The Effect of Clomiphene Citrate Male Infertility

The Effect of Clomiphene Citrate Male Infertility. tn RAYMOND C. MELLINGER, M.D., and ROBERT J. THOMPSON, M.D. CLOMIPHENE CITRATE, an analog of the nonsteroidal estrogen TACE,* has proved effective in the induction of ovulation in infertile women. There is no general agreement about the mechanism of this drug action. In animals, clomiphene is gonadotropin-inhibiting and can manifest either estrogenic or antiestrogenic effects.4, 6 In human subjects, however, gonadotropin inhibition is rarely observed except in hypogonadal subjects with markedly increased gonadotropins prior to treatment. In fact, augmentation of the gonadotropin titer, associated with evidence of gonadal stimulation, has been observed in many patients. The antiestrogenic properties of the drug have been demonstrated in estrogen-treated agonadal women, and it seems likely that the gonadotropin-augmenting property of clomiphene is dependent upon this block of estrogenic effects at the cell site." Administration of clomiphene to oligospermic males has been reported to produce augmented gonadotropin excretion, a variably increased sperm count and increased levels of urinary estrogen and 17-ketosteroids. 1,,, The number of reported cases is small, and contrary to the experience in infertile women, restoration of fertility has not yet been reported. PROCEDURE Studies of 18 infertile men are reported. Two subjects were eunuchoid, with congenital absence of gonadotropins but without other evidence of pituitary disease. The third, a 16-year-old boy, was studied for delayed From the Division of Endocrinology and the Department of Obstetrics and Gynecology, Henry Ford Hospital, Detroit, Mich. Presented at the 21st Annual Meeting of the American Society for the Study of Sterility, San Francisco, Calif., Apr. 2-4, 1965. The investigation was partially supported by a grant from The William S. Merrell Company, Cincinnati, Ohio. The authors are indebted to.r ohn E. Johnson, Jr., M.D., who made available clomiphene citrate. *William S. Merrell Company. 94

VOL. 17, No.1, 1966 CLOMIPHENE IN INFEIITILE MALES 95 puberty and a unilateral undescended testis. In all 3 cases, an androgenic response to a previous course of chorionic gonadotropin proved the hormonal capacity of the testes, although all 3 patients were clinically hypogonadal. In 2 patients with azoospermia, testicular biopsy demonstrated maturation arrest of spermatogenesis. Both subjects were endocrinologically normal except for very high gonadotropin titers. The remaining 13 patients presented varying degrees of oligospermia with or without deficient sperm motility. In none of these subjects was there evidence of testicular disease except for 1 patient who had undergone a unilateral operation for an undescended testis. In 2 oligospermic subjects, gonadotropins were clearly above normal, while the remaining 11 had normal gonadotropins. Clinical evidence indicated normal androgenic function of the testis in all 13 subjects. Total gonadotropins were determined by the mouse uterus assay. FSH assay by ovarian weight augmentation of the chorionic gonadotropin-treated rat and LH assay by the hypophysectomized rat ventral-prostate weight were performed in selected cases. Urinary 17-ketosteroids were assayed by a modified Drekter method. Fractionation of urinary 17-ketosteroids was performed by gas-liquid chromatographic separation of the trimethylsilyl ether derivatives isolated after enzyme hydrolysis and solvolysis. Testosterone glucuronide in urine was likewise determined by gas-liquid chromatography of the trimethylsilyl ether derivative after enzyme hydrolysis and multiple paper chromatography. Multiple semen analyses were performed on specimens collected in the clinic after a period of abstinence of 3 days. Sperm counts were made within 2 hr. of collection. RESULTS Increased urinary gonadotropins were demonstrated in 10 of 11 subjects whose pretreatment level was within the normal range. Quantitation of FSH and LH by simultaneous assay with standard ovine gonadotropins failed to reveal a consistent disproportionate augmentation of either moiety (Fig. 1). Three of the 4 subjects with elevated gonadotropins in the pretreatment assays were not found to have any significant change in the level with treatment although suhstantial increase occurred in the fourth. Similarly, the 2 congenitally deficient subjects failed to secrete gonadotropins even with prolonged therapy. The young man with delayed puberty showed a definite increase in gonadotropin excretion with therapy, but puhertal changes were only slightly advanced.

