Luteinizing hormone-independent rise of progesterone as the physiological trigger of the ovulatory gonadotropins surge in the human.

The current ovarian cycle paradigm postulates that ovulation is triggered by a critically sustained elevation of estradiol. However, an in-depth look into the published data reveals considerable uncertainty about the relative roles of progesterone and estradiol in the ovulation process.This review provides compelling evidences that the role of estradiol in ovulation has been misinterpreted and that the true physiological trigger of ovulation is a luteinizing hormone-independent preovulatory progesterone surge in the circulation to approximately 0.5 ng/mL. Furthermore, the current work reconciles the ability of progesterone to trigger ovulation, with its well-established ability to block ovulation during pregnancy, or when administered in the form of a synthetic progestin in birth control formulations and with experimental data that estradiol benzoate triggers ovulation in the complete absence of progesterone.

Preclinical pharmacological profile of nomegestrol acetate, a synthetic 19-nor-progesterone derivative.

BACKGROUND: Nomegestrol acetate (NOMAC), a synthetic progestogen derived from 19-nor-progesterone, recently completed clinical trials for use with 17beta-estradiol in a new monophasic combined oral contraceptive. In this review, published as well as previously unpublished preclinical studies that detail the effects of NOMAC on estrogenic, progestogenic, and androgenic systems, as well as mineralocorticoid, glucocorticoid, bone, and metabolic indices are described. METHODS: In vitro assays to determine NOMAC structure-activity relationships used tissue derived from rat uteri. Transactivation profiles were performed using Chinese hamster ovary (CHO) cells transfected with cDNAs encoding human steroid receptors. Estrogenic and anti-estrogenic activities were monitored in vivo in rats as well as in vitro in human breast cancer cells. Standard in vivo techniques were used in rats to determine progestational activity; antigonadotropic, androgenic, mineralocorticoid, and glucocorticoid activities; as well as effects on bone and other metabolic indices. Ovulation inhibition was monitored in rats and primates. NOMAC's effects on cardiovascular systems were determined in dogs and primates. RESULTS: NOMAC was without significant agonistic or antagonistic activity for estrogen receptor alpha or beta in vitro, and inhibited ovulation in rats and monkeys (2.5 mg/kg and 1 mg/kg, respectively). NOMAC lacked androgenic, antimineralocorticoid, glucocorticoid, and metabolic activity and exhibited moderate anti-androgenic activity in rats. NOMAC did not affect bone mineral density (BMD) in rats or hemodynamic and electrophysiologic parameters in dogs and primates. CONCLUSIONS: NOMAC is a selective progestogen structurally similar to progesterone that has modest anti-androgenic activity and does not affect lipid or carbohydrate metabolism, BMD, or many cardiovascular parameters in selected animal models.

Synthetic progestins differentially promote or prevent 7,12-dimethylbenz(a)anthracene-induced mammary tumors in sprague-dawley rats.

Recent clinical trials show that combined oral dosing with estrogen and progestin increases the incidence of breast cancer in postmenopausal women. Similarly, in a rat model system of mammary carcinogenesis, the synthetic progestin medroxyprogesterone acetate (MPA) decreases latency and increases incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors. The goal of this study was to compare the effects of four clinically relevant progestins, MPA, norgestrel (N-EL), norethindrone (N-ONE), and megestrol acetate (MGA), on DMBA-induced mammary carcinogenesis in the rat. The experimental protocol involved implantation of 60-day release progestin pellets four weeks after rats were treated with DMBA. In contrast to the effect of MPA, N-ONE, and N-EL, but not MGA, blocked DMBA-dependent carcinogenesis and a dose-dependent effect on tumor growth was shown for N-EL; MGA did not alter tumor growth. Histopathologic studies showed extensive hyperplastic lesions in mammary tissue of progestin-treated animals. Furthermore, following treatment with N-EL or N-ONE, immunohistochemical staining for vascular endothelial growth factor in hyperplastic mammary tissue was lower than in animals treated with DMBA plus MPA or DMBA alone. Expression of vascular endothelial growth factor receptor-1, estrogen receptor alpha, and progesterone receptor was also lower in hyperplastic mammary tissue in N-EL-, N-ONE-, and MGA-treated animals. Interestingly, N-EL stimulated progression of existing mammary tumors in DMBA/MPA-treated rats, suggesting stage-specific effects of N-EL in this model. Because N-EL and N-ONE prevent tumor growth in the early stages of DMBA-induced mammary carcinogenesis in rats, these progestins may have potential as chemopreventive agents in women with no history of breast disease or family history of breast cancer.

