Reversible decreases of fertility in male Sprague-Dawley rats treated orally with finasteride, a 5α-reductase inhibitor

Reproductive Toxicology, Vol. 5, pp. 337-346, 1991

0890-6238/91 $3.00 + .00 Copyright © 1991 Pergamon Press pie

Printed in the U.S.A.

• Original Contributions

REVERSIBLE DECREASES OF FERTILITY IN MALE SPRAGUE-DAWLEY RATS TREATED ORALLY WITH FINASTERIDE, A 5a-REDUCTASE INHIBITOR L. DAVID WISE, DAVID H. MINSKER, MARK A. CUKIERSKI, ROBERT L. CLARK, SRINIVASA PRAHALADA, JOSEPH M . ANTONELLO, JAMES S. MACDONALD, a n d RICHARD T. ROBERTSON Merck Sharp & Dohme Research Laboratories, Department of Safety Assessment, West Point, Pennsylvania Abstract -- Finasteride, a 5~-reductase inhibitor, was investigated for its effects on fertility in male rats as part of its preclinical safety assessment. Studies were initiated when the male Sprague-Dawley rats were either young (4 to 6 weeks old) or mature (15 weeks old). Treatment duration ranged from 6 to 32 weeks. Each male was cohabited with two untreated females at various periods during and after treatment. Litter parameters were evaluated on either day 14 or 20 of gestation. Males were necropsied at the end of treatment or 7 to 11 weeks following the end of treatment. The major findings of these studies were that 1) young rats given 20 to 80 mg/kg/day of finasteride first showed mild to moderate decreases in fertility after 12 weeks of treatment, whereas mature males (given only 80 mg/kg/day) did not show a similar decrease until 24 weeks of treatment, 2) fewer copulatory plugs and atrophy of prostates and seminal vesicles were associated with t'masteride treatment, 3) the decreased fertility was only partial (ie, fertility index did not decrease below 48% of control in any study) and was not due to decreases in mating, 4) formation of copulatory plugs, organ weights, and fertility returned to normal levels after at least 6 weeks of drug withdrawal, and 5) the testes showed no histologic or weight changes that would explain the effect on fertility. These results show that the decreased fertility in male rats was associated with flnasteride-induced inhibition of accessory gland secretions, an expected pharmacologic effect. Key Words: finasteride;5-alpha-reductase;rat fertility;reproductivetoxicity.

icle, epididymis, and penis in immature male rats (4); and growth of the above tissues in immature castrate male rats supplemented with testosterone (5,6); prostatic growth in dogs (7); growth of the fetal gubernaculum in rats (8); nipple differentiation in the neonatal rat (9); and virilization of external genitalia in fetal rats (10,I1) and guinea pigs (12). These processes are, therefore, largely under DHT control. To our knowledge there has been only one published report in which the reproductive function in laboratory animals was assessed with this class of enzyme inhibitor (13). In that study a compound with 5oL-reductase inhibitory activity (N,N-diethyl-4-methyl-3-oxo-4aza-5a-androstane-1713-carboxamide, also known as 4-MA), which also possesses antiandrogen activity, had no effect on fertility when mature male rats were treated sc for 67 days with dose levels of 10 and 50 mg/kg/day. Significant decreases in the weights of the ventral prostate (down to 44% of control) and seminal vesicles (down to 34% of controls) were seen at the end of treatment. It is likely that the duration of treatment and/or

INTRODUCTION Finasteride (also known as L-652,931, MK-0906, and Proscar®) is a 4-azasteroid with potent and specific inhibitory activity against 5a-reductase, the enzyme responsible for the conversion of testosterone (T) to 5adihydrotestosterone (DHT). The compound does not appear to interfere with androgen receptor binding or androgen transport (1). Finasteride has been shown to decrease serum DHT levels in men (2), and is in development for the treatment of benign prostatic hyperplasia. In laboratory animals, agents with 5a-reductase inhibitory activity have been shown to alter the following processes in vivo: prostatic differentiation and growth in neonatal rats (3); growth of the prostate, seminal ves-

Address correspondence to Dr. L. David Wise, Merck Sharp & Dohme Research Laboratories, Safety Assessment (W45-115), West Point, PA 19486. Received 11 June 1990; Accepted 28 July 1990. 337

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Reproductive Toxicology

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Table 1. Outline of study animals, treatments, and timings of cohabitations and necropsies

Study Protocol parameters

1

Age of males at start of 4--6 treatment (weeks) Males per group 19-20 Weight range at start of 75-150 treatment (g) Finasteride dose levels 0, 20, 40, 80 (mg/kg/day) Weeks of treatment prior 12 to cohabitation Total weeks of treatment 27 Recovery period prior to -final cohabitation (weeks) Male necropsy, weeks after 0 end of treatment Age of males at necropsy (weeks) 31-32

2

3 6

15 180-231

15 30a 435-535

0, 80

0, 80

6, 12

6, 12, 24, 30

14 6

32 6

11.3 31

0, 7 47, 54

a14 and 15 males per group were necropsied immediately and 7 weeks after treatment, repectively.

the low number of animals (N = 6) were inadequate to observe a significant effect on fertility. The purpose of the present investigations was to examine the effects of finasteride on the reproductive organs, mating, and fertility of male rats. In addition, the reversibility of the observed effects was examined.