96 MELLINGER & THOMPSON FERTILITY & STERILITY The 17-ketosteroid excretion was augmented in 6 of 7 subjects with normal pretreatment gonadotropin titers who were studied before and during therapy. The increase was as much as double control levels, and the mean increment exceeded 50%. In 2 of 3 subjects with elevated gonadotropins, 17-ketosteroids increased, but in the 2 individuals with congenital absence of gonadotropins there was no significant change. A slight rise was evident in the patient with delayed puberty (Fig. 2). 5.0 4.0 Mg. per = = 3.0 CLOMIPHENE CONTROL 24 HIS. 2.0 1.0 0 J Fig. 1. FSH (top) and LH ( bottom) excretion by infertile men before and during clomiphene administration. Levels are expressed as equivalent to ovine standards..', 1.2 = CLOMIPHENE CONTROL [EJ 0,8 Mq. per 24 Hr., 0,6 04 0.2 o... ~.~ rn~ ~~.. r;:81 ru Fig. 2. Excretion of 17-ketosteroids by infertile men before and during clomiphene administration. Bars of upper row are for 7 subjects who had normal pretreatment gonadotropins. Those in lower row are for 3 subiects (left) who had elevated gonadotropins and 3 (right) with congenital gonadotropin deficiency.

VOL. 17, No.1, 1966 CLOMIPHENE IN INFERTILE MALES 97 The response of urinary testosterone glucuronide to clomiphene treatment was measured in 4 subjects, 1 of whom responded with significantly increased 17-ketosteroids. Two of these demonstrated a very striking augmentation of testosterone excretion, the third subject had a more modest although definite increase, and the fourth exhibited no significant change (Fig. 3). In all of these 4 subjects, quantitative fractionation of the urinary 17-ketosteroids has been accomplished with isolation of androsterone, etiocholanolone, dehydroisoandrosterone, 11-hydroxyandrosterone, 11-hydroxyetiocholanolone, 11-keto androsterone, and 11-ketoetiocholanolone. When control specimens were compared to treatment specimens, there was little change in any of these fractions except dehydroisoandrosterone, which increased slightly in 3 subjects and fell substantially in the fourth. No correlation between the 17-ketosteroids and urinary testosterone is apparent (Table 1). In the 4 azoospermic subjects, 2 with congenital absence of gonadotropins and 2 with maturation arrest, there was no spermatogenic response. In 10 of the 13 oligospennic subjects, slight to marked increases in sperm counts were observed (Fig. 4). In only 1 of these individuals was the enhanced count sustained throughout the course of clomiphene therapy. In 3 subjects, there was a progressive decrease in sperm count, reaching azoospermia in 1. The counts returned to pretreatment level while medication was continued in 1 case and after it had been discontinued in the other 2. Despite an augmentation of the total sperm count, in no case was there an enhancement of motility, and the total motile count was rarely increased significantly. The number of abnormal sperm forms was not affected by the treatment. Representative sperm counts are shown in Fig. 5. One oligospermic sub- Fig. 3. Urinary testosterone glucuronide excretion by 4 infertile men before and during clomiphene administration. Testosterone ug/iooomg. Creatinine

TABLE 1. Levels of Urinary Steroids in 4 Infertile Male Subjects 17-Ketosteroids and component fractions Testosterone Total Gonadotropin (p.g./gm. (mg./gm. A E DHA 11-OHA 11-OHE 11-0A 11-OE (M.U.) creatinine) creatinine) (0/0 ) (0/0 ) (0/0 ) (0/0 ) (0/0 ) (0/0 ) (0/0 ) Control >16 <64 20.2 11.6 28.7 49.7 7.0 4.1 3.8 0.3 6.4 Clomiphene >64 53 18.7 31.3 46.9 8.6 3.8 3.0 0.8 5.7 Control >64 20.3 16.9 22.1 48.0 15.8 4.1 4.5 1.3 4.1 Clomiphene >64 173 14.2 18.3 46.0 17.1 5.7 5.1 2.0 5.8 Control >16 <64 36.6 12.0 30.2 38.4 7.5 8.8 2.5 6.9 5.7 Clomiphene >16 <64 39.1 14.7 30.2 39.7 9.5 10.6 2.2 2.8 5.0 Control >16 <64 25.6 15.5 40.6 17.4. 26.0' 6.3 3.7 2.3 3.7 Clomiphene >64 122 12.2 44.0 29.7 10.2 10.2 3.1 2.7