Role of nonhuman primate models in the discovery and clinical development of selective progesterone receptor modulators (SPRMs).

Selective progesterone receptor modulators (SPRMs) represent a new class of progesterone receptor ligands that exert clinically relevant tissue-selective progesterone agonist, antagonist, partial, or mixed agonist/antagonist effects on various progesterone target tissues in an in vivo situation depending on the biological action studied. The SPRM asoprisnil is being studied in women with symptomatic uterine leiomyomata and endometriosis. Asoprisnil shows a high degree of uterine selectivity as compared to effects on ovulation or ovarian hormone secretion in humans. It induces amenorrhea and decreases leiomyoma volume in a dose-dependent manner in the presence of follicular phase estrogen concentrations. It also has endometrial antiproliferative effects. In pregnant animals, the myometrial, i.e. labor-inducing, effects of asoprisnil are blunted or absent. Studies in non-human primates played a key role during the preclinical development of selective progesterone receptor modulators. These studies provided the first evidence of uterus-selective effects of asoprisnil and structurally related compounds, and the rationale for clinical development of asoprisnil.

Dynamics of progestogenic activity in serum following administration of Ligusticum chuanxiong.

Many women are using botanical alternatives for menopausal hormone replacement therapy (HRT) because current progestins, compounds with progesterone activity, have adverse risk profiles. However the development of phyto-progestins for HRT is hampered by the absence of basic pharmacokinetic/pharmacodynamic (PK/PD) data due to the lack of methods to capture summated effects of the numerous compounds that contribute to bioactivity in vivo. In this study, we explored the utility of progesterone receptor (PR)-driven bioassays to track changes in serum progestogenic activity following administration of traditional Chinese medicinal herb, Ligusticum chuanxiong, with potent progestogenic activity. Sensitive and specific (>300-fold) increases in progestogenic activity were observed when HeLa cells transfected with PR and a PR-driven promoter were exposed to the progestogenic drug, medroxy-progesterone acetate (MPA), suggesting the utility of the bioassay to measure progestogenic effects for PK/PD studies. Progestogens were administered to male Sprague-Dawley rats and serum extracted for measurement of progestogenic activity. Effect-time studies indicate that injection of MPA and L. chuanxiong extract raised area-under-curve of progestogenic activity in sera by 8.2-fold (p<0.001) and 4.5-fold (p<0.01) respectively, compared to sera from rats administered vehicle only. Administration of MPA and L. chuanxiong extract by the oral route resulted in a 5.4 (p<0.001) and 2.3-fold (p=0.07) increase respectively. Our data suggest that PR-responsive reporter gene bioassays can measure bioavailability of compounds, known and unknown, of complex botanicals for hormone replacement therapy. L. chuanxiong extracts exert progestogenic activity in vivo, and may have utility for progesterone-replacement therapy.

Hormonal properties of norethisterone, 7alpha-methyl-norethisterone and their derivatives.

