[CANCER RESEARCH 47, 2340-2343, May 1, 1987)
Ovarian Toxicity of Cyclophosphamide
Irradiation in the Rat1
Alone and in Combination with Ovarian
J. Jarrell,2 E. V. Young Lai, R. Barr, A. McMahon, L. Belbeck, and G. O'Connell
Departments of Obstetrics and Gynecology fJ. J., E. V. Y. L., A. M., G. O.J, Pediatrics [R. BJ, and Pathology fL. BJ, McMaster University,
Hamilton, Ontario L8N 3Z5, Canada
ABSTRACT
The effects of radiation and chemotherapy on gonadal function are
relevant to the morbidity induced by such treatments. Cyclophosphamide
given i.p. to rats on Day 30 of age delayed vaginal opening, prevented
vaginal cyclicity, and caused a reduction in serum estradiol and proges
terone. Antrat follicular atresia increased in a dose-dependent fashion in
response to Cyclophosphamide (0 mg/kg, 53.5%; l mg/kg, 673%; 50 mg/
kg, 65.7%; 100 mg/kg, 73.9%; 150 mg/kg, 92.2%). Despite such altera
tions in ovarian function, serum gonadotrophins did not rise. The concur
rent administration of 0, 20, 30, 40, 50, and 60 Gy of radiation to the
exteriorized ovaries in rats receiving 50 mg/kg Cyclophosphamide induced
widespread loss of primordial, preantral, and healthy antrat follicles
associated with reduction in serum progesterone and estradiol. Such
irradiation induced dose-related increases in serum follicle-stimulating
hormone and luteinizing hormone. Parenteral Cyclophosphamide and
local irradiation appear to induce ovarian toxicity by different mecha
nisms.
INTRODUCTION
The role that antineoplastic agents play in the induction of
sterility is a topic of increasing interest (1-9). The fully devel
oped clinical picture in women in response to some agents is
that of premature ovarian failure and its associated hypergonadotrophic hypogonadal state, which has suggested that the pri
mary target of toxicity is the ovarian follicle complex (10).
Recently, acute dosing experiments with high doses of Cyclo
phosphamide were shown to cause a reduction in ovarian folli
cles over a 3-day period in Sprague-Dawley rats (11). Other
investigators have shown in a similar strain of rats that Cyclo
phosphamide resulted in a loss of medium-large follicles when
the drug was administered chronically at 5 mg/kg/day for 21
days after a loading dose of SO mg/kg (12). In addition, a
depressing effect of Cyclophosphamide in the hypothalamic
pituitary axis in cycling rhesus monkeys has recently been
reported (13).
It was the objective of these investigations to further evaluate
the reproductive toxicity of increasing doses of Cyclophospha
mide on Sprague-Dawley rats, using the parameters of organ
weights, follicle counts, and serum gonadotrophins, estradiol
and progesterone levels. Since irradiation also has effects on
ovarian function, it was of interest to study the combined effects
of Cyclophosphamide and direct ovarian irradiation which were
evaluated with respect to the same parameters.
were given food and water ad libitum. For the Cyclophosphamide doseresponse curve, animals received 0,1, 50, 100, 150, or 200 mg/kg body
weight Cyclophosphamide i.p. on Day 30 (5/group). They were sacri
ficed on Day 44. The animals were examined every day for vaginal
opening, and vaginal smears were taken to assess estrous cycles.
Animals in the combined treatment group were irradiated 24 h
following the injection of Cyclophosphamide (50 mg/kg) by methods
previously described (14, 15). Briefly, the animals were anesthetized
with pentobarbital and, through a mid-line incision, both ovaries were
exteriorized after ligation of the ovarian artery. The ovaries and the
uterus were withdrawn from the abdomen and secured to a lead box.
The animal was protected from irradiation by the lead box which had
walls 1 cm thick. Irradiation was carried out as previously described
with doses of radiation of 10 Gy/min to the ovary with a General
Electric Maxitron 250. The distance from the columnator was 17 cm.
Time and humidity were adjusted to deliver the appropriate dosimetry
to the ovaries (20, 30, 40, 50, 60 Gy).