METHODS These studies were carried out under Good Laboratory Practice Regulations for nonclinical safety assessment studies.

Animals Sprague-Dawley rats (CrhCD(SD) BR, obtained from Charles River Breeding Laboratories, Wilmington, MA) were maintained under standard laboratory conditions in accordance with standards established by the Federal Animal Welfare Act and the Institute for Laboratory Animal Resources. Rats were housed individually in wire-bottomed cages in climate controlled rooms with 12 h light/dark cycles. Food (Purina certified chow) and drinking water were provided ad libitum.

Drug Finasteride (also known as L-652,931, MK-0906, and Proscar ®) is chemically designated as N-(1,1-dimethylethyl)-3-oxo-4-aza-5oL-androst-l-ene- 17[3-carboxamide. In all studies the dosing formulations were prepared daily in the vehicle, 0.5% aqueous methylcellulose (Dow Chemical Company, Pittsburgh, PA, or Fisher Scientific Company, Pittsburgh, PA), at concentrations appropriate to deliver 5 mL/kg body weight to each animal. Control animals received 5 mL/kg/day of the ve-

hicle. Analyses of drug content in the various dosing preparations were conducted periodically during the studies; all concentrations were within at least 10% of the expected concentrations. The drug has been shown to be stable under these dosing conditions.

Assessment of fertility Three sequential studies were conducted to assess the effects of finasteride treatment on male rat fertility. In the first two studies, young (4 to 6 weeks old) male rats, sexually immature at study initiation, were utilized, while in the third study treatment was initiated in adult (15 weeks old) sexually mature rats. A general outline of these studies is shown in Table 1. All male rats were weighed twice per week during treatment and weekly during recovery until termination. Each rat was examined daily for mortality and physical signs of toxicity. To assess reproductive performance, each male was cohabited with two untreated female rats at the periods stated in Table 1. Cohabitation was limited to a maximum of 16 nights, except during the last two cohabitations of Study 3 where mating was limited to 8 nights. Treatment of the males continued during the cohabitation periods, except during recovery in Studies 2 and 3. Female rats were 10 to 12.5 weeks of age at the time of mating. A vaginal lavage was obtained daily from each female and examined microscopically for the presence of sperm. The presence and number (Studies 2 and 3) of copulatory plugs were also noted. The day of finding sperm in the vaginal lavage was considered day 0 of gestation. Each presumed pregnant rat was observed for physical signs and weighed on days 0, 7, and 14 (and day 20 in Study 1) of gestation, and ob-

Fertility assessment of finasteride in male rats • L. D. WISEEl"AL

339

Table 2. Summary of reproductive performance of male rats treated with finasteride beginning at 4 to 6 weeks of age (Study 1) or 6 weeks of age (Study 2)

Endpoints Males cohabited Males that mated Fertile males, % Mean day of mating° Mating incidenced'c Females with plugs/ females mated, %d Plugs/mated femaled Fecundity indexd'f Fertility indexd's

Study 1a (mg/kg/day) 20 40

Control 20 18 85 4.8 85 69 . 92 78

19 18 89 3.8 87 50* .

. 78 68

Study 2 80

(6) b

Control (12) (14+R c)

19 17 79 5.2 76 47*

20 18 90 4.8 82 47*

15 15 93 3.0 97 80

15 15 100 3.9 97 87

15 15 93 3.5 100 97

15 15 87 3.1 83 37**

15 15 73 5.0 87 57*

15 15 100 4.5 93 77*

68* 50*

75* 60*

1.0 60 57

1.8 80 77

2.5 90 90

0.53* 73 60

1.0" 53* 47*

2.9 97 90

.

(6)

80 mg/kg/day (12) (14+R c)

aResults after 12 weeks of dosing. bNumbers in parentheses refer to weeks of dosing prior to cohabitation. CR = recovery period of 6 weeks. dparameter calculated on a per male basis. ~Mating incidence = mated females/females cohabited ( × 100). fFecundity index = pregnant females/mated females ( x 100). ~Fertility index = pregnant females/females cohabited ( x 100). *P --- 0.05, **P -< 0.01. (Difference from the concurrent control group is statistically significant.) *P -< 0.05, *P --<0.01. (Significant decreasing trend).

served for mortality daily. On day 14 (Studies 2 and 3) or day 20 (Study 1) of gestation, females were euthanized with C02 gas and the numbers of corpora lutea, resorptions, and live and dead embryos or fetuses were recorded. In Study 1, live fetuses were weighed and given external gross examinations.