Norethisterone (NET) is a progestagenic compound with very weak androgenicity and estrogenicity. These low androgenic and estrogenic activities may be attributed to NET itself or induced by metabolites of NET. In order to improve the bioactivity of NET, the effects of a 7alpha-methyl substitution were studied. Thus this study has two objectives: first the comparison between biological activities of NET and 7alpha-methyl-NET (MeNET), and second the biological activity of tentative metabolites of NET and those of MeNET. The metabolites consist of a 3-keto-, 3alpha- or 3beta-hydroxy-group located next to a carbon 4 to 5 double bond (Delta(4)) or a 5alpha-hydrogen atom. The 7alpha-methyl substitution was of special interest as it prevents 5alpha-reduction. The biological activities of NET, MeNET and their potential metabolites were assessed by in vitro binding, transactivation and proliferation assays on progesterone (PR), androgen (AR), estrogen (ER) and glucocorticoid (GR) receptors and by in vivo progestagenic McPhail, androgenic Hershberger, estrogenic Allen-Doisy tests and combined estrogenic and progestagenic ovulation inhibition tests. NET is a compound with five- to eight-fold weaker PR binding and transactivation activities than the reference compound Org 2058 (100%) and two-fold stronger than progesterone. Binding and transactivation activities of NET for AR (DHT=100%) are 3.2 and 1.1%, respectively, for ER none (E2=100%) and for GR below 1% (DEX=100%). MeNET is 1.5- to two-fold less progestagenic and ten- to 20-fold more androgenic than NET, while it does not show activity for ER and GR. The relative binding affinity of 5alpha-NET was seven-fold lower for PR and 1.5-fold higher for AR than for NET, while in transactivation assays 5alpha-NET was only active at levels below 1% for all tested receptors. 3beta-Hydroxy-(5alpha-reduced)-metabolites showed clear ER binding and transactivation activities, while 3alpha-hydroxy-(5alpha-reduced)-metabolites did hardly possess these characteristics. These hydroxy metabolites did not bind or activate other receptors. Substitution of 7alpha-methyl to NET metabolites led to similar characteristics, but with higher activities for AR and ER and weaker activity for PR. The outcome of in vivo tests showed a remarkable effect for MeNET. Progestagenic activity in rabbits appeared for NET equipotent to or eight-fold higher than for MeNET, after subcutaneous or oral treatment, respectively. On the other hand, MeNET showed in rats a ten-fold higher androgenicity and eight-fold higher estrogenicity than NET. Ovulation inhibition was induced at very low oral or subcutaneous dose levels, being 120- or ten-fold lower than for NET, respectively. The estrogenicity can also be induced by 3alpha- or 3beta-hydroxy metabolites of MeNET, which are 15 or even more than 40-fold stronger than those of NET, respectively. In conclusion, after the introduction of a 7alpha-methyl substituent to NET an increased estrogenicity and androgenicity and a reduced progestagenic activity was found. The in vivo estrogenicity is mainly due to 3beta-hydroxy-MeNET and to a lesser extent to 3alpha-hydroxy-MeNET, while the androgenicity and progestagenicity are most likely caused by MeNET itself. Since the 7alpha-methyl substituent inhibits 5alpha-reductase, 5alpha-reduced MeNET metabolites can be excluded from biological activities. As MeNET is a very effective ovulation inhibitor, due to its mixed progestagenic and estrogenic profile, a further reduction of androgenicity of MeNET may yield new contraceptives with an attractive profile for contraception.

The antigonadotropic activity of a 19-nor-progesterone derivative is exerted both at the hypothalamic and pituitary levels in women.

We have previously shown in postmenopausal women that a 19-nor-progesterone derivative, nomegestrol acetate (NOMA) had a strong antigonadotropic activity and that this effect was not mediated via the androgen receptor. The aim of the present study was to further assess the action of this progestin on gonadotropin secretion in women. To demonstrate at which level of the hypothalamo-pituitary-ovarian axis the gonadotropin inhibition was exerted, 10 normally cycling (NC) women, 3 women with a gonadotropin-independent ovarian function [McCune-Albright (MCA) syndrome], and 5 women with functional hypothalamic amenorrhea (FHA) participated in the study. NC women were treated orally with 5 mg NOMA for 21 days, after one control cycle. Plasma estradiol (E2) and progesterone, LH, and FSH levels were measured during each cycle. A frequent sampling study (every 10 min for 4 h), followed by a classic GnRH test (100 microg, i.v.), was performed on day 11. Women with MCA were studied before, during NOMA, and after long-acting GnRH agonist administration. In women with FHA, pulsatile GnRH (20 microg s.c., every 90 min) was given for two cycles with or without NOMA (5 mg for 21 days). In all NC women, ovulation was suppressed by NOMA. Mean plasma LH levels, LH pulse frequency, and the LH response to exogenous GnRH were significantly decreased. In MCA, neither NOMA nor GnRH agonist modified multiple ovarian cysts on ultrasound or plasma E2, levels which remained elevated, ruling out a direct ovarian effect. In FHA, pulsatile GnRH administration recreated a normal ovulatory menstrual cycle. Addition of NOMA prevented the increase of plasma E2, decreased the amplitude of LH pulses, and prevented ovulation. In view of this unexpected action of NOMA at the pituitary level, seven samples of normal human female pituitaries were tested for the presence of progesterone receptor (PR) using a double labeling immunocytochemical technique. The presence of PR was detected in the seven human pituitary tissues. In addition, PR was found to be expressed only in gonadotroph cells. In conclusion, NOMA, a 19-nor-P derivative, has a potent antigonadotropic activity exerted at the hypothalamic level, inhibiting ovulation in NC women. In women with FHA, NOMA decreased the gonadotropin stimulation induced by pulsatile GnRH administration. According to the presence of PR in gonadotroph cells of normal human pituitaries, 19-nor-progesterone derivatives may also act on the gonadotropin secretion at the pituitary level.

Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

Effect of nutritional management, trace mineral supplementation, and norgestomet implant on attainment of puberty in beef heifers.

We conducted a study to evaluate the influences of nutritional management, trace mineral supplementation, and exogenous progesterone on attainment of puberty in beef heifers. Heifers (n = 180) were assigned at weaning to blocks and treatments. Treatments included two dietary regimens (corn silage vs pasture + oatlage), trace mineral supplementation, and puberty induction strategy (with or without progestin implant). Heifers that received pasture + oatlage were managed on grass-legume pastures from October 14 until December 14 and were then placed in pens and fed an oatlage-based diet through May 1994. Heifers fed the corn silage-based diet were housed in pens throughout the study. Norgestomet was implanted in half of the heifers on April 11 for 10 d. Progestin implant increased (P < .05) the number of heifers that had attained puberty by the end of the study, compared with nonimplanted heifers (89% vs 71%). Trace mineral supplementation did not affect percentage of heifers that reached puberty before the implant period. Plasma copper levels were below recommended levels in heifers fed oatlage-based diets without trace minerals. We conclude that heifers can be placed on regrowth in irrigated pastures during the fall and still make acceptable gains for attainment of puberty the following spring and that progestin treatment can aid in inducing heifers to reach puberty.

Development of a progestin-based estrus synchronization program: I. Reproductive response of cows fed melengestrol acetate for 20 days with an injection of progesterone.

We designed two experiments to determine the efficacy of an estrus control system in cows that combined long-term progestin exposure (20 d) with an acute increase in progesterone concentration. In Exp. 1, cows (n = 30) were fed either melengestrol acetate (MGA; .5 mg x cow(-1) x d(-1)) or ground ear corn (MGA carrier) for 20 d. On d -15 (last day of MGA feeding = d 0), cows were administered 25 mg of PGF2alpha to regress the corpus luteum (CL) and establish an environment conducive to the development of persistent follicles. To synchronously regress persistent follicles, cows fed MGA (n = 15) were injected with 200 mg of progesterone on d -2 (MGA-P), and the cows fed the MGA carrier were not treated (CONT; n = 15). Cows in the CONT group were artificially inseminated 12 h after detection of spontaneous estrus from d -20 to d 8. Estrus was observed, and all cows in the MGA-P group were artificially inseminated during the period of estrus synchronization (SYNC; d 1 to 8). No difference in conception rate was observed between treatments. In Exp. 2, postpartum cows (n = 113) received either the MGA-P (n = 56) or CONT (n = 57) treatment. More (P < .05) cows were observed in estrus during SYNC in the MGA-P (50%) than in the CONT (28%) group. Of the cows in the MGA-P group that were not observed in estrus during SYNC, 50% were in estrus for the first time 23 to 29 d after MGA withdrawal (SYNC2), suggesting that these cows ovulated without observable estrus during SYNC. Estrus was observed for the first time during SYNC2 in more (P < .05) cows in the MGA-P (25%) than in the CONT (7%) group. Conception rate at the synchronized estrus, pregnancy rate, and interval to first service and pregnancy were similar between treatments. We conclude that administration of MGA-P results in the synchronization and(or) induction of a fertile estrus in cows.

Development of a progestin-based estrus synchronization program: II. Reproductive response of cows fed melengestrol acetate for 14 days with injections of progesterone and prostaglandin F2alpha.