r VOL. 17, No.1, 1966 99 CLOMIPHENE IN INFERTILE MALES ject, whose course is represented in Fig. 6, responded to clomiphene with initially augmented sperm counts which returned to control levels by the twentieth week of continuous therapy. Testosterone administration induced azoospermia, but no "testicular rebound" was observed until clomiphene ABSOLUTE COUN.T C!J ii Count Clomlph.~. 1 ~ " Go II) Fig. 4. Absolute sperm counts (upper row) and motile sperm counts (lower row) in infertile men, showing highest control count and highest count during clomiphene therapy. was readministered. The resulting augmentation of sperm numbers, however, became progressively less with continued therapy. In no instance did the wives of these subjects conceive during or after clomiphene therapy. DISCUSSION Administration of clomiphene to oligospermic male subjects resulted in marked testicular stimulation. Only 2 individuals with congenital absence of gonadotropins and 1 oligospermic male with normal gonadotropins failed to evidence testicular response. It is believed that the source of testicular stimulation is the augmented release of total pituitary gonadotropin. We have been unable to confirm the claim of Heller and Moore that the drug induces a disproportionate augmentation of LH. Testicular stimulation can be demonstrated by both hormonal and germinal cell responses. Increases in both urinary estrogens and 17-ketosteroids in males treated with clomiphene have been reported by others.1, 2 Increases in 17-ketosteroids occurred in most of our subjects. In addition, a very striking augmentation of urinary testosterone glucuronide was found in 3 of 4

100 MELLINGER & THOMPSON FERTILITY & STERILITY 560 500 450 4 350 Sperm 300 Millions 250 0-------0 200 /.----/. Absolute Count M / ee Absolute Moti lity 500 450 - - Absolute Count 0------_.. M / ee. A-'-A Absolute Motility Sperm Millions 500 450 4 350 Sperm 300 Millions 250 ----- Absolute Count 0-----<> M / ec &---4 Absolute Motility 200 150 100 Fig. 5. Variations in ~erm counts and motility during clomiphene administration (shaded bar) to 3 infertile men.

VOL. 17, No.1, 1966 CLOMIPHENE IN INFERTILE MALES 101 subjects studied. Fractionation of the urinary 17-ketosteroids failed to show a uniform augmentation of any C 19 0 2 or C 19 0 3 steroids. Substantial increases in androsterone and etiocholanolone, which might be expected if testosterone secretion were the major steroid response to clomiphene, were SPERM MILLIONS 90 0----0 ABSOLUTE MOTI L1TY ABSOLUTE COUNT ~-.-~ MIce o 10 20 30 40 50 TIME IN WEEKS 60 70 80 Fig. 6. Effect of clomiphene on sperm counts of oligospermic subject. Drug yielded greater augmentation of sperm numbers after testosterone suppression of spermatogenesis. Augmenting effect was not sustained. not observed in this study. One subject who responded to clomiphene with a marked increase in urinary testosterone glucuronide was shown to have substantially decreased DHA in the same urine. Augmented sperm counts could be demonstrated at least transiently in most oligospermic males with normal gonadotropins. The augmentation was sometimes striking, but in no case was it accompanied by increased motility of the spermatozoa. There was no increase in abnormal forms. Most of these oligospermic subjects had a low initial percentage of motile sperm, and in no case was fertility restored by the use of the agent. In 1 individual the greatest response to clomiphene seemed to follow testicular suppression with testosterone. In 3 subjects, severely decreased sperm counts resulted. Two of these had elevated pretreatment gonadotropins, and all 3 were in the severe oligospermic range. The severe suppression of spermatogenesis was not permanent. The mechanism of action of clomiphene remains in dispute. Since it can