All animals were killed on Day 44, and the weights of whole body,
pituitary, adrenals, uteri, and ovaries were determined. One ovary was
placed in Davidson's fixative for histology and stained with hematoxylin
and eosin, and the other ovary was placed in liquid nitrogen. Trunk
blood was also collected for the measurement of serum estradiol,
progesterone, FSH,3 and LH.
Radioimmunoassay. Serum LH and FSH were measured by homol
ogous double antibody radioimmunoassay techniques using reagents
provided by the National Institute of Arthritis, Diabetes, and Digestive
and Kidney Diseases (16). Estradiol and progesterone were extracted
from serum with diethyl ether, and dried ether extracts were subjected
to radioimmunoassay using specific antibodies to estradiol and proges
terone, respectively (14).
Histology. A single observer, blinded to groups and previously inter
nally validated, evaluated every third section (7 ¿im)
for primordial and
preantral follicles and every filth section for antral follicles, both healthy
and atretic, through one entire ovary of each animal. The ovarian
follicles were classified according to the method of Peterson and Peters
(17) utilizing a direct measurement of maximal diameter with a Bioquant Digitizing System as previously described (14, 15). Antral folli
cles were assessed for the presence of atresia utilizing the criteria of
Byskov (18). Only follicles in which there was an oocyte present in the
cross-section were evaluated. All corpora lutea in each ovary were
counted.
Statistical Analysis. The evaluation of statistical differences utilized
the analysis of variance, the Student two tailed t test, and Duncan's
multiple range test. Nonparametric data were evaluated with the Kruskall-Wallace and Mann-Whitney U tests. Statistical significance was
accepted at P< 0.05 (19).
RESULTS
MATERIALS AND METHODS
Female Sprague-Dawley rats aged 22 days were purchased from
Charles River and were placed on a 14-h light/10-h dark cycle. They
Received 9/16/86; revised 1/28/87; accepted 2/2/87.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by a grant from the National Cancer Institute of Canada. A
preliminary communication of these data was made at the Society for Gynecolog
ical Investigation, Toronto, 1986.
•'
To whom requests for reprints should be addressed, at Department of
Obstetrics and Gynecology, McMaster University Medical Centre, 1200 Main
St. W., Hamilton, Ontario L8N 3Z5, Canada.
Cyclophosphamide. Only one animal in the 1-mg/kg body
weight group displayed a pattern of vaginal cyclicity similar to
the normal 4-day pattern of control animals. All others dem
onstrated long periods of diestrus or absent proestrus days.
Vaginal opening was delayed significantly in the 150-mg/kg
group; rank sum, Kruskall-Wallis test, 0 mg/kg, 40.0; 1 mg/
kg, 82.5; 50 mg/kg, 68.0; 100 mg/kg, 99.0; 150 mg/kg, 140.5
days; H = 13.56, P < 0.01. Six animals died from chemother' The abbreviations used are: FSH, follicle-stimulating hormone; LH, lutein
izing hormone.
2340
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OVARIAN EFFECTS
OF CYCLOPHOSPHAMIDE
AND RADIATION
Table 1 Effects ofcyclophosphamide on whole-body weight and pituitary, adrenal, ovarian, and uterine weights
Dose(mg/kg
±8.3°(a)
150
155.2 ±8.4
130.2 ±9.0
124.8 ±29.0
100
ISORat(g)148.8 106.9 ±32.2 (c)Pituitary
1Mean ±SE. a, b, P < 0.05; a, c, P < 0.01.
6.9
5.6
3.7
3.4
ovary
(mg)53.4
adrenal
(mg)41.1
(mg)6.5
body wt)0
±2.8 (a)
±1.4
±1.5
±1.5
±0.8 (c)Combined
47.8
36.0
33.6
29.9
(mg)326.7
±5.3 (a)
±7.0
±4.8
53.2 ±8.1
±5.5
38.3 ±10.5
33.3+ 12.1
±2.4
±4.9 (b)Combined 21.1 ±0.7
(a)
(b)
(b)
(c)Uterus
±78.2 (a)
249.9 ±67.7
131.8±43.0(c)
82.6 ±22.5 (c)
82.6 ±22.5 (c)
Table 2 Effects ofcyclophosphamide on follicle population and ovarian follicular atresia
Dose(mg/kg)0
antrat61.4
±315'
228
933 ±346
1
288
1252 ±386
50
1169 ±269
305
100
150Primordial1376849 ±472Preantral281 226
" Mean ±SE. a, b, P< 0.05; a, c, P< 0.01; a, d, P<
±82
±73
±104
±72
±150Healthy
0.001.