Necropsy and histopathology All males were euthanized by exsanguination under ether or CO 2 asphyxiation at the times specified in Table 1. All males in Study 1 underwent a complete gross examination, and selected organ weights were recorded. Numerous tissues from all animals were fixed in 10% neutral buffered formalin, while the testes and epididymides were fixed in Bouin's solution. Samples of most organs and tissues were embedded in paraffin, sectioned at 6 izm, and stained with hematoxylin and eosin for light microscopic examination. All males in Studies 2 and 3 underwent limited postmortem examinations consisting of gross observations, weights (except epididymides), and histology of the testes, epididymides, prostate, and seminal vesicles with coagulating glands.

Statistical methods In Studies 2 and 3, treatment differences in the fertility, fecundity, mating (see Table 2 footnotes for definitions), and copulatory plug indices were assessed within each cohabitation using a randomization test (14). For each parameter, the within-male response outcomes (0/2, 0/1), 1/2, and (2/2, 1/1) were scored as 0,

0.5, and 1, respectively. Treatment differences in timeto-mating per mated female, number of copulatory plugs per mated female, numbers of implants and live fetuses per pregnant female, and preimplantation and postimplantation loss were assessed within each cohabitation on either the untransformed or rankit transformed (15, 16) data using an analysis of variance (ANOVA) model for a nested design. Treatment differences in testes, prostate, and seminal vesicle weights were assessed on the rankit transformed percent of body weight data using a one-way ANOVA. In Study 1, since the treatments included three graded doses of finasteride and a vehicle control group, the NOSTASOT (NO-STAtistical-Significance-Of-Trend) sequential trend testing approach (17) was used to assess the effects of finasteride on litter, organ weight, and reproductive performance parameters. Trends in fertility, fecundity, mating, and copulatory plug indices were assessed within the NOSTASOT framework using the extended Mantel-Haenszel test statistic (18). With the exception of fetal weight, all statistical tests were one-sided. Hypothesis testing was performed at the 0.05, 0.01, and 0.001 levels of significance. RESULTS

Mortality, adverse physical signs, and body weight change There were no deaths, adverse physical signs, or effects on body weight gain related to drug treatment in any of the 3 studies. In Study 3, rats given 80 mg/kg/

340

Reproductive Toxicology

Volume 5, Number 4, 1991 Study 2

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Fig. 1. Fertility indices from studies of fnasteride-treated male rats. Values presented as percent of control. Asterisk above column denotes statistically signifcant difference from control: *P -< 0.05, **P <-- 0.01, ***P -< 0.001. R = recovery period.

copulatory plugs, either below the wire cage floor or in the vagina, was significantly decreased in the finasteridetreated groups (38%, 32%, and 32% below the concurrent control in the 20, 40, and 80 mg/kg/day groups, respectively). In Study 2 the copulatory plugs found beneath the wire-bottomed cage floor and/or in the vagina were counted. The average number of plugs per mated female was significantly decreased in the finasteride-

day did not show a significant change in mean body weight gain during the dosing period.

Treatment starting with sexually immature males (4-6 weeks old) 1. Evaluation of copulatory plugs and mating (Table 2). In Study 1 the percentage of mated females with

Table 3. Summary of litter parameters from female rats cohabited with finasteride-treated males beginning at 4 to 6 weeks of age (Study 1) or 6 weeks of age (Study 2) Study 1"

Study 2 b

(mg/kg/day) Endpoints Total females Pregnant females Not pregnant females Did not mate Preimplantation loss, % Implants/female Resorptions Dead fetuses Resorptions/implants, % Live fetuses/female Live fetal weight

Control

80 mg/kg/day

Control

20

40

80

(6)

(12)

(14+R c)

(6)

(12)

(14+R c)

40 31 3 6 17.9

38 26 7 5 19.6

38 19 10 9 17.0

40 24 9 7 24.4

30 17 12 1 13.1

30 23 6 1 15.6

30 27 3 0 9.3

30 18 7 5 18.1

30 14 12 4 17,7

30 27 1 2 4.0

13.7 18 0 4.1 13.1 3.73

13.4 13 1 4.0 12.8 3.65

14.5 12 0 4.2 13.9 3.75

13.3 29 0 9.2** 12.1 3.62

13.7 11

12.7 14

13.8 20

12.4 15

125 6

14.1 14

5.0 13.1

5.0 12.1

7.2 13.1

5.8 11.6

3.6 12.6

3.6 13.6

"Females cesarean-sectioned on day 20 of gestation. bFemales cesarean-sectioned on day 14 of gestation. ¢R = recovery period of 6 weeks. **P <-- 0.01. (Difference from the concurrent control group is statistically significant.)