102 MELLINGER & THOMPSON FERTILITY & STERILITY be shown to inhibit,the cellular effects of estrogen on vaginal epithelium in women, 5 it is postulated that the augmented gonadotropins result from pituitary release through a similar estrogen-inhibiting mechanism. The postulate is strengthened by the frequently observed vasomotor flushes in women receiving the drug. The hypothesis that clomiphene is a direct gonadal stimulant seems unlikely, inasmuch as the resulting increase in estrogen secretion should lead regularly to suppressed gonadotropins. Direct hypothalamic stimulation of gonadotropin release, another postulated mechanism, involves the drug in two complex actions, hypothalamic stimulation and estrogen antagonism. We believe these are related properties. Most of these oligospermic male subjects responded to clomiphene with measurable increases in urinary gonadotropins. However, in the total absence of gonadotropins, no stimulation was observed. Augmentation of gonadotropins was associated with increased germinal and hormonal function of the testes. If the primary function of clomiphene is estrogen antagonism, these results suggest that testicular estrogen is a major controlling influence of gonadotropin secretion in the male. The data do not support the "utilization" hypothesis of urinary gonadotropins, 3 inasmuch as the obviously increased testicular function under these circumstances does not reduce the urinary gonadotropins. Although no hot flushes were observed in any male subjects, it seems entirely likely that clomiphene does serve as an estrogen antagonist in the male. Marked increases in urinary estrogens have been reported, but no case of gynecomastia has yet been observed; nor has there been any other clinical manifestation of this increased estrogen titer. SUMMARY Clomiphene citrate was administered to 18 infertile males. Augmented urinary gonadotropins, both FSH and LH, were observed in 10 of 11 subjects with normal pretreatment levels. Two individuals with congenital absence of gonadotropins failed to respond to the drug. One case of delayed puberty had a moderate gonadotropin response. Augmented sperm counts, irregularly sustained and not accompanied by increased motility, were observed in 10 of 13 oligospermic subjects. The remaining 3 subjects had a marked decrease in sperm counts which subsequently returned to normal. Two subjects with maturation arrest of spermatogenesis failed to respond with sperm maturation. Increased urinary 17-ketosteroids were observed in 6 of 7 subjects with normal gonadotropins. In 3 of 4 subjects, 1 of whom had maturation arrest of spermatogenesis and elevated gonadotropins, a marked increase in urinary testosterone glucuronide occurred. There were

VOL. 17, No.1, 1966 CLOMIPHENE IN INFERTILE MALES 103 no significant side effects from the therapy, but in no case was fertility demonstrated. Henry Ford Hospital Detroit, Mich. REFERENCES 1. HARKNESS, R. A., BELL, E. T., LORAINE, J. A., and MORSE, W. 1. The effects of clomiphene on endocrine function in normal men. ]. Endocrinol. 31:53, 1964. 2. HELLER, C. G., and MOORE, D. J. Dichotomy between total gonadotropins (HPG) and ICSH excretion produced by clomiphene. Clin. Res. 11:111, 1963. 3. HELLER, C. G., PAULSON, C. A., MORTIMORE, G. E., JUNeK, E. C., and NELSON, W. O. Urinary gonadotrophins, spermatogenic activity and classification of testicular morphology-their bearing on the utilization hypothesis. Ann. New York Acad. Sc. 55:685, 1952. 4. HOLTKAMP, D. E., GRESLIN, J. G., ROOT, C. A., and LERNER, L. J. Gonadotrophin inhibiting and anti-fecundity effects of chloramiphene. Proc. Soc. Exper. BioI. & Med. 105:197, 1960. 5. THOMPSON, R. J., and MELLINGER, R. C. The effects of clomiphene citrate in patients with pituitary gonadal disorders. Am.]. Obst. & Gynec. In press. 6. VAN MAANEN, E. F., GRESLIN, J. G., HOLTKAMP, D. E., and KING, W. M. Endocrine and other biologic effects of chloramiphene. Fed. Proc. 20:419, 1961. Change in Journal Reference Style As is indicated under "Information for Authors," references in articles in the Journal should henceforth be prepared in accordance with the abbreviations used in Index Medicus, published by the U. S. Department of Health, Education, and Welfare, in cooperation with the National Library of Medicine. The abbreviation style follows that adopted by the A.M.A. when it ceased publishing the Quarterly Cumulative Index Medicus. While the next few issues of the Journal may carry some bibliographies in the old style, authors are urged to use the Index Medicus system in preparing references for Journal articles.