DANAZOL SUPPRESSES LUTEAL FUNCTION IN VITRO AND IN VIVO

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1 FERTILITY AND STERILITY Copyright 1980 The American Fertility Society Vol. 33, No.5, May 191!0 Printed in U.SA. DANAZOL SUPPRESSES LUTEAL FUNCTION IN VITRO AND IN VIVO KEITH M. HENDERSON, PH.D.* BENJAMIN K. TSANG, PH.D.t Department of Obstetrics and Gynaecology and Department of Physiology, University of Western Ontario, University Hospital, London, Ontario, Canada N6A 5A5 Danazol inhibited chorionic gonadotropin-stimulated progesterone production by pregnant rat luteal cells in vitro in a dose-dependent fashion. Spectral studies indicated that the inhibition was a consequence of danazol's interfering with the functioning of mitochondrial cytochrome P-450, an essential component of the enzyme system involved in progesterone biosynthesis. Danazol also suppressed luteal function in vivo, serum levels of progesterone being reduced by 50% to 70% when danazol (50 mg/kg) was administered thrice daily to rats from days 10 to 15 of pregnancy. Since danazol (30 f.lm) also inhibited progesterone production by human luteal cells in vitro and was dominant to the luteotropic action of chorionic gonadotropin, it is suggested that danazol may have some potential as an interceptive agent in humans. Fertil Steril33:550, 1980 Danazol (17a-pregn-4-en-20-yno(2,3-d)isoxazol-17-0l), a synthetic analog of ethinyltestosterone, is used in a number of clinical situations, 1 including the therapy of endometriosis,2 cystic mastitis,3 precocious puberty,4 and menorrhagia,5 and has been proposed as a male 6 and female 7 contraceptive. Its mode of action is generally attributed to a suppressive effect on pituitary gonadotropin release. 8 Recent studies, however, have indicated that danazol can also inhibit the activity of steroidogenic enzymes in the ovary, testis, and adrenal. 9, 10 Of particular interest in this respect is the observation that danazol could inhibit progesterone production by ovarian cells in vitro.11 This finding raises the possibility that danazol may have some potential as an interceptive agent, i.e., that it may interrupt pregnancy after implantation if it can inhibit ovarian progesterone production when pregnancy is sup- Received November 26, 1979; revised January 25, 1980; accepted January 28, *Present address and address for reprint requests: Department of Biochemistry, University of Western Australia, Nedlands, Western Australia tpresent address: Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont., Canada KIA OR ported by progesterone secreted. by the corpus luteum. This possibility was examined in vitro using human and pregnant rat luteal cells in culture and in vivo using pregnant rats. MATERIALS AND METHODS Reagents. Danazol capsules (Danatrol) were donated by the Department of Clinical Research, Winthrop Laboratories, Aurora, Ont., Canada. In addition to danazol, these capsules contain starch, lactose, talc, and magnesium stearate. For the in vivo studies the encapsulated product was administered by subcutaneous (sc) injection as a suspension in sesame oil. For the in vitro studies, danazol was extracted from the encapsulated product by shaking the equivalent of 200 mg of danazol (two capsules) in 15 ml of redistilled ethanol for 30 minutes in a metabolic shaker. The ethanolic extract was filtered and the concentration of danazol was determined spectrophotometrically, assuming that the molar extinction coefficient for danazol in ethanol at 286 nm was 11,300 (Merck Index). Animals. Adult female Sprague-Dawley rats weighing 250 to 300 gm were purchased from Bio Breeding Laboratories, Ottawa, Onto Pairs of fe-