Fertility a s s e s s m e n t o f finasteride in m a l e rats • L, D. WISE Er AL

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Fig. 2. Relative reproductive organ weights of male rats treated with 80 mg/kg/day of finasteride. Relative (% of body weight) weights of testes, prostate, and seminal vesicles are presented as percent of control. Asterisk(s) above column denotes statistically significant difference from control: *P -< 0.01, **P -< 0.001. R = recovery period. treated group during the treatment period (0.53 and 1.0 plugs per female compared to 1.0 to 1.8 in the control group after 6 and 12 weeks of treatment, respectively). The percentage of mated females with plugs was also significantly decreased in rats administered 80 mg/kg/ day (54% and 34% below control after 6 and 12 weeks of treatment, respectively). There was no apparent effect on the libido of finasteride-treated males, as evident in the incidence of mated females (determined by the presence of sperm in the vaginal lavage). Likewise, the mean day of mating did not show any meaningful delays compared to the concurrent control groups.

2. Evaluation of fertility. Significant decreasing trends in the fecundity index (pregnant females per mated females, per male) were observed (see Table 1 and Figure 1) through the 40 and 80 mg/kg/day groups in Study 1 (26% and 18% below the control, respectively). The decrease observed in the 20 mg/kg/day

group (15% below control) approached statistical significance (P = 0.07). Decreases in fertility indexes (pregnant females per females cohabited, per male) were observed in the three groups (13%, 36%, and 23% below control in the 20, 40, and 80 mg/kg/day groups, respectively), but trend analysis was significant only through the two higher dose groups. The findings from the 80 mg/kg/day group in Study 1 were essentially repeated in Study 2. After 6 weeks of treatment in Study 2, there were no apparent effects on the fecundity or fertility indexes of the finasteridetreated group compared to the concurrent control group. The control group at this cohabitation had low fecundity and fertility indexes (60% and 57%, respectively), which may have been due to the young age (12 weeks) and/or inexperience of the males. As in Study 1, after 12 weeks of treatment with 80 mg/kg/day there were significant decreases in fecundity and fertility indexes (34% and 39% below control, respectively).

342

Reproductive Toxicology

Volume 5, Number 4, 1991

Table 4. Summary of reproductive performance of male rats treated with finasteride beginning at 15 weeks of age--Study 3 Weeks of Dosing Endpoints Males cohabited Males that mated Fertile males, % Mean day of matingb Mating incidenceb'c Females with plugs/ females mated, %b Plugs/mated femaleb Fecundity index~'d Fertility indexb'c

6

12

Control 24

30

31 + Ra

6

30 30 97 3.4 95 83

30 30 97 3.3 100 90

30 29 93 3.5 95 91

29 29 93 3.7 93 95

15 14 87 3.6 90 89

30 30 90 3.6 97 75

29 29 93 5.2** 90* 79

29 28 72 4.5 90 75*

29 29 55 4.0 84 83

15 15 93 4.1 87 83

2.5 78 75

2.8 87 87

2.2 84 80

2.4 88 84

2.2 75 70

1.5"* 72 70

1.5*** 84 76

1.3"* 52*** 47***

1.5"* 45*** 40***

2.3 87 73

12

80 mg/kg/day 24 30

31 + Ra

aR = recovery period of 6 weeks. bparameter calculated on a per male basis. CMating incidence = mated females/females cohabited ( x 100). dFecundity index = pregnant females/mated females ( x 100). eFertility index = pregnant females/females cohabited ( × 100). *P - 0.05, **P ~ 0.01, ***P ~ 0.001, (Difference from the concurrent control group is statistically significant.)

3. Evaluation

of litter and fetal parameters.

Administration o f finasteride to male rats beginning at 4 to 6 weeks o f age did not adversely affect preimplantation loss, the number of implants or live fetuses per litter, the resorption rate, or the average fetal weight (Study 1 only) (see Table 3). There was a significant increase in the percent resorptions per litter in Study 1 at the 80 mg/kg/day dose level; however, this was not considered to be an effect of drug treatment since there was no similar effect at this dose level in Study 2 at either 6 or 12 weeks of drug treatment, and the rate was within the limits of historical control values for this laboratory (0% to 10%).

4. Evaluations after 6-week recovery period. Following 6 weeks of no drug treatment, the males in Study 2 were again cohabited with untreated females. The percent of mated females with copulatory plugs following 6 weeks of drug withdrawal (77%) was increased relative to the week 6 and 12 observations (37% and 57%, respectively), but remained less than the concurrent control group (97%). The fecundity and fertility indexes returned to control levels after this recovery period. Litter and fetal parameters were comparable to the concurrent controls (Tables 2 and 3, Figure 1). Treatment starting when males were sexually mature (15 weeks) 1. Evaluation of copulatory plugs and mating. In this study (Study 3) the average number of plugs per mated female was significantly decreased during the treatment period in the finasteride-treated group (1.3 to 1.5 plugs per female compared to 2.2 to 2.8 in the control) (see Table 4). The percentage o f mated females in

the finasteride-treated group that had at least one copulatory plug was also slightly decreased during the treatment period. The most pronounced decrease was observed after 24 weeks of treatment (18% below concurrent control). The percentage of mated females in the finasteride-treated group having at least one copulatory plug was within 7% of the control group after the recovery period. The mating indices in the finasteride-treated group were comparable to the control group at all cohabitations.