29.8
40.6
34.8
2.3
apy-induced toxicity at 100 mg/kg (1), 150 mg/kg (1), and 200
mg/kg (4).
There was a parallel decline in whole-body weight, pituitary
weight, and adrenal weight (Table 1). There was a relative as
well as absolute decrease in the ovarian and uterine weights
when expressed per 100-g/body weight. These changes were
largely dose related and significant at 50 to 100 mg cyclophosphamide/kg body weight and above.
With respect to the follicle counts, there was no significant
change in the number of primordial and preantral follicles
present in the ovary after cyclophosphamide (Table 2). In
contrast, there was a prominent dose-related reduction in the
number of healthy antral follicles which was statistically signif
icant at the 150-mg/kg dose. Additionally, an increase in the
number of atretic antral follicles fell short of statistical signifi
cance (Table 2). There was a significant increase in the per
centage of antral atresia noted at the 1-mg/kg dose and above.
There was a significant reduction in the number of corpora
lutea at the 50-mg/kg dose and above.
The effects ofcyclophosphamide administration on the mean
maximal antral follicle diameter are represented in Table 3.
Where there is no effect of cyclophosphamide on atretic antral
follicles, there was a dose-related reduction in the mean maxi
mal diameter of healthy antral follicles, significant at the 150mg/kg dose. The effects of cyclophosphamide on the size dis
tribution of antral follicles are demonstrated in Table 4. At 150
mg/kg, the drug produced a significant decrease in the number
of antral follicles of all diameters from <200 urn to >600 ^m,
and the most profound effect was noted in small antral follicles
<200 juin. In contrast, there was a tendency for atretic antral
follicles <500 i/m to increase in response to all doses of cyclo
phosphamide (Table 4). The proportion of atresia increased
significantly in follicle ranges of <200, 200 to 300, 300 to 400,
and 400 to 500 /im.
The effects of cyclophosphamide on the serum levels of LH,
Table 3 Effects ofcyclophosphamide on the mean maximal diameters of antral
follicles dim)
Dose
(mg/kg
bodywt)0
1
SO
100
150Healthy
1Mean ±SE. a, b,/><0.05.
antral260.9
±39.7* (a)
237.6 ±13.8
224.3 ±14.6
206.3 ±21.3
165.4 ±1.5 (b)Atretic
antral304.7
±45.9
301.1 ±48.9
280.3 ±42.8
27 1.0 ±20.7
256.4 ±23.1
trai55.6
an
±31.5 (a)
±11.0
±18.4
±26.9
±2.1 (b)Atretic
64.4
73.4
79.6
75.0
±17.0
±22.4
+ 23.1
±15.6
±54.9%of
atresia53.5
67.3
65.7
73.9
92.2
±1.9 (a)
+ 5.5 (b)
±3.9 (b)
±6.9 (b)
±6.7 (d)Corpora
lutea10
15
5
4
2
±2 (a)
±2
±1 (b)
±1 (c)
±1 (d)
FSH, estradici, and progesterone are presented in Table 5.
There was no increase in the levels of either gonadotrophin
above control levels with increasing doses ofcyclophosphamide.
There was, however, a significant dose-related reduction in
serum estradiol and progesterone in animals treated with 50100 mg cyclophosphamide/kg.