We tested the efficacy of an estrus control system designed to provide optimal control of follicular development. In Exp. 1, postpartum cows (n = 133) and yearling heifers (n = 57) were fed either .5 mg x female(-1) x d(-1) of melengestrol acetate (MGA) or the carrier for MGA from d -13 to d 0 (d 0 = last day of MGA feeding). All females received 25 mg of PGF2alpha (i.m.) on d -13 and 0. On d -6, cows and heifers fed MGA were administered an i.m. injection of progesterone (200 mg; MGA/P4), and those fed the corn carrier (2XPGF2alpha) received no progesterone. Beginning on d 1, females were bred by AI from d 1 to at least d 5. During the estrus synchronization period (d 1 to d 5), more (P < .05) postpartum cows were observed in estrus (70.1 vs 42.4%), the timing of estrus was more (P < .05) precise, conception rate was similar, and pregnancy rate was higher (P < .05) in the MGA/P4 than in the 2XPGF2alpha treatment. More (P < .05) cows that were anestrous at the beginning of the breeding season were in estrus during the synchronization period in the MGA/P4 (55.8%) than in the 2XPGF2alpha (28.6%) treatment. In heifers, estrus was synchronized in over 90% of females, and neither conception nor pregnancy rate during the synchronization period differed between treatments. In Exp. 2, postpartum cows (n = 122) and heifers (n = 84) received treatments (MGA/P4 or 2XPGF2alpha) as described for Exp. 1 with one exception. In the MGA/ P4 treatment, progesterone was administered on d -7 rather than d -6. Females were bred by AI from d 1 to 5. The estrus response and conception rate during the synchronization period did not differ between treatments for either cows or heifers. We conclude that the progestin-based estrous synchronization system used in this study effectively synchronized an estrus of normal fertility in cyclic cows and induced a majority of anestrous cows to reinitiate estrous cycles.

Urinary 6 beta-hydroxycortisol and D-glucaric acid excretion rates are not affected by lansoprazole treatment.

Lansoprazole has been shown to induce cytochrome P450 1A (CYP1A) and CYP3A enzymes in human hepatocytes in vitro. In this study, urinary excretion of 6 beta-hydroxycortisol (6 beta-OHF) and D-glucaric acid (D-GA) were used to investigate the potential enzyme-inducing property of lansoprazole in vivo. Twenty-four healthy female volunteers (aged 19-35 years), who were taking oral contraceptives containing 0.03 mg ethinylestradiol and 0.15 mg levonorgestrel, were randomized in a cross-over design for the treatment with either 60 mg lansoprazole or placebo once daily during 2 subsequent menstrual cycles. Urinary excretion rates of 6 beta-OHF and D-GA were measured at days 14 and 21 of the menstrual cycles. Median pretreatment urinary excretion of 6 beta-OHF (212 and 218 micrograms/d, n = 24) and D-GA (20.1 and 32.7 mumol/d) did not significantly differ. Upon treatment median excretion of 6 beta-OHF was 255 and 241 micrograms/d (n = 23), and that of D-GA was 25.5 and 33.8 mumol/d, respectively. Thus, the relatively high dose of 60 mg/d lansoprazole failed to statistically significantly alter urinary excretion of 6 beta-OHF and D-GA, indicating that therapeutic doses of lansoprazole might not exhibit a phenobarbital-like induction in vivo.

An amino-terminal truncated progesterone receptor isoform, PRc, enhances progestin-induced transcriptional activity.