2. Evaluation of fertility. There was no effect on fecundity or fertility of the finasteride-treated males after 6 and 12 weeks of treatment (Table 4 and Figure 1). Decreases in the fecundity and fertility indexes became apparent after 24 and 30 weeks of treatment, at which time each index was significantly (P <_ 0.001) below the control value. The fertility index decreased to 52% below control after 30 weeks of treatment, At the 24and 30-week cohabitations, there was an increase in the number o f finasteride-treated males that mated but failed to produce a pregnancy. After 6 and 12 weeks o f drug treatment, only 3 and 2 males, respectively, failed to produce a pregnancy compared to 1 control male, but thereafter the number of these males increased to 7 (25%) at 24 weeks and to 13 (45%) at 30 weeks, compared to 2 (7%) in the control group. The 7 nonfertile males at 24 weeks also did not produce a pregnancy after 30 weeks. Three of these 7 males were subsequently cohabited with females after the recovery period, and 2 produced a pregnancy. 3. Evaluation of litter parameters. No meaningful effects of finasteride treatment were observed on litter parameters in Study 3 (Table 5). The percent preimplan-

Fertility assessment of finasteride in male rats • L. D, W~SEE'r At

343

Table 5. Summary of litter parameters from female rats cohabited with finasteride-treated males beginning at 15 weeks of age--Study 3 Weeks of Dosing Control Endpoints Total females Pregnant females Not pregnant females Did not mate Preimplantation loss, % Implants/female Resorptions/ implants, % Live fetuses/ femaleb

80 mg,/kg/day

6

12

24

30

31 + R a

6

12

24

30

31 + R a

60 45 12 3 4.6

60 52 8 0 11.2

60 48 9 3 10.1

58 49 5 4 11.0

30 21 6 3 15.5

60 42 16 2 10.7

58 44 8 6 19.5

58 27 25 6 23.3*

58 23 26 9 12.7

29 21 4 4 28.2*

14.4 5.9

14.3 6.7

14.0 6.7

13.2 6.3

14.4 5.3

13.2 9.4

12.3 9.6

12.8 5.2

13.3 6.2

11,5' 4.8

13.6

13.4

13.0

12.4

13.6

12.5

11.7

12.0

12.7

11.0"

aR = recovery period of 6 weeks. *P -< 0.05. FD(Difference from the concurrent control group is statistically significant.)

tation loss at 24 weeks was significantly (P -< 0.05) increased; however, this was not considered to be biologically meaningful since no such increase was observed after continued treatment to 30 weeks.

4. Evaluations after 6-week recovery period. Following a 6 week period of no drug treatment, the remaining males (15 per group) were again cohabited with two females each. The results of this cohabitation show that all previously affected parameters (plugs per mated female, fecundity index, and fertility index) returned to control levels (see Tables 4 and 5, Figure 1). Percent preimplantation loss, mean implants per female, and mean live fetuses per female were statistically different from the control values, but these effects were not considered biologically meaningful since previous cohabitations during chronic dosing had shown no similar effects. Necropsy and histopathology Study 1. Necropsy of the males was performed after 27 weeks of treatment, 15 weeks after the fertility assessment was conducted. The prostate and seminal vesicles of most rats in all drug-treated groups were reduced in size at necropsy (Figure 2). There were significant decreasing trends in the average weights of these organs (prostate: 37% to 51% below control, seminal vesicles with coagulating glands: 57% to 60% below control; calculated as the percent of body weight) through all doses tested. There was no effect on the size, weight, or histomorphology of the testes. Microscopically, an increase in the incidence and severity of prostatic and seminal vesicular atrophy was seen in all drug-treated groups. Within the 20 males per group, prostatic atrophy was seen in 6 control rats

(graded very slight to slight) compared to 16, 19, and 19 rats (graded very slight to marked) in the 20, 40, and 80 mg/kg/day groups, respectively. This change was characterized by either a reduction in the amount of prostatic tissue or an increase in the number of individual prostatic glands with flattened epithelium or both over that commonly seen in control rats of this age. Very slight to slight atrophy of seminal vesicles was observed in 3 of 20 control animals, compared to very slight to marked atrophy in 13, 16, and 16 rats in the 20, 40, and 80 mg/kg/day groups, respectively. This change was characterized principally by a reduction in the amount of seminal fluid, with no alteration of the morphology of the epithelial components of the gland. There was no effect on the histomorphology of the coagulating gland. A histomorphologic change, however, was observed in the head of the epididymis of most finasteride-treated animals and is described under Study 3 below.