Cyclophosphamide plus Irradiation. Cyclophosphamide plus
radiation altered the pattern of vaginal cyclicity, producing
periods of diestrus of up to 3 to 4 days and frequent patterns
of multiple estrus days. Estrus was commonly encountered
following diestrus. In these experiments, the cyclophosphamide-only control animals demonstrated similar abnormalities,
and no statistical differences were apparent. There was a sig
nificant reduction in uterine weights at 60 Gy and a highly
significant decline in ovarian weights (Table 6). There was no
change in body weights. The slight variation in control in
uterine weights may reflect nonsynchronous cyclicity at the
time of sacrifice.
Cyclophosphamide plus irradiation produced a dramatic de
cline in primordial follicles at the 20-Gy dose which was main
tained at all doses (Table 7). There was a similar although not
as severe decline in preantral and healthy antral follicles. No
response of atretic antral follicles was noted.
Animals treated with cyclophosphamide and irradiation had
significantly lower serum estradiol (Table 8). The serum FSH
and LH increased significantly with doses of radiation of 60
Gy.
DISCUSSION
The results of these investigations provide additional descrip
tive information concerning the effects of a potent alkylating
agent, cyclophosphamide, on the reproductive system of the
female rat. The findings of altered cyclicity and abnormal
follicular development in response to cyclophosphamide are
consistent with the clinical reports of similar abnormalities in
women treated with similar doses (20, 21) and monkeys treated
during normal ovulatory cycles (13).
The dose-related reductions in total numbers of follicles and
mean maximal diameters of healthy antral follicles <200 urn
are consistent with the reports of Ataya et at. (12) in which the
drug effect occurred only in follicles >30 urn. The current
findings suggest the possibility of a period of developmental
susceptibility within these experimental conditions. As the drug
was administered 14 days prior to sacrifice and the period for
2341
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OVARIAN EFFECTS
OF CYCLOPHOSPHAMIDE
AND RADIATION
Table 4 Effects of cyclophosphamide on the size distribution of antrat follicles fom)
No. of follicles
<200
Dose
Healthy01SOtoo150Atretic0150100ISO23.6
(a)12.8
±11.6"
5.919.8
±
8.618.0
±
12.11.7
±
(b)12.8
±1.5
±6.716.4
14.722.4
±
14.723.6
±
5.726.0
±
±17.319.4
200-<300
300-<400
400-<500
500-<600
11.810.4
±
5.015.4
±
±7.911.8±
10.30.6
0.518.4
±
±9.74.6
1.94.4
±
2.94.2
±
4.70.0
±
0.010.2
±
5.61.4
±
1.12.0
±
2.80.4
±
0.90.0
±
0.04.2
±
1.30.6
±
0.60.2
±
0.50.0
±
0.00.0
±
0.02.4
±
1.40.0
±
0.00.0
±
0.00.0
±
0.00.0
±
0.03.4
±
12.918.0
±
4.426.0
±
9.231.4
±
±4.623.0
±16.111.0
5.415.6
±
±4.213.4
±8.515.0
±7.215.0
±13.15.6
1.39.4
±
3.56.2
±
3.74.8
±
0.88.7
±
±7.10.8
I.I3.8±
3.33.8
±
2.72.2
±
1.61.7
±
±2.91.0
2.31.2
±
1.30.8
±
0.82.2
±
2.30.3
±
±0.6
>600
1Mean ±SE. a,b,/'<0.05.
Table 5 Effects of cyclophosphamide on serum gonadotrophin and steroid levels
Dose(mg/kg)0I
(ng/ml)84.3
±10.8°
(pg/ml)22.7
(ng/ml)13.8
±4.8 (a)
±33.1
±3.2 (a)
12.4 ±1.7
78.3 ±9.6
25 1.8 ±63.3 31.7 ±5.3
94.1 ±12.3
189.3 ±48.1
6.2 ±1.9(b)
9.3 ±1.7
50
100
73.6 ±9.2
155.9 ±36.8 11.2±3.5(b)
4.8 ±0.8 (c)
9.7 ±1.1 (b)Progesterone
133.1 ±26.4Estradiol
5.9 ±1.2 (c)
ISOLH 77.9 ±10.6FSH(ng/ml)130.9
' Mean ±SE. a, b, P < 0.05; a, c, P < 0.01.