2 Vol. 33, No.5 DANAZOL AND LUTEAL FUNCTION 551 males were caged with adult male rats, the animals being housed under constant temperature and lighting conditions and fed pellet food and water ad libitum. Vaginal smears were taken each morning. Day 0 of pregnancy was designated as the day spermatozoa were found in the smear. At day 10 of pregnancy a blood sample (-1 mt) was obtained from each animal, under light ether anesthesia, from a tail vein and the animal was given its first sc injection of either danazol in 0.2 ml of sesame oil or 0.2 ml of oil alone. The injections were repeated at 8- or 24-hour intervals through to day 15 of pregnancy. Additional blood samples were obtained at 48-hour intervals through to day 18 of pregnancy. The blood samples were stored overnight at 4 C and the serum was collected and stored at - 20 C until assayed for progesterone by radioimmunoassay (RIA). Serum was similarly obtained from adult ovariectomized female rats undergoing a similar regimen of injections and blood samplings. These provided a "control" group to ensure that the levels of progesterone measured in the serum of the pregnant rats by RIA could not be accounted for by cross-reactivity of danazol present in the serum with the progesterone antiserum. Superovulation was induced in immature female rats obtained from the Small Animal Breeding Station, University of Edinburgh, by sc injection of 50 IU of pregnant mare serum gonadotropin (PMS) (Gestyl; Organon, Morden, Surrey, United Kingdom) followed 3 days later by an sc injection of 25 IU of human chorionic gonadotropin (hcg) (Pregnyl; Organon, Morden). The gonadotropins were administered in 0.2 ml of phosphate-buffered saline. The animals were used 7 to 8 days after the second injection; they were housed under constant temperature and lighting conditions and fed pellet food and water ad libitum. Luteal Cell Cultures. Human corpora lutea (CL) were obtained from the ovaries of patients undergoing ovariectomy for various gynecologic disorders. Rat CL were dissected out ofthe ovaries of rats killed on day 10 of pregnancy. The technique for obtaining dispersed luteal cells was the same for both species. The CL were freed of any adherent connective tissue and chopped into pieces approximately 2 mm in diameter in Hanks' balanced salt solution devoid of magnesium and calcium and. supplemented with 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid (HEPES) buffer (20 mm), glutamine (2 mm), penicillin (50 units/ mi), streptomycin (50 tj..g/ml), and amphotericin B (0.625 tj..g/ml). All reagents were obtained from Flow Laboratories, Mississauga, Canada. The chopped tissue was washed twice with this medium (HBS-HGA) and incubated for 20 minutes at 37 C with stirring in HBS-HGA containing 0.2% collagenase (type II, Sigma Chemical Co., St. Louis, Mo.). The medium was decanted and the released cells were collected by low-speed centrifugation and stored at 4 C in HBS-HGA. The remaining fragile tissue fragments were reincubated in 0.2% collagenase in HBS-HGA at 37 C, and final dispersal of cells was achieved by drawing the suspension through a 2-ml syringe tip and a series of needles (18- to 22-guage), the cells being collected by centrifugation. All of the released cells were pooled together, filtered through two layers of sterile gauze, and washed four times to remove any traces of collagenase. An aliquot was taken to determine the total cell number, using a hemocytometer, and cell viability was determined using nigrosin dye. Replicates of -5 x 10 5 "live" cells were cultured at 36 C in a humidified incubator on 15-mm diameter round plastic cover slips (Thermanox: Lux Scientific Corporation, Newbury Park, Calif.) in 1 ml of culture medium consisting of 10% (v/v) calf serum and 90% Minimum Essential Medium (modified) with Earle's salts and supplemented with glutamine and antibiotics, as above, and non-essential amino acids. The gas phase was 5% CO 2-95% O 2, Exogenous danazol was added daily throughout the culture period as 0.5 tj..m to 150 tj..m in 10 tj..i of ethanol; control cultures received 10 tj..i of ethanol only. The culture medium was replaced daily and stored at - 20 C until RIA of steroids. At the end of the culture period the cells were washed and stained with hematoxylin and eosin. The number of cells remaining was estimated by counting the number of cells within a 0.3-sq mm area of 12 sampling points on the cover slip, the total number of cells being estimated by extrapolation. Control cultures containing added amounts of danazol, but no cells, allowed corrections to be made for any cross-reaction of the danazol with the steroid antisera. Radioimmunoassays. Progesterone was measured in petroleum ether-extracted aliquots of serum and culture medium, and 1713-estradiol was measured in diethyl ether-extracted aliquots of culture medium using specific radioimmunoassays described and validated previously. 12, 13 The progesterone antiserum was prepared against 11a-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin (ESA). The 1713-