Study 2. In order to examine the reversibility of the previously described organ weight and histologic changes males were necropsied 11 weeks after the cessation of 14 weeks of treatment with 80 mg/kg/day of finasteride. As shown in Figure 2, mean prostate and seminal vesicle weights of drug-exposed males increased to within 82% of the concurrent control (a 61% and 58% increase, respectively, above the weights in Study 1). Prostatic atrophy was noted in 7 of the 15 finasteride-treated animals and in 3 of 15 control animals in Study 2. As described under Study 1, the atrophy was seen as flattening of the acinar epithelium as well as loss of cytoplasmic secretory granules. The mean seminal vesicle (including the coagulating gland) weights and histomorphology had recovered to within the nor-

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ReproductiveToxicology

real range in the finasteride-treated group. As observed previously, the testes weight and histomorphology were unremarkable. As will be described below, vacuolation in the head of the epididymis was seen histologically in all animals.

Study 3. Males that were sexually mature at the beginning of finasteride treatment (80 mg/kg/day for 32 weeks) had a statistically significant (P -< 0.001) decrease in average prostatic weight (58% below control relative to body weight) and seminal vesicular weight (80% below control relative to body weight). As in Study 1, the prostatic and seminal vesicular atrophy were seen in most finasteride-treated males. The prostatic atrophy was observed in 10 of 14 treated males, compared to 2 of 14 control males; the atrophy was characterized by decreased luminal secretion with very slight to moderate degrees of glandular epithelial attenuation. The atrophic change in seminal vesicle was observed in 14 of 14 treated males, compared to 1 of 14 controls, and was characterized by a marked decrease in luminal fluid and very slight to slight attenuation of the glandular epithelium. Microscopically, the coagulating gland showed no remarkable changes. After a 7-week recovery period without drug administration, average prostate and seminal vesicle weights were within control range and were not statistically different from controls. The microscopic atrophic changes described above for the prostate and seminal vesicles were not observed in finasteride-treated animals examined after the recovery period. There were no weight differences of the testes attributed to finasteride treatment, and microscopically there was no evidence of an effect on spermatogenesis. Sections of epididymal head and tail from all rats were examined by light microscopy. In Study 3, the head of the epididymis had a slight discoloration at necropsy. Microscopically, the head of the epididymis in all finasteride-treated animals had a very slight to slight epithelial vacuolation. Oil red O-stained cryosections were negative for neutral lipids, and PAS-stained, paraffin embedded sections were negative for polysaccharides (glycogen), mucopolysaccharides, mucoproteins, glycoproteins, and glycolipids. Similar microscopic changes were also noted in finasteride-treated animals from Studies 1 and 2, including those animals examined after the drug withdrawal periods. There were no similar microscopic changes in the tail of the epididymides. DISCUSSION The results of the present studies demonstrate that the fertility of male rats is partially decreased after prolonged oral treatment with finasteride at dose levels of 20 mg/kg/day and greater. The effect on fertility is only partial in that the fertility index did not decrease below

Volume 5, Number4, 1991 approximately 50% of the control group even after 30 weeks of treatment with 80 mg/kg/day. In addition, individual males that did not produce a pregnancy at one cohabitation would sometimes produce l or 2 pregnancies at the next cohabitation despite continued drug treatment. During the period of reduced fertility, the mean number of copulatory plugs and/or percent of mated females with plugs was reduced, and mean weights of prostates and seminal vesicles were lower than the control. These effects were reversible in that following a 6-week drug-free period, the accessory gland weights, mean number of copulatory plugs, and fertility returned to within the normal range. The changes in the prostate and seminal vesicles are expected pharmacologic effects of a 5a-reductase inhibitor, since the growth of these organs is under DHT control (5, 7). Our hypothesis to explain this decreased fertility relies on the earlier work of others regarding the critical role of the copulatory plug in rodent reproduction (see below), as well as the lack of any changes in the histology or weight of the testes or in litter parameters (ie, litter size or resorption rate). The prostate and seminal vesicle weight decreases were most probably associated with reduced secretions from these organs. The diminished secretions result in suboptimal copulatory plug size and/or fit in the female reproductive tract. Ineffective copulatory plug fit would not allow sperm transport to the uterus, and a mated female would not become pregnant. This hypothesis is supported by all of the major findings from the present studies (ie, decreases in mean number of copulatory plugs and accessory sex organ weights, no histologic effects in the testes, and return to normal values following drug removal). It is known that the seminal vesicles of rats secrete soluble proteins, which are coagulated in a reaction initiated by vesiculase, an enzyme produced by the coagulating gland located in the medial aspect of the seminal vesicles. The proteins coagulate in the vagina at the time of mating to form the copulatory (also called seminal) plug. The size and placement of the copulatory plug is known to be important in the rat for transport of sperm through the cervix (19-21). Infertility can be induced in male rats if the coagulating gland or the seminal vesicles are surgically excised (22,23). Additional studies conducted in this laboratory make it clear that following finasteride treatment there are gradual decreases in prostate and seminal vesicle weights, which are seen after at least 6 weeks (ie, mean absolute prostate weight 23% below control). However, the effects on fertility are not seen until 12 weeks after treatment initiated in sexually immature animals when prostate weight is estimated to be near 40% below control, and not until 24 weeks after treatment initiated in

