Steroid metabolism in primates. XVIII. Return to higher levels of 17-ketosteroid excretion in urine after ending a long-term treatment with mestranol/chlormadinone acetate in the baboon]

6 weeks after terminating a long-time application of the ovulation inhibitor Ovosiston (mestranol/chlormadinone acetate) in female baboons, excretion of 17-ketosteroids in urine is still decreased. 6 months after ceasing the preparation, urinary 17-ketosteroid excretion resembles that of the control group.     

Affinities of progestogen and estrogen receptors in rabbit uterus for synthetic progestogens

The affinities of progestogen and estrogen receptors of rabbit uterus for a number of synthetic progestogens in clinical use and some analogues have been measured. Progesterone, 17-hydroxyprogesterone caproate, the chlormadinone, megestrol and cyproterone acetates had similar affinities for the progestogen receptor. Medroxyprogesterone acetate and particularly DU-41164 possessed much higher affinities. 17-hydroxyprogesterone or other agents with a free 17-hydroxyl had much lower receptor affinities than the corresponding 17-esters.Of the nortestosterone derivatives tested, norethisterone equalled progesterone in affinity for the progestogen receptor while d-norgestrel and Wy-4355 were more active. Norethynodrel and ethynodiol diacetate had much lower receptor affinities than progesterone. These results are discussed in relation to possible metabolic bioactivation.Only norethynodrel and ethynodiol (free alcohol) showed marked affinity for the estrogen receptor.     

The metabolic fate of chlormadinone acetate in the baboon.

The metabolic fate of chlormadinone acetate (17alpha-acetoxy-6-chloro-4, 6-pregnadiene-3, 20-dione; CAP) was studied in intact and biliary fistula baboons. The steroid was labeled with 3H at position 1 and with 14C at the carboxyl moiety of the 17alpha-acetate, thus affording the opportunity to ascertain the loss of the 17alpha-acetoxy group and the fate of both labels. The averages of the radioactivity excreted, given as percentages of the amounts injected, and the standard deviations were as follows: In the urine of intact animals after 6 hours, 5.7 +/- 0.2% and 5.5 +/- 0.7% of the 3H and 14C were recovered, respectively. After 6 days, there was 17.5% of the 3H and 16.2% of the 14C in the urine plus 15.3% of the 3H and 16.4% of the 14C in the feces. In baboons with biliary fistulas, the total radioactivity excreted was 7.8 +/- 0.7% of the 3H and 11.6% of the 14C in the urine, and 30.9 +/- 4.4% of the 3H and 30.7% of the 14C in the bile after 6 hours. Glucosiduronates were the predominant conjugates in the urine and bile. The similarity in the urinary excretion of radioactivity in the first 6 hours in intact and biliary fistula animals, the relatively low excretion of radioactivity in the bile and after 6 days in the urine, and the low fecal excretion suggest that the metabolites of CAP are not involved in an extensive enterohepatic circulation in the baboon. Deacetylation of the 17alpha-acetate in CAP was detected in the early collection periods of the urine and bile and constituted a very small percentage of the injected compound. No significant oxygenation of CAP at position 1 was detected. The metabolism of CAP is discussed and compared to our previously reported data on the metabolism of progesterone, ethynodiol diacetate and medroxyprogesterone acetate and the data on other progestogens reported in the literature. It appears that the excretion of CAP is significantly slower in the baboon than that of the other progestogens. The amounts of glucosiduronates of CAP and/or its metabolites formed in vivo are less than those formed with the other progestogens. Also, the extent of deacetylation of the 17alpha-acetate of CAP is much less than that of the 3beta-acetate of ethynodiol diacetate.     

Mutagenicity of some contraceptive drugs in Drosophila melanogaster

Combinations of 3 progestins, ethynodiol diacetate, norethynodrel and norgestrel, and 2 oestrogens, ethinyloestradiol and mestranol, were fed to larval Oregon-R fruit flies. None of the steroids studied induced X-linked recessive lethal mutations above the control level in Drosophila melanogaster.     