3 552 HENDERSON AND TSANG May 1980 TABLE 1. Effect of Danazol on kcg (1 IUlml}-Stimulated Progesterone Production by Rat Luteal Cells Cultured for 24 Hours Treatment Untreated hcg aloneb hcg IJ.M danazol hcg + 5 IJ.M danazol hcg + 50 IJ.M danazol hcg IJ.M danazolb Progesteronea ng/los cells 25 ± ± ± ± 6 99 ± 4 c 83 ± 7 d % Inhibition None None aeach value is the mean ± standard eror of the mean off our replicate cultures. bcellular levels of progesterone in cultures treated with hcg alone and hcg IJ.M danazol were < 1 ng/lo S cells. CSignificantly different from hcg-treated cultures (P < 0.05). dsignificantiy different from hcg-treated cultures (P < 0.01). estradiol antiserum was generated against 17~estradiol-6-carboxymethy loxime-bsa. Danazol, at the concentrations studied, showed no cross-reactivity with either antiserum. The sensitivities of the progesterone and 17~-estradiol assays were 25 pg and 5 pg/tube, respectively. Recovery, of steroids from serum and culture medium was monitored by the. addition of trace amounts of tritiated steroid ( cpm) to the samples prior to their extraction. The intra- and interassay variations of the assays were <10% and <17%, respectively. Preparation of Luteal Mitochondria. Superovulated immature female rats were killed by cervical dislocation. The ovaries were removed, trimmed free of fat, and homogenized at 4 C in 10% (w/v) 0.25 M sucrose (ph 7.0) using a Teflonglass homogenizer. After sedimentation of nuclei and cell debris at 650 gay for 10 minutes, the mitochondrial fraction was pelleted at 8500 gay for 15 minutes. The mitochondrial fraction was washed by resuspension in one-half the original volume of 0.25 M sucrose and resedimented as before. The final pellet was taken up in 10 mm potassium phosphate buffer (ph 7.4) containing 5 mm MgC1 2 and 100 mm sucrose. Spectral Studies. Spectral measurements were made using a Unicam SP800 ultraviolet-visible wavelerigth spectrophotometer. The mitochondrial suspension was divided between sample and reference cuvettes, and a baseline of equal light absorbance was obtained. Danazol difference spectra were obtained by adding varying amounts of danazol in 5 ILl of ethanol to the sample cuvette and equal amounts of ethanol to the reference cuvette and scanning the spectrum from 510 to 370 nm. The ethanol concentration in the cuvettes never exceeded 1.4% (v/v). Mitochondrial protein concentrations in these studies were approximately 1 mg/ml as determin~d by the method of Lowry et al. 14 Statistical Analysis. Analysis of variance and Duncan's New Multiple Range test 15 were used to test for statistical significance of differences due to treatment. The data were transformed to logarithms to eliminate heterogeneity of variance when necessary, as determined by Bartlett's test. 1S RESULTS Effect of Danazol on Rat Luteal Cells. Table 1 shows the effect of various concentrations of danazol on human chorionic gonadotropin (hcg; A.P.L., Ayerst Laboratories, New York, N. Y.) stimulated progesterone production by rat luteal cells obtained at day 10 of pregnancy and cultured for 24 hours. While failing to abolish completely the stimulatory effect ofhcg, danazol did significantly reduce progesterone production when added to the cultures at concentrations of 50 and 150 ILM (P < 0.05 and 0.01, respectively). Lower concentrations of danazol (0.5 and 5 ILM) had no significant effect (P > 0.05). No detectable amounts of 17~-estradiol were produced by any of the cultures. At concentrations of 0.5 to 50 ILM, danazol had no effect on either cell numbers or cellular morphology, as observable by light microscopy, and so the inhibition observed with 50 ILM danazol was most likely due to a direct Days in culture TABLE 2. Effect of Danazol (30 11M) on Steroid Production by Human Luteal Cells in Vitro Progesteronea 17f3.Estradiola Control Danazol % Inhibition Control Danazol % Inhibition ng/los cells pg/ios cells 1 81 ± 6 58 ± 3b ± ± 47 C ± 4 35 ± 2 c ± 8 78 ± 8 c 73 aeach value is the mean ± standard error of the mean of four replicate cultures.