Table 6 Effects of cyclophosphamide administration and ovarian irradiation on
weightControl
ovarian and uterine
(g)154.2
wt
(mg)48.4 wt
(mg)21 wt
±6.3°
±4.4 (a)
1.3 ±15.1 (a)
51.X ±4.5 (d)
Cyclophosphamide
140.1 ±4.4
247.5 ±33.5 (d)
CTX*-20Gy
223.2 ±34.6
140.3 ±2.7
33.3 ±1.9
CTX-30 Gy
136.2 ±4.3 26.8 ±2.6 (b, e) 192.5± 17.9
230.5 ±27.9
CTX^OGy
151.7 ±1.3
25.9 ±1.7(b, 0
CTX-50 Gy
141.9 ±1.0
25.6 ±3.7 (b, 0
20 1.9 ±35.9
CTX-60GyRat
149.5 ±5.1Ovarian
17.5±2.3(c, 0Uterine
147.7 ±10.9 (b, e)
" Mean ±SE between control and cyclophosphamide with radiation (a, b, P <
0.05; a, c, P < 0.01) and cyclophosphamide control with cyclophosphamide with
radiation (d, e, P < 0.05; d, f, P < 0.01).
" (IV cyclophosphamide treatment.
complete follicle maturation has been calculated to be 19 days
(22), the actual target may be the primordial or small premurai
follicle. Such findings are consistent with the work of Shiromizu
and Mattison (11) who reported that primordial germ cells are
the target in mice and to a lesser degree in rats.
It was interesting that doses of cyclophosphamide which
resulted in loss of vaginal cyclicity, reduction in organ weight,
and a reduction in serum steroids did not produce an elevation
in serum gonadotrophins. It appeared that either cyclophos
phamide did not have a significant effect on total ovarian FSH
suppression or that it may have had an effect on the hypothalainopi iuitary axis. Irradiation of the cyclophosphamide-1 rea tod
animals was done to determine if the additional ovarian lesion
could cause an elevation in serum FSH.
Previously reported experiments from this laboratory (14)
have shown that ionizing radiation alone in doses of 20 to 30
Gy to the exteriorized ovary resulted in no change in the onset
of puberty, no alteration in vaginal cyclicity, despite doserelated reductions in primordial, preantral, and healthy antral
follicles when compared to control or sham-irradiated rats. The
serum FSH was found to be increased with no change in serum
LH, reflecting continued normal serum estradici and proges
terone (14).
The combination of cyclophosphamide and radiation pro
duces a different picture of the reproductive status; the animals
did not cycle and had a delayed puberty as defined by vaginal
opening. The elevation of serum FSH and LH was greater when
cyclophosphamide was administered, reflecting greater ovarian
damage as evidenced by a significant reduction in serum pro
gesterone and estradici. It would appear that radiation and
cyclophosphamide act by way of different mechanisms to cause
significant disruption of reproductive function in the rat. Fur
ther studies will be helpful to determine if such mechanisms
are additive or synergistic. Cyclophosphamide may alter the
endogenous radioprotectant properties of healthy ovarian tis
sues such as Superoxide dismutase, glutathione, glutathione
peroxidase, and catalase, thereby promoting free radical dam
age within ovarian cells (23).
The current observations suggest, within these experimental
conditions, that animals treated with cyclophosphamide con
tinue to have an inhibition of the hypothalamopituitary-ovarian
axis, possibly as a consequence of continued ovarian inhibit!
production as serum estradici and progesterone values were
reduced (24, 25). Previous reports have suggested that antral
fluid from large follicles may be an important source of inhibin
(26), and yet despite the elimination of virtually all healthy
antral follicles with 150 mg/kg cyclophosphamide, the serum
FSH did not rise. The increase in number of atretic follicles
would not appear to maintain such inhibition as atresia has
been associated with a decreased ability to secrete inhibin (27).