Hemostatic changes induced by exercise during oral contraceptive use

Exercise-induced changes in hemostatic measurements were studied in 25 women. Twelve of the subjects were not using oral contraceptives and the remainder were using Demulen (ethynodiol diacetate (1 mg) and ethinyl estradiol (0.05 mg)). Exercise on a treadmill induced similar changes in both groups, but during the use of Demulen the levels of fibrinogen and plasminogen were higher, antithrombin level was lower, and the recalcified clotting and dilute whole blood lysis times were shorter in group 2 than in the corresponding samples obtained from the nonpill users.     

Steroid metabolism in primates. XV. Excretion of C19 and C21 steroids in the urine of baboons (Papio hamadryas) after long-term treatment with a combination of mestranol-chlormadinone acetate

The urinary excretion of C21- and C19-steroids was investigated in female babons (Papio hamadryas) treated with the ovulation inhibitor Ovosiston (mestranol + chlormadinone-acetate), in comparison with an untreated control Group Urinary C21-steroid excretion was not significantly altered by Ovosiston. 17-Ketosteroids were decreased, predominantly 11-oxygenated compounds.     

Steroid hormones and the fertilizing capacity of spermatozoa

The effects of eleven different steroid hormones on $\text{in vitro}$ development of fertilizing capacity by hamster sperm were examined. Capacitation of epididymal sperm occurred only in the presence of female genital tract secretions. Fertilizing ability of sperm was poor if estradiol-17β, cortisol, chlormadinone acetate, medroxyprogesterone acetate, or megestrol acetate were present in the incubation medium at 10^?5M, whereas similar concentrations of estradiol-17α, progesterone, norethisterone acetate, ethynodiol diacetate, or norgestrel had little effect. Testosterone was a weak inhibitor of capacitation. Capacitation activity of female uterus and oviduct washings was higher at estrus than diestrus. This activity was reduced by treating intact animals with progesterone, cortisol, or testosterone, but increased by estradiol-17β or HCG. Estradiol-17α has no effect. Activity was low in pregnant or ovariectomized hamsters. Treatment of ovariectomized animals with estradiol-17β increased capacitation activity, but estradiol-17α, HCG or progesterone treatment was ineffective.     

A comparative study of the estrogenic potential of two synthetic estrogens (mestranol and ethinylestradiol)

The purpose of this study was to establish the relative estrogenicity of ethinylestradiol and mestranol and to establish .05 and .1 mg of mestranol as standard reference preparations for the comparative study of other compounds. Patients were selected from a large group of primary amenorrhea, secondary amenorrhea, and postmenopausal women. All required hormonal replacement therapy. Patients with chromosomal abnormalities or those with diminished or abnormal response were excluded. Vaginal cytology was used as the main method for the assessment of the response. Before treatment all had either an atrophic pattern (Maturation Index = 50-50-0) or a hypertrophic pattern (MI=5-95-0) with a mean value of 10-87-3 for patients with primary amenorrhea and 18-79-3 in the other 2 groups. Patients with a proliferative intermediate cell pattern and postmenopausal women thought to have continuing endogenous estrogen activity were omitted. Ethinylestradiol or mestranol were given orally in doses of .025, .05, and .1 mg for a period of 10 days during which frequent vaginal smears were made. When cell patterns showed extremely marked cytolysis of the glycogen-containing intermediate cells and a low percentage of karyopyknotic superficial cells these patients were excluded. 40 women were finally included. The effects of estrogen on the vaginal epithelium in all 40 women after 10 days therapy were quite uniform. In those cases showing delayed regression of estrogenic effects 4 mg of chlormadinone acetate was given daily for 10 days. These patients then showed a reduction in the superficial cell percentage accompanied by a shift to the left in the maturation index such as 0-40-60 before to 0-95-5 after treatment. A few women had an insignificant reduction in the superficial cell percentages. Changes in the percentages of the intermediate type of cells were not significant. The regression was considered to be complete when the smear contained mainly small intermediate cells and parabasal cells with numerous leukocytes and some necrotic debris. Both estrogen preparations produced the same vaginal response at .05 mg doses. Tablets of .05 and .1 mg mestranol were chosen for further study. The increase in the superficial cell index to 30 (SD plus or minus 18.5) following treatment with .05 mg and the increase to 54.1 (SD plus or minus 26.7) following .1 mg have been adopted as standard vaginal responses. The term estrogenic effect used in vaginal cytology is taken to mean the local vaginal effect induced by estrogens.     