4 Vol. 33, No.5 DANAZOL AND LUTEAL FUNCTION 553 Day. in culture TABLE 3. Effect of Danazol (30 JJM) on Steroid Production by Human Luteal Cells Cultured in the Presence of hcg (1 IUlml) Progesterone" 17~E.tradiol" Untreated bcgb bcg + danazol Untreated bcg' bcg + danazol ngll(/ cells pgll(/ cells 1 53 ± 8 93 ± 3 58 ± 4d 776 ± ± ± ± ± 7 27 ± 2d 539 ± ± ± 54' GEach value is the mean ± standard error of the mean of four replicate cultures. bhcg stimulation of progesterone production was significant at P < "bcg did not stimulate 17p-estradiol production (P > 0.05). dsignificantly different from correspon4ing hcg-treated culture (P <: 0.01). -Significantly different from corresponding untreated and hcg-treated cultures (P < 0.01). 'Significantly different from corresponding untreated (P < 0.05) and hcg-treated (P < 0.01) cultures. biochemical interaction with the cellular systems responsible for progesterone biosynthesis. In those cultures receiving danazol at a concentration of 150 IJ.M, there were 25% fewer cells remaining after 24 hours of culture as compared with the cultures receiving hcg alone. Thus, at this high concentration, danazol was beginning to have a deleterious effect on cell viability in addition to its direct inhibition of cellular progesterone production. Effect of Danazol on Human Luteal Cells. Danazol at a concentration of 30 IJ.M inhibited basal progesterone production and abolished hcg-stimulated progesterone production by human luteal cells (Tables 2 and 3). 17~-Estradiol production was also inhibited by danazol, with the inhibition being greater than that of progesterone production (Tables 2 and 3). In addition, the inhibitory effect of danazol-on progesterone production in particular-was more pronounced during the 2nd day of culture than during the 1st day of culture. Danazol at this concentration (30 IJ.M) did not affect the number of cells remaining at the end of the culture period, relative to the control cultures, nor were there any morphologic differences between the control and treated groups on examination by light microscopy. Thus, the inhibition of progesterone and estradiol production was most likely the result of a direct biochemical action of danazol on the luteal cells. Mitochondrial Spectral Changes Induced by Danazol. Spectral studies by Barbieri et al.lo indicate that danazol inhibits testicular steroid production by interfering with the functioning of microsomal cytochrome P-450, an essential component of the microsomal enzyme system involved in androgen synthesis. Cytochrome P-450 is also an essential component of the mitochondrial enzyme system involved in cholesterol side-chain cleavage, the rate-limiting step in progesterone production. Spectral studies were therefore performed to determine whether danazol could interact with mitochondrial cytochrome P-450 and thereby provide a possible biochemical mechanism by which danazol inhibited luteal progesterone production. In view of the large numbers of animals that would have been required to prepare adequate quantities of mitochondria from luteal tissue of pregnant rats for spectral studies, it was decided to use mitochondria prepared from the ovaries of immature rats superovulated with ~ '" ~ 0 ; -< <I WAVELENGrH (n m) FIG. 1. Spectral changes produced by the addition of danazol (25 to 100 p.m) to rat luteal mitochondria.

5 554 HENDERSON AND TSANG May r;j t> o-l -< > ~ ~ 2.2 (J f:l 11 -< ~ 1/1 Ii!! ~ CONTROL A... DANAZOL (125 mg/ kg/day) CONTROL Can'ROL ~CONTROL INJECTIONS... DANAZOL (150 mg/ kc/day) B "'---""'(6) (6) (6) ) DAY OF PREGNANCY FIG. 2. Effect of danazol on serum progesterone levels in pregnant rats. A, Danazol administered once daily; B, danazol administered thrice daily (50 mg/kg/injectionl. Results are means ± standard error of the mean. Numbers in parentheses refer to the number of animals in each group. regimen ofpms and hcg. The addition of danazol to these mitochondrial preparations produced type II difference spectra characterized by a trough between 390 and 400 nm and a peak at -425 nm (Fig. 1). These spectra are characteristically produced by substances which interact with the substrate-binding sites on cytochrome P-450 and thereby inhibit the binding of the natural substrate,17 in this instance cholesterol. A Hanes plot ls of the changes in absorbance produced by the different concentrations of danazol gave an apparent dissociation constant for the danazol binding of 25 flm. Effect of Danazol Administration on Serum Progesterone Levels in Pregnant Rats. To test the possibility that danazol might suppress luteal progesterone production in vivo, serum progesterone levels were monitored in pregnant rats receiving danazol in sesame oil or oil alone on a daily or thrice-daily basis from days 10 to 15 of pregnancy. The