Previously we reported the identification of two unique progesterone receptor (PR) messenger RNA transcripts that encode a smaller PR isoform, termed the C-receptor (PRc). These two PR transcripts encode a protein that is N-terminally truncated, so that it lacks the first zinc finger of the DNA binding domain, but still contains a complete hormone binding region with sequences for dimerization and nuclear localization. We also have demonstrated the existence of a 60-kDa progestin-specific binding protein in progestin target cells using a monoclonal antibody directed to the C-terminus of PRs, suggesting that these two novel transcripts generate a truncated form of PR. In this paper, we address the hypothesis that the C-receptor arises from the initiation of translation of a methionine C-terminal to the methionine start sites that generate the larger 94-kDa A and 116-kDa B human PR isoforms. The studies shown here support the postulate that another downstream in-frame methionine within the PR-coding region can serve as a translation initiation site for the generation of a third PR protein. A partial PR complementary DNA, lacking the translation start sites for B- and A-receptors was translated in vitro. The synthetic protein product bound [3H]progestins and unlabeled progestins. The antiprogestin RU486 also competed for this binding. Transfection of this partial PR complementary DNA into PR-negative HeLa cells resulted in progestin-specific binding activity. Because the third PR isoform lacks the first zinc finger of the DNA binding domain, but contains sequences for dimerization, we reasoned that the C-receptor isoform would be transcriptionally in-active and not bind DNA directly. Surprisingly, however, in the presence of A- and/or B-receptors, we found that C-receptors can modulate the transcriptional activity of A- and/or B-receptors using a reporter gene. These studies emphasize that multiple receptor isoforms may have distinct biological properties, and that the truncated C-receptor may play a role in explaining some of the pleiotropic effects of progestins.

Effect of gonadal steroids on bone and other physiological parameters of male broiler chickens.

Comparative studies of the effects of estradiol, progesterone, testosterone, cholesterol, and megestrol on juvenile chickens were carried out to determine their effects on bone and other physiological parameters. The chickens were implanted at 6 wk of age with ethylene-vinyl acetate copolymers containing steroids equivalent to a weekly dose of 10 mg/kg body weight for 3 consecutive wk. Estradiol caused a gain in body weight and relative liver weight but suppressed the growth of comb and testis. It also increased several serum variables, including triglycerides, cholesterol, calcium, phosphorus, and iron, and reduced testosterone levels. Testosterone produced an increase in comb weight and decreased both testicular and bursal weights. Growths of testis and comb were suppressed in progesterone-implanted chickens, as was the level of serum testosterone. Megestrol stimulated liver growth and increased serum testosterone levels. The lengths, relative weights, diaphyseal diameters, and ash percentages of both femur and tibia did not change significantly due to any treatment except that estradiol reduced tibial weight. Both progesterone and megestrol increased fibular growth plate alkaline and tartarate-resistant acid phosphatase activities. Other steroids did not affect these or the levels of calcium and of phosphorus of the fibular growth plate. Only testosterone caused a marked increase in the breaking strengths of both femur and tibia in all three parameters, i.e. load at yield, Young's modulus, and stress at yield responses. These findings suggest that the effects of steroids on bone in juvenile chickens may be limited.

Inhibition of ovulation by progestin analogs (agonists vs antagonists): preliminary evidence for different sites and mechanisms of actions.

Continuous administration of the antiprogesterone RU486 inhibits ovulation in women and in monkeys; in this regard RU486 may act as a progestin agonist rather than as an antagonist. We compared the site(s) and mechanism(s) of RU486-induced ovulation inhibition with those of levonorgestrel (LNG). Six regularly menstruating cynomolgus monkeys each received placebo, RU486 (1 mg/kg/d) or LNG (2 g/kg/d) i.m. between days (cd) 2-22 of three separate menstrual cycles. Serum levels of estradiol (E2), progesterone (P4), androstenedione, LH and FSH were analyzed by RIAs in daily blood samples. Basal and GnRH-stimulated (1 and 50 g of GnRH i.v. 2 h apart) secretion of LH and FSH was assessed using serial blood samples collected for 12 h on cd 10. Mean cycle length was prolonged by RU486 and LNG treatments from 32 d to 70 d and 52 d, respectively (p < 0.02). Ovulation was inhibited in five of the six primates during RU486, and in all six during LNG treatment. During RU486 treatment, serum E2 levels were similar to those of the control cycle; despite peaks of E2 secretion, no LH peaks were seen. In contrast, E2 concentrations were profoundly suppressed during LNG treatment (p < 0.005). The reduction in serum E2 was accompanied by lower levels of androstenedione, and suppressed ratio of E2/androstenedione (p < 0.02) suggesting both reduced synthesis and aromatization of androgen precursors during administration of LNG. Consequently, LNG treatment was associated with higher levels of serum FSH and LH (p < 0.001; 1-way ANOVA). Similarly, as during the luteal phase of the menstrual cycle, the amplitude of basal LH-pulses was increased during LNG treatment (p < 0.05), whereas RU486 treatment did not affect basal LH secretion. The GnRH-stimulated release of LH was similar during the placebo, RU486 and LNG cycles; enhanced release of FSH was seen during administration of LNG. Thus, in the present model system, RU486 seems to inhibit ovulation mainly at the level of hypothalamus, possibly by interfering with the steroidal positive feedback signals from the ovary. However, LNG inhibits ovulation differently, most likely via direct progesterone-like effects on folliculogenesis and the hypothalamus. The pituitary does not appear to be the major site of action(s) of RU486 or LNG. Thus, the differential mechanisms of ovulation inhibition by RU486 and LNG seem to result from lesser intraovarian impact of RU486 as well as dissimilar influences on tonic gonadotropin secretory levels. We conclude that when inhibiting ovulation, RU486 does not act as a progestin agonist, but rather, functions through a hypothalamic mechanism(s), which might be unique to RU486 as a progesterone antagonist.