The metabolic fate of medroxyprogesterone acetate in the baboon.

The metabolic fate of medroxyprogesterone acetate (6alpha-methyl-17a lpha-acetoxyprogesterone; MAP)was studied in intact baboons and in those with bile fistulas. The steroid moiety was labeled with tritiated hydrogen at positions 1 and 2 and the 17alpha-acetate with carbon-14, thus affording the opportunity to ascertain the loss of the 17-acetoxy group and the fate of both labels. Following the iv administration of labeled MAP only a small percentage (less than 15%) of the administered dose was recovered in the urine in 7 hours in intact baboons, as well as in the urine of baboons with biliary fistulas. Higher amounts of radioa ctivity were excreted in the bile (approximately 25%), amounting to almost double the percentage excreted into the urine. The similarity in the urinary excretion of radioactivity in intact and biliary fistula animals indicates that, even though a substantial biliary excretion of the labeled MAP occurred, the amount involved in an enterohepatic circulation is probably small. Glucosiduronates were the predominant conjugates, both in the urine and bile. The loss of the 17alpha-acetate group appeared to be rather extensive, ranging 30-70% among different co njugated and unconjugated metabolities of MAP. The degree of in vivo hydrolysis of the axial 17alpha-acetate of MAP, though extensive appeared to be of a significantly lesser magnitude than that exhibited toward the equatorial 3beta and 17beta acetate groups of labeled ethynodiol diacetate injected into baboons. The deacetylation of the 17alpha-acetate in MAP was similar to that observed in humans given the drug. Oxygenation of MAP at positions 1 and/or 2 appeared to be rather minimal (less than 5%).     

Metabolic fate of ethynodiol diacetate in the baboon.

The enterohepatic circulation and metabolism of ethynodiol diacetate (3beta,17beta-diacetoxy-17alpha-ethynyl-estr-4-ene) in baboons were studied following the intravenous injection of this contraceptive steroid labeled with 14C (4-position) and with 3H (in either the 3- or 17-acetoxy moieties). Bile and urine from four baboons with biliary fistulas and urine from four intact baboons were collected for 7 hours. On the average, 40% and 44% of the injected dose were excreted in the bile and urine, respectively. Only 48% was recovered in the urine of intact baboons. Analysis of these excretion rates indicates an insignificant enterohepatic circulation of this compound. The steroid was excreted mostly (over 80%) as a glucosiduronate in urine and bile. Very little excretion of the 3-acetoxy compound was detected in the urine or bile at any time interval. 17-Monoacetoxy compounds, however, were detected both in urine and bile, suggesting a difference in the rate of in vivo hydrolysis of the 17beta- vs. the 3beta-acetate.     

Contraceptive treatment with chlormadinone and its effect on the endometrium. A histological investigation

To study the effectiveness of chlormadinone acetate as an oral contraceptive and its effect on the endometrium, 42 women took .5 mg of chlormadinone acetate daily for a total of 424 cycles. 19 patients became pregnant and represent Group 1, while 18 patients with constant cycles were identified as Group 2. Histological investigations of Group 1 revealed abnormal decidual and placental structures in 17 of the 19 women in this group. Group 2 was studied for the effect of luteal supplementation on the endometrium. Mucosa patterns showed numerous deviations from the normal pattern. Investigations into the endometrium showed chlormadinone to be a cause of disturbance in the endometrium, whereby normal placentation is checked to a greater extent than nidation. This would appear to be supported by a number of pathological findings in the fetal membranes of Group 1.     