results, shown in Figure 2, are expressed as a percentage of the serum progesterone concentrations found in the blood sample taken on day 10 of pregnancy immediately prior to the first injections of danazol or vehicle. These serum progesterone concentrations were not significantly different between each of the experimental groups (P > 0.05), the values (mean ± standard error of the mean) being as follows: control, 61 ± 7 ng/ml; 62.5 mg of danazol/kg/day, 52 ± 4 ng/ml; 125 mg of danazol/kg/day, 59 ± 4 ng/ml for data in Figure 2A, and control,52 ± 7 ng/ml; 150 mg of danazollkg/day, 43 ± 5 ng/ml for data in Figure 2B. The data were transformed to logarithms to eliminate heterogeneity of variance. Once-daily injections of danazol at 62.5 or 125 mg/kg of body weight produced a slight, but nonsignificant (P > 0.05, 3 x 4 mixed model analysis of variance), reduction in serum progesterone concentrations (Fig. 2A). Litter sizes of the control and treated groups were not significantly different from each other (P > 0.05), their sizes (mean ± standard error of the mean) being as follows: control, 11 ± 1 pups; 62.5 mg/kg/day, 10 ± 1 pups; 125 mg/kg/day, 10 ± 1 pups. Similar studies with up to 500 mg/kg of danazol injected once daily also produced no significant effect on either serum progesterone levels or litter size.19 When danazol was administered every 8 hours on a thrice-daily basis at a dosage of 150 mg/kg/day from days 10 to 15 of pregnancy, there was a pronounced, significant -reduction in serum progesterone levels (P < 0.01, 2 x 4 mixed model analysis of variance), as is shown in Figure 2B. There was also a slight, although nonsignificant (P> 0.05), reduction in the mean litter size of the danazol-treated animals (8 ± 2 pups) as compared with the control animals (11 ± 1 pups). DISCUSSION The results of the present study demonstrate that danazol effectively suppresses luteal function both in vitro and in vivo. The in vitro studies with human and pregnant rat luteal cells indicate that danazol can inhibit both basal and chorionic gonadotropin-stimulated progesterone production. In addition, danazol inhibited 17f3-estradiol production by human luteal cells, and it was found that the inhibition of both progesterone and 17f:3-estradiol production by human luteal cells was more pronounced during the 2nd day of culture than during the 1st day of culture, indicating a cumulative effect. These inhibitory effects of

6 Vol. 33, No.5 DANAZOL AND LUTEAL FUNCTION 555 danazol on steroidogenesis in vitro are most likely the results of a direct biochemical interaction with the cellular systems responsible for steroid production since, in the studies with both human and rat luteal cells, danazol at concentrations of 30 J.LM and 50 J.LM, respectively, inhibited steroid production without affecting either cell numbers or' cellular morphology. Only at the highest concentration tested (150 J.LM), in the studies with rat luteal cells, did danazol show an additional deleterious effect on cell viability as indicated by a reduction in the number of cells remaining at the end of the culture period. An insight into the possible biochemical mechanisms by which danazol inhibited luteal steroidogenesis is provided by the appearance of type II difference spectra on incubation of danazol with luteal mitochondria. This indicates that danazol can interact with the substrate-binding sites of cytochrome P-450, an essential component of the mitochondrial enzyme system involved in cholesterol side-chain cleavage, the-rate-limiting step in progesterone biosynthesis. Thus danazol may inhibit luteal progesterone production through interfering with the binding of cholesterol to cytochrome P-450. Such an action would also account for the inhibition of 1713-estradiol production by human luteal cells. Interestingly, the calculated apparent dissociation constant for danazol binding of 25 J.LM is comparable to the concentration of danazol required to inhibit effectively steroid production by the luteal cell cultures. The finding that danazol could inhibit chorionic gonadotropin-stimulated progesterone production by luteal cells in vitro raised the possibility that danazol might similarly interfere with luteal function in vivo if administered to rats during the second half of pregnancy, when luteal function is maintained by a luteinizing hormone-like chorionic gonadotropin produced by the placenta. 