PIP: Researchers administered a placebo, 1 mg/kg/day of RU-486, and 2 g/kg/day of levonorgestrel (LNG) to six regularly cycling cynomolgus monkeys (Macaca fascicularis) during days 2-22 of three separate treatment cycles in order to compare the site(s) and mechanism(s) of ovulation inhibition of RU-486 with those of LNG. One rest cycle separated the placebo and RU-486 cycles and at least two menstrual cycles separated the RU-486 and LNG cycles to ensure complete clearance of RU-486. Both RU-486 and LNG significantly prolonged the mean cycle length (from 32 days to 70 days and 52 days, respectively; p 0.02). During RU-486 treatment, five of the six monkeys did not ovulate, while during LNG treatment all six monkeys did not ovulate. Serum estradiol (E2) levels during RU-486 corresponded with those during the control cycle. There were peaks of E2 secretion during RU-486, but no peaks of luteinizing hormone (LH). E2 levels fell significantly during LNG treatment (p 0.005). Androstenedione levels also decreased significantly (p = 0.001) during LNG treatment, as well as the ratio of E2/androstenedione (p 0.02), suggesting that LNG inhibits aromatase activity. LNG treatment increased serum follicle stimulating hormone (FSH) and LH (p 0.001). Just like during the luteal phase of the menstrual cycle, the amplitude of basal LH pulses increased during LNG treatment (p 0.05). RU-486 did not alter basal LH secretion. LNG treatment amplified release of FSH. Neither RU-486 nor LNG affected the gonadotropin-releasing hormone stimulated release of FSH. These findings suggest that RU-486 inhibits ovulation largely at the hypothalamus level, perhaps by obstructing the steroidal positive feedback signals from the ovary. LNG likely inhibits ovulation through direct progesterone-like effects on folliculogenesis and the hypothalamus. Neither progestin analog seems to act at the pituitary level. In conclusion, RU-486 does not function as a typical progestin agonist but through a hypothalamic mechanism or mechanisms that are probably unique to RU-486.

Release characteristics, ovarian activity and menstrual bleeding pattern with a single contraceptive implant releasing 3-ketodesogestrel.

The properties of a single contraceptive subdermal implant releasing 3-ketodesogestrel were assessed in fifteen women over twelve months. Serum levels of 3-ketodesogestrel were monitored regularly following insertion and after removal. The mean serum level of 3-ketodesogestrel was 245 pg/ml after 72 h (steady state) and 176 pg/ml after twelve months. All volunteers demonstrated ovulation inhibition throughout the study. Transient oestradiol peaks occurred during the study. No luteal activity was noted. The cervical mucus was rapidly rendered hostile to sperm migration. Two women withdrew from the study during the first six months for medical reasons. Both volunteers cited bleeding irregularity as the main cause, one complaining of oligomenorrhoea, the other of prolonged bleeding/spotting episodes. A small but significant increase in weight was noted during the study period.