Fertility assessmentof finasteridein male rats • L. D. W~sEEr AL sexually mature animals when prostate weight is approximately 50% below control. The decreases in seminal vesicle weights were slightly greater than the decreases in prostate weight at these times. Since the effect on fertility was only partial, as discussed above, these effects on accessory glands would thus appear to be at or near a threshold for affecting copulatory plug size and/or function and thereby affecting fertility. Decreased accessory gland weights appeared earlier in rats treated when they were sexually immature than in rats treated when they were sexually mature. This would be expected since the rate of growth of the prostate and seminal vesicles is greatest during this period (24). The available data indicate that the decreased fertility was not due to either decreased ability to mate or adverse effects on spermatogenesis. There were no consistent effects on indices of mating (ie, mean day of mating and mating incidence) during any of the three studies. It is interesting to note that another 5a-reductase inhibitor was previously shown to attenuate the stimulatory effect of testosterone propionate on penile erections displayed by castrated male rats when restrained in a supine position; however, there was no effect on mating behavior with receptive females (25). That spermatogenesis was not affected by finasteride treatment is supported by the findings that showed that there were no effects on testes weight or histomorphology (also supported by lack of effects in rats and dogs treated for up to one year in other studies, data not presented) and that complete reversibility to normal fertility was seen after a short (within 6-week) drug-free period. Recently George and coworkers (4) also showed that finasteride had no effect on testicular histology or daily sperm production in rats treated from birth to week 4 with 35 mg/kg/day and declining doses from week 4 to 7 (down to approximately 15 mg/kg/day). Additional information regarding the lack of an effect on spermatogenesis, specifically the ability of sperm from "infertile," finasteride-treated rats to fertilize, is presented in the following article in this issue (26). Evidence indicates that DHT-mediated secretion of glycoproteins by the epididymis, and subsequent coating of the spermatozoa by these secretions, is required for complete maturation of the rat spermatozoa (27). The head (caput) of the epididymis has been shown to have a high concentration of 5a-reductase (28). Epididymal sperm collected from mice that had been treated for approximately 2 weeks with another less specific 5ct-reductase inhibitor had a low rate of fertilization as measured either in vitro (29) or after intrauterine inseminations (30). The microscopic changes in the head of the epididymis could have been related to the decreased fertility in the drug-treated rats; however, the duration of treatment with finasteride required to produce an effect on fertility suggests that the epididymis is not the

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likely target. Subsequent studies performed in this laboratory have shown that the microscopic changes can be seen after as few as 2 doses of finasteride (80 mg/kg/ day). The total sperm transit time in the epididymis of rats is less than 2 weeks. Any drug-related effect on fertility related to epididymal changes should have occurred when rats were mated after 6 weeks of treatment as in Study 3; however, fertility was not affected until 24 weeks of treatment. In addition, the vacuolar changes in the head of the epididymis persist after drug withdrawal and the return of normal fertility. The epididymal vacuolar change in rats appears to be a species specific phenomenon; this is emphasized by other studies conducted in this laboratory in which similar changes in the epididymis were not seen in mice, rabbits, or dogs irrespective of dose or duration of treatment. The "no-adverse-effect" level for the effects on fertility and organ weights in rats was not determined in these studies. Another male fertility study of finasteride (data not presented) has shown accessory sex organ weight decreases after 30 weeks at the lowest dose levels tested (0.1 and 3.0 mg/kg/day) and associated slight decreases (<20%) in fertility. Nevertheless, given the requirement for the copulatory plug in rodent reproduction (but not in human reproduction) and the findings of Cukierski and coworkers in the accompanying paper (26), these effects are not considered to represent a hazard to the intended clinical population. REFERENCES 1. Brooks JR, Berman C, Primka RE,, Reynolds GF, Rasmusson GH. 5~-reductase inhibitory and anti-androgenic activities of some 4-azasteroids in the rat. Steroids. 1986;47:1-19. 2. Stoner E, Ca'egg H, Otterbein J, et al. Administration of MK0906, a 4-azasteroid compound, results in marked suppression of serum dihydrotestosterone in healthy men. Clin Res. 1987;35: 402A. 3. George FW, Peterson KG. 5et-dihydrotestosterone formation is necessary for embryogenesis of the rat prostate. Endocrinology. 1988; 122:1159-64. 4. George FW, Johnson L, Wilson JD. The effect of a 5a-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology. 1989;125:2434-8. 5. Brooks JR, Baptista EM, Berman C, et al. Response of rat ventral prostate to a new and novel 5ct-reductase inhibitor. Endocrinology. 1981;109:830-6. 6. Blohm TR, Laughlin ME, Benson HD, et al. Pharmacological induction of 5ct-reductase deficiency in the rat: separation of testosterone-mediated and 5et-dihydrotestosterone-mediated effects. Endocrinology. 1986;119:959-66. 7. Wenderoth UK, George FW, Wilson JD. The effect of a 5a-reductase inhibitor on androgen-mediated growth of the dog prostate. Endocrinology. 1983;113:569-73. 8. George FW. Developmental pattern of 5ct-reductase activity in the rat gnhernaculum. Endocrinology. 1989;124:727-32. 9. Imperato--McGinley J, Binienda Z, Gedney J, Vaughan ED. Nipple differentiation in fetal male rats treated with an inhibitor of the enzyme 5et-reductase: definition of a selective role for dihydrotestosterone. Endocrinology. 1986; 118:132-7. 10. Imperato-McGinley J, Binienda Z, Arthur A, Mininberg DT, Vaughan ED, Quimby FW. The development of a male pseudohermaphroditic rat using an inhibitor of the enzyme 5ctreductase. Endocrinology. 1985;116:807-12.