2o Moreover, the ability to over-ride the luteotropic action of chorionic gonadotropin would be an essential requirement of any potential interceptive agent designed to terminate early pregnancy. in humans when it is supported only by progesterone secreted by the corpus luteum under the influence of hcg. Thrice-daily administration of danazol from days 10 to 15 of pregnancy at a dose of 50 mg/kg reduced serum progesterone levels by 50% to 70% from days 12 to 18 of pregnancy. Although one cannot state unequivocally that this arose solely from a direct inhibition of luteal function, it is perhaps the most likely explanation, being consistent with the in vitro findings. Moreover, since litter sizes were not significantly reduced, it is unlikely that a toxic effect of danazol on the developing pups affected luteal function. In addition, although pituitary gonadotropin production is inhibited by danazol 8 this would not affect luteal function during the second half of pregnancy, pituitary gonadotropins being required only during the first half of pregnancy in the rat. In contrast to the effect of thrice-daily administration of danazol (50 mglkg), once-daily administration at doses of up to 500 mg/kg from days 10 to 15 of pregnancy had no significant effect on either serum progesterone or litter size. Studies in man, however, have indicated that danazol is rapidly degraded in vivo and that by 8 hours after administration it can no longer be detected in plasma. 21 The failure of the once-daily injections of danazol to reduce serum progesterone levels significantly may be due to the fact that, although high doses were administered, rapid metabolism of danazol results in circulatory levels too low to bring about a sustained inhibition of luteal progesterone production. More frequent injections of a lower dose of danazol (50 mg/kg every 8 hours) probably allows maintenance of a circulatory level of danazol sufficiently elevated to bring about the observed sustained inhibition of luteal steroidogenesis. Although danazol administered thrice daily at a dose of 50 mg/kg reduced serum progesterone levels by 50% to 70%, pregnancy was not terminated. However, the normal circulating serum progesterone levels of pregnant rats exceeds by a wide margin the minimal requirements for maintenance of gestation, serum progesterone levels as low as 20% of initial values being compatible with undisturbed gestation. 22 Whether danazol could interfere with human luteal function to reduce serum progesterone levels sufficiently to terminate pregnancy in its early stages, when it is maintained by progesterone secreted by the corpus luteum alone, is speculative. However, the results of the present in vitro studies with human luteal cells are encouraging in this respect, since danazol is capable of effectively inhibiting both basal and hcg-stimulated steroidogenesis; further clinical studies may therefore be worthwhile. In a recent study in nonpregnant women, danazol administration failed to shorten the luteal phase, although there was some reduction in plasma progesterone levels. 23 The failure to reduce plasma progesterone levels sufficiently to induce premature menstruation may have been due to dana-

7 556 HENDERSON AND TSANG May 1980 zol's being administered only on a once-daily basis. The results of the present study indicate that for maximal effectiveness danazol should be administered more frequently. It could be argued that the dose of da,nazol required to reduce serum progesterone levels in pregnant rats (150 mg/kg/day) would be excessive if applied directly to humans. Studies with other steroids, however, indicate that dosages expressed oil a milligram per kilogram basis in the rat are good predictors of the total clinical dose that would be required in humans. 24 By this reckoning, a dosage of 150 mg/day would be considerably less than the dosages presently used clinically (400 to 800 mg/day) to treat endometriosis. 25 In conclusion, the present findings demonstrate that danazol can inhibit luteal function in vitro and in vivo and can suppress the luteotropic action of chorionic gonadotropins. Thus, danazol may have some potential as an interceptive agent by virtue of these luteolytic characteristics. Clinical studies to assess this potential seem worthwhile. Acknowledgments. The authors are grateful to Dr. D. T. Armstrong for providing laboratory facilities made possible through grants to him by the Canadian Medical Research Council and the World Health Organization. This study was performed in part while K. M. H. was in receipt of a Canadian Medical Research Council Research Fellowship. Spectral studies were performed in the laboratory of Professor G. S. Boyd, Department of Biochemistry, University of Edinburgh, while K. M. H. was employed as a Medical Research Council (British) Research Fellow in that laboratory. Antisera to progesterone was generously supplied by Dr. H. R. Behrman (Yale University) and to17~-estradiol by Dr. G. Niswender (Colorado State University). The technical assistance of Linda Henderson is gratefully acknowledged. REFERENCES 1. 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