Steroid effects on human endometrial glycoprotein biosynthesis.

Human endometrium from the secretory phase of the menstrual cycle was incubated with 3H- and 14C-labelled glucosamine and [3H]leucine. Incorporation into secreted extracellular glycoprotein and accumulation of the label into the microsomal fraction were measured. When oestradiol or progesterone were added to the medium, medroxyprogesterone acetate (MPA), ethynodiol diacetate and chlormadinone acetate reduced incorporation of glucosamine and MPA reduced incorporation of leucine into glycoprotein. MPA reduced the amount of glucosamine in the microsomal fraction and also had an effect on amino acid transport within the endometrial cells, as indicated by intracellular alpha-aminoisobutyric acid space measurements. These results and the ratios of 3H and 14C in the microsomal fraction and secreted protein suggest that MPA has a primary effect in decreasing amino sugar incorporation and a secondary effect in reducing amino acid incorporation into glycoprotein.     

LH excretion during the ovulatory cycle and during therapy with various estrogen-gestagen combinations

Immunochemical determination of urinary LH was carried out in 7 normally ovulating women and in 25 women treated with various combined, sequential, and depot hormonal contraceptives. In ovulatory cycles without hormone treatment an LH peak was always observed at midcycle. During treatment with Ovosiston, OZN, and Quinestrol-norethisterone acetate, no LH peak was seen. In women receiving sequential preparations (mestranol-chlormadinone acetate, estrone cyanate-chlormadinone acetate), elevated LH levels were observed during estrogen medication. LH excretion was suppressed after administration of chlormadinone acetate. LH levels were also slightly elevated before and after medication with Quinestrol-chlormadinone acatate (1 pill per month).     

Alteration in circulating LH level and in cervical mucus induced by mid-cycle progesterone in chimpanzees

Administration of 200 micrograms mestranol/day (oestrogen) to ovariectomized chimpanzees caused a rapid decrease of circulating gonadotrophin concentrations to values similar to those in intact females. Administration of 2 mg chlormadinone acetate (progestagen) resulted in a prompt and significant rise in LH and FSH. This rise was accompanied by alteration in the physical characteristics and electrolyte composition of the cervical mucus which were the same as those observed around the time of ovulation. These results suggest a role of preovulatory progesterone secretion in relation to changes associated with ovulation in the chimpanzee.     

Radioimmunoassay of norethindrone (17 alpha-ethynyl-17 beta-hydroxy-4-estren-3-one) and ethynyl-estradiol (17 alpha-ethynyl-1,3,5, (10)-estratien-3, 17 beta-diol). Application to human plasma determination of norethindrone after oral administration of this steroid.

The experiment conditions for the evaluation of Norethindrone (17 alpha-Ethynyl-17 beta-hydroxy-4-estren-3-one, NET) and Ethynyl-estradiol (17 alpha-ethynyl-1, 3, 5 (10) estratrien-3, 17 beta-diol, EE) by radioimmunoassay are described. A minimal quantity of 25 pg of these two steroids could be evaluated using different reduced metabolites of NET, very little cross reaction is observed with 200 pg of these metabolites. No effect was observed with estradiol for the EE-antiserum. The NET-antiserum was used to evaluate this steroid and ethynodiol diacetate after oral administration to female volunteers. Maximal values in the plasma (2-3% of the administered dose) was found between 1-3 h after administration and at 24 h a concentration of 0.1-0.3% still remained in the plasma.     

Effects of a Low-oestrogen Oral Contraceptive on Urinary Excretion of Luteinizing Hormone and Ovarian Steroids

The urinary gonadotrophin and ovarian steroid excretion pattern was studied in five women taking an oral contraceptive formulation consisting of mestranol 50 μg and norethisterone 1 mg. Both the pretreatment and post-treatment cycles were normal. The ovulatory peak of luteinizing hormone (LH) during the treatment cycles was uniformly suppressed, but LH continued to be excreted within the normal range. In one fifth of the treatment cycles there was a pronounced and sustained rise of oestrogen output in the absence of ovulation, and in many of the other treatment cycles oestrogen levels suggested that active ovarian steroidogenesis was taking place.     