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11. Clark RL, Antonello JM, Grossman SJ, et al. External genitalia abnormalities in male rats exposed in utero to a 5a-reductase inhibitor. Teratology. 1990;42:91-100. 12. Connolly PB, Resko JA. Role of steroid 5a-reductase activity in sexual differentiation of the guinea pig. Neuroendocrinology. 1989;49:324-30. 13. Brooks JR, Berman C, Hichens M, Primka RL, Reynolds GF, Raasmusson GH. Biological activities of a new steroidal inhibitor of A4-5c~-reductase (41309). Proc Soc Exp Biol Med. 1982;169:67-73. 14. Edington ES. Randomization tests. New York: Marcel Dekker; 1980. 15. Harter HL. Expected values of normal order statistics. Biometrika. 1961;48:151-65. 16. Conover WJ, Iman RL. Rank transformation as a bridge between parametric and nonparametric statistics. American Statistician. 1981;35:124-33. 17. Tukey JW, Ciminera JL, Heyes JF. Testing the statistical certainty of a response to increasing doses of a drug. Biometrics. 1985 ;41:295-301. 18. Mantel N. Chi-square tests with one degree of freedom; extensions of the Mantel-Haenszel procedure. J Am Star Assoc. 1963 ;58:690-700. 19. Matlhews M, Adler, NT. Systematic interrelationship of mating, vaginal plug position and sperm transport in the rat. Physiol Behav. 1978;20:303-9. 20. Toner JP, Atlas AI, Adler NT. Transcervical sperm transport in the rat: the roles of pre-ejaculatory behavior and copulatory plug fit. Physiol Behav. 1987;39:371-5. 21. Blandau RJ. On the factors involved in sperm transport through

Volume 5, Number 4, 1991 the cervix uteri of the albino rat. Am J Anat. 1945;77:253-72. 22. Queen K, Dhabuwala CB, Pierrepoint CG. The effect of the removal of the various accessory sex glands on the fertility of male rats. J Reprod Fert. 1981;62:423-6. 23. Cukierski MA, Sina JL, Prahalada S, Robertson RT. Effects of seminal vesicle and coagulating gland ablation on fertility in male rats. Reprod Toxicol. 1991;5:342-52. 24. Scheer H, Robaire B. Steroid A4-5c~-reductase and 3a-hydroxysteroid dehydrogenase in the rat epididymis during development. Endocrinology. 1980;107:948-53. 25. Bradshaw WG, Baum MJ, Awh CC. Attenuation by a 5et-reductase inhibitor of the activational effect of testosterone propionate on penile erections in castrated male rats. Endocrinology. 1981;109:1047-51. 26. Cnkierski MA, Sina JL, Prahalada S, et al. Decreased fertility in male rats administered the 5a-reductase inhibitor, finasteride, is due to deficits in copulatory plug formation. Reprod Toxicol. 1991;5:353-62. 27. Eddy EM, Vernon RB, Muller CH, Hahnel AC, Fenderson BA. Immunodissection of sperm surface modifications during epididymal maturation. Am J Anat. 1985;174:225-37. 28. Saksena SK, Lau IF, Chang MC. The inhibition of the conversion of testosterone into 5a-dihydrotestosterone in the reproductive organs of the male rat. Steroids. 1976;27:751-7. 29. Cohen J, Ooms MP, Vreeburg JTM. Reduction of fertilizing capacity of epididymal spermatozoa by 5a-steroid reductase inhibitors. Experientia. 1981;37:1031-32. 30. Lau IF, Saksena SK, Chang MC. Antifertility effect of 3-oxo-4androstene-17[3 carboxylic acid in male mice. Arch Androl. 1979;2:179-81.