat testicle from doxorubicin-related drug toxicity using TCI during bolus doxorubicin administration . Regional drug-delivery approaches have been useful in other organs to limit drug toxicities . For over 30 years, isolation-perfusion of the extremities has been an accepted technique which permits very high drug levels to be delivered to a region of the body involved with cancer . Innovative approaches which regionalize drug delivery to lung, liver, brain, and other organs have been reported . The testicular anatomy is certainly favourable for a similar approach . A potential difficulty of this regional drug exclusion technique would be that testicular metastases
or second primary testicular cancers [31) would receive decreased treatment . However, such metastases are distinctly uncommon [32], and' second primary testis cancers occur in only about 1-2% [33] or less [34] of testicular cancer patients . An obvious exception would be involvement of the testicle with leukaemia, which reportedly occurs in 5-15% of patients (35] . However, secondary treatments can reliably eradicate testicular relapses . In childhood lymphoma patients, testicular involvement is not seen in early-stage disease and is only seen in 3% of patients overall . Most instances are unilateral, and salvageable [36] . High-dose chemotherapy and marrow autotransplantation will probably be applied increasingly in the future and carries the risk of even greater testicular injury . TCI may prove useful in these patients . The use of other testicular toxins (such as radiopharmaceuticals) [37] represents another area of hazard which might be suitable for the application of TCI . We are aware that hormonal manipulation of susceptibility of spermatogenic epithelium to toxic agents has been reported - using LHRH [38] and testosterone [39], for example . These approaches have fallen short of their theoretical promise, just as there has been incomplete testicular protection with TCI as reported here . These approaches are not mutually exclusive, suggesting that combined hormonal manipulation and TCI at the time of drug delivery may be valuable . By diminishing the metabolic demand of spermatogenic cells, such medical therapy may not only partially protect against cytotoxicity of the chemotherapy agents, but may also protect against the ischemic injury of TCI . Doxorubicin, an anthracycline, is a common component of many multi-agent treatment regimens for various cancers afflicting young men . Certain pharmacological aspects of doxorubicin metabolism suggest that mechanical testicular circulatory isolation may be worthwhile . When doxorubicin is administered as a single i .v . bolus, its plasma concentration decays as a double exponential function of time for the first 48 h . While the plasma T 1/2 alpha is estimated to be 20 min in the rat [40], in man this is only 4-7 min [40, 41] . This suggests that short periods of testicular circulatory isolation may be adequate to avoid doxorubicin-induced toxicity . We assayed fertility on day 56±2 for several
Doxorubicin-induced infertility reasons . The spermatogenic cycle in rats is normally 48 days [42] ; it may be longer after exposure to a cytotoxic insult such as ionizing irradiation
[43] .
Fifty-six days should provide enough time for surviving stem cells to progress through the pathway of maturation and produce a new generation of spermatozoa . This time span is also short enough to
149
7. DeVita VT, Chapter 13 in DeVita VT, Hellman S, Rosenberg SA (ads), Cancer. Principles and Practice of Oncology (2nd Ed .) Philadelphia : Lippincott, 1985 . 8. Kreuser ED, Harsch U, Hetzel WD, et al. Gonadal toxicity in patients with testicular cancer after chemotherapy. EurJCancer Clin Onco11986 ; 22 : 2899. Sorio R, Tirelli U, Zagonel V, Monfardini S . Sexual function among adult patients with malignant lympho-
minimize regeneration of surviving stem cells .
mas undergoing combination chemotherapy .
Spermatogenic cells which were already differen-
ASCO 1987 ; 6 : 204 .
tiated at the time of doxorubicin administration
Proc
10 . Roeser HP, Stocks AE, Smith AJ . Testicular damage
should have left the testis by this time . We thus felt
due to cytotoxic drugs and recovery after cessation of
that day 56 would provide a good point to assess
therapy . Aust NZJ Med 1978 ; 8 : 250 . 11 . Schilsky RL, Lewis BJ, Sherins RJ, Young AC . Gonadal
the value of TCI in fertility preservation . This study emphasizes the difficulty of single-dose
dysfunction in patients receiving chemotherapy for
doxorubicin administration for establishment of a
cancer . Ann lnt Med 1980 ; 93 : 109 . 12 . Charak BS, Gupta R, Mandrekar P, et al. Testicular
testicular injury, since a dose of 5 mg kg - ' body weight produced approximately 10% animal mortality by day 56, whereas 50% dose reduction abolished the effect on fertility . Nonetheless, TCI appeared to provide partial protection from doxorubicin-induced infertility in our first experiment. We find this particularly exciting since, prior to this work, we had carried out several pilot mating studies of rats treated with doxorubicin at 5-6 mg
dysfunction
after
cyclophosphamide
vincristine-
procarbazine-prednisolone chemotherapy for advanced Hodgkin's disease . Cancer 1990 ; 65 : 1903-6 . 13 . Austin GJS, Pektasicles D, Bagshawe KD, et al. Fertility after chemotherapy for male and female germ cell tumours . lntjAndrol 1987 ; 10 : 389 . 14 . Horstman MG, Meadows GG, Yost GS . Separate mechanisms for procarbazine spermatotoxicity and anticancer activity . Cancer Res 1987 ; 47 : 1547 .
kg - ' and observed universal infertility (unpublished) .
15 . DaCunha MF, Meistrich ML, Nader S . Absence of testi-
It is also noteworthy that TCI alone did not diminish
cular protection by a gonadotropin-releasing hormone analogue against cyclophosphamide-induced testicular
fertility . These studies are encouraging enough that we have begun preliminary trials using a similar clamping
approach,
not requiring
surgery, to
regionalize drug delivery in humans .
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