Sex chromatin in women treated with steroid hormones

3 groups of women, aged 15-71 years, were examined: a control group of 17 healthy women and 11 women with blood complications who had nver received steroid treatments, a 2nd group of patients with complications similar to the 1st groups' who were treated with the steroid preparation Enkorton-Polfa (for 5-10 days at 20-50 mg daily), and a 3rd group of 20 patients with similar prolonged complciations from 4 weeks to 5 years) who were treated with the steroid preparation Enkorton-Polfa in daily doses of 10-160 mg. Sex chromatin from these 3 groups was studied using the method of Sanderson and Stewart and the results compared. A lower percentage of sex chromatin bodies was found in those treated with steroids. Significant statistical differences were found in the comparison of the standard deviations of sex chromatin count: Group 1, + or -13%; Group 2, + or -10% before treatment and + or -5% during treatment; and Group 3, + or -8%.     

Effect of some progestational steroids on lactation in Egyptian women. I. Milk yield during the first year of lactation.

The effect of monthly injectios of 300 mg Depo-Provera or 150 mg Deladroxone, and of daily oral administration of .5 mg chlormadinone acetate or .3 mg quingestanol acetate on lactation was studied in Egyptian women during the 1st year of lactation. Women receiving Depo-Provera had the highest milk yield, followed by those taking Deladroxone and quingestanol acetate. The milk yields while taking these hormonal preparations were higher than in untreated controls. However, those women taking chlormadinone acetate had lower milk yields than untreated controls. The increased milk yield is probably due to the progestagenic activity, and minimal estrogenicity, of these drugs.     

The feedback effects of sex steroid hormones on pituitary gonadotropin release in Turner's syndrome and Klinefelter's syndrome.

The present study was designed to elucidate the feedback relationship between the release of pituitary gonadotropins and sex steroid hormones in Turner's syndrome and Klinefelter's syndrome. LH-RH stimulation test was employed to evaluate the effects of sex steroids on the release of gonadotropins. The release of gonadotropins in response to LH-RH as well as in baseline level was suppressed after the treatment with estrogen (mestranol 0.08 mg/day) for 10 days, followed by the treatment of the same period with estrogen (mestranol 0.08 mg/day) and progesterone (chlormadinone acetate 2.0 mg/day) in combination in both syndromes. The inhibitory effect of the combined treatment was greater than that of the treatment with estrogen alone. Administration of testosterone propionate (25 mg/day) for 3 days resulted in suppression of the release of both gonadotropins in baseline level and in response to LH-RH in both syndromes, but the suppressive effect appeared to be less complete as compared with that of estrogen or estrogen-progesterone. It was thus verified that the feedback interaction between the pituitary gonadotropin release and sex steroids such as estrogen, estrogen-progesterone or testosterone was operative in the same fashion in the patients with Turner's syndrome and Klinefelter's syndrome.     

Repair replication in human cells studied by sodium iodide isopycnic centrifugation of DNA in a fixed-angle rotor

A method for purification of ethynyl steroids from biological fluids has been developed using silver-sulfoethyl cellulose column chromatography. Ethynyl steroids were applied in methanol, and the firm binding allowed the columns to be washed with methanol, ethyl acetate, or diethyl ether to remove endogenous materials. Nonethynylated steroids did not bind to silver-sulfoethyl cellulose. Release of ethynylated steroids was achieved with a saturated NaCl/methanol solution. Dehydration or de-ethynylation of ethynyl estradiol, ethynodiol diacetate, and ethynodiol was not observed. The utility of this technique for purification of ethynyl steroid metabolites from the urine of a beagle metabolizing norethynodrel was demonstrated.