The exquisite sensitivity of primordial follicles and preantral
follicles to radiation and the subsequent increase in serum FSH
suggests smaller follicles may be equally if not more important
Table 7 Effects of cyclophosphamide (50 mg/kg) administration and irradiation of the ovaries on ovarian follicles
antral38
antral60.3
±531.7°(a)
±53.0 (a)
±6 (a)
±18.2
Control
1485.3 ±175.4 (d)
277.8 ±32.6 (d)
42 ±6.5 (d)
62.8 ±9.2
Cyclophosphamide
Cyclophosphamide-20 Gy
14.8 ±5.5 (b, e)
33.0 ±8.6 (c, f)
141.8 ±32.9
52.0 ±7.3
Cyclophosphamide-30 Gy
50.3 ±15.6(c, 0
105.5 ±9.3
11.8±3.6(b,e)
48.0 ±13.9
Cyclophosphamide-40 Gy
17.3± 10.9(c, f)
58.8 ±3.5 (c)
5.5 ±0.6 (c, 0
41.8 ±2.6
Cyclophosphamide-50 Gy
83.3 ±16.2(b)
10.0 ±3.3 (c, 0
35.8 ±22.7 (c, 0
55.0 ±9.8
Cyclophosphamide-60 GyPrimordial1702.618.8 ±10.3 (c, 0Preantral206.4 85.6 ±19.9 (b)Healthy
2.0 ±1.1 (c,f)Atretic
34.6 ±10.0
* Mean ±SE compared between control and cyclophosphamide with radiation (a, b, P < 0.05; a, c, P < 0.01 ) and cyclophosphamide control with cyclophosphamide
with radiation (d, e. P < 0.05; d, f, P < 0.01 ).
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OVARIAN EFFECTS OF CYCLOPHOSPHAMIDE
AND RADIATION
Table 8 Effects of cyclophosphamide and ovarian irradiation on serum steroid and gonadotrophin levels
(pg/ml)31.2
±8.3°
(a)
±6.2 (a)
Control
±14.9 (a)
±53.0 (a)
15.8±7.0(d)
Cyclophosphamide
9.0 ±2.6 (d)
55.3 ±7.6 (d)
267.5 ±98.6 (d)
CTX*-20Gy
36.1 ±12.5
13.5 ±2.2
51.8 ±4.9
328.0 ±112.6
CTX-30Gy
8.4 ±1.9 (h)
16.6 ±3.1
61.8 ±4.9
384.0 ±161.2
CTX-40Gy
12.1 ±2.2
11.8±2.2(b)
72.0 ±5.8
807.0 ±213.9
CTX-50 Gy
14.8 ±7.2
8.3 ±2.7 (b)
66.0 ±9.9
692.5 ±182.4
CTX-eOGyEstradiol
6.7±2.1(c,e)Progesterone(ng/ml)19.8
4.1 ±0.9(c)LH(ng/ml)59.0112.8 ±34.7 (b, e)FSH(ng/ml)330.0
1354.0 ±238.4 (b, f )
* Mean ±SE compared between control and cyclophosphamide with radiation (a, b, P< 0.05; a, c, P < 0.01) and cyclophosphamide control with cyclophosphamide
with radiation (d, e, P < 0.05; d, f, P < 0.01).
* (IV cyclophosphamide treatment.
in the regulation of serum FSH than larger follicles under these
conditions. Alternatively, the follicular fluid of atretic antral
follicles induced by these conditions may still provide signifi
cant quantities of inhibin. However, despite the loss of ovarian
FSH inhibition when cyclophosphamide-treated rats underwent
ovarian irradiation, an additional deleterious effect of cyclo
phosphamide in the hypothalamus may be present due to the
loss of vaginal cyclicity and pubertà ! delay, consistent with the
findings of Pfeiffer et al. (13) in the rhesus monkey. Further
studies of possible hypothalamic-pituitary function in response
to cyclophosphamide appear warranted.
The evaluation of the reproductive toxicity of chemotherapeutic agents may clarify the putative protective capacity of
gonadotrophin-releasing
hormone analogues in a variety of
tissues (28-31).
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1987 American Association for Cancer Research.
Ovarian Toxicity of Cyclophosphamide Alone and in
Combination with Ovarian Irradiation in the Rat
J. Jarrell, E. V. Young Lai, R. Barr, et al.
Cancer Res 1987;47:2340-2343.
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