PIP: 15 sterilized women participated in a clinical trial of the implant Implanon (Organon), a single ethylene vinyl acetate rod containing 60 mg 3-ketodesogestrel (3-KDG), the metabolite of desogestrel. The rod is 40 mm long, 2 mm in diameter and is packaged in its inserter. In this trial the implants were treated to simulate the 2nd year of use. The study subjects underwent intensive hormone and ultrasound monitoring for 72 hours after insertion, twice weekly for 6 weeks and at 6-month intervals. 13 women completed 6 months, 7 completed 12 months, and 5 continued the trial 24 months. There were no complications related to insertion or removal. 3-KDG levels rose to a steady state of 245 pg/ml by 72 hours, then fell to a mean of 17 pg/ml at 12 months. 90 pg/ml of 3-KDG is the critical serum level for anovulation. After removal, 3-KDG declined to 54 pg/ml in 3 days. Follicle development tended toward small follicles or those larger than 10 mm. There was no luteal activity, and LH, FSH and progesterone remained in the follicular phase range. Estradiol levels were not low enough to risk osteoporosis. There was no significant change in serum sex hormone binding globulin. Systolic blood pressure decreased significantly at 12 months; mean weight gain was 3.7 kg (range from loss of 4 kg to gain of 22 kg); a variety of bleeding irregularities were recorded by individual women.

Effects of low-dose oral contraceptives on female whole saliva.

The composition and flow rate of paraffin-stimulated whole saliva were analysed in 22 women, of whom 11 used oral contraceptives and 11 did not. Ten men served as the controls. The salivary samples were collected during one month (oral contraceptive users and men), or during one menstrual cycle (non-users). The saliva analyses included flow rate, pH, buffer effect, sialic acid, thiocyanate, peroxidase, lysozyme, amylase, immunoglobulins A, G and M, total protein, mutans streptococci, lactobacilli, yeasts and total numbers of aerobic bacteria. The salivary buffer effect of oral contraceptive users was significantly (p less than 0.005) higher than that of non-users. All the other constituents showed intra- and interindividual variation in all groups, but with no apparent hormone-dependency.

PIP: The flow rate and composition of whole saliva were analyzed in 11 women using low dose oral contraceptives in comparison with 11 menstruating women and 10 men. Paraffin-stimulated whole saliva samples were collected Monday, Wednesday and Friday mornings for 1 cycle or 1 month in all subjects, checked for pH and buffer effect (Dentobuff method, Orion Diagnostics, Espoo, Finland, a measure of bicarbonate content) immediately, and frozen for later assay of salivary lysozyme, amylase, peroxidase, thiocyanate, sialic acid, total protein, IgA, IgG, IgM, Mutans streptococci, Lactobacilli, yeasts and aerobic bacteria. The oral contraceptives taken were Marvelon (Organon, Holland) by 4 subjects, Microgynon (Leiras, Finland) by 1, and Trikvilar (Leiras) by 6. The only significant differences between subject groups of cycle phases was a higher salivary buffer effect in oral contraceptive users than that seen in non-users, who resembled male controls. There was a wide individual variation in most values, but less variation in pH and buffer effect. Salivary buffer effect, which is correlated with HCO3-content and salivary flow, is also higher in late pregnancy.

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat.

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of estrogen and progestogen on the ascorbic acid status of female guinea pigs.

Female, adult guinea pigs were fed a low ascorbic acid diet ad libitum. Oral administrations of either estinyl (5 micrograms) or progestogen (250 micrograms) in combination with 5 mg of ascorbic acid (minimum requirement) daily for 21 d, resulted in significantly lower (P less than 0.05) concentrations of ascorbic acid in plasma, liver, adrenals and urine than in animals receiving only 5 mg of the vitamin. None of these animals showed any clinical signs of ascorbic acid deficiency. Clinical manifestations of scurvy were exhibited, however, when animals receiving no ascorbic acid supplement were treated with the steroid hormones for 7 d. All of these animals died by d 10. On the other hand, the animals receiving neither ascorbic acid nor the steroids remained free from any signs of scurvy, except one (out of six), which died by d 12. In vitro studies revealed a markedly higher rate of oxidation of ascorbic acid in the presence of either estinyl or progestogen than in untreated controls. These results were further supported by a higher level of plasma ceruloplasmin in animals receiving a combination of estrogen and progestogen than in animals receiving no hormones. An in vivo dose-related effect of ascorbic acid indicated that the steroid-mediated lowering effect of the vitamin status could be counteracted by increasing the dose of ascorbic acid from 5 to 10 mg/d for 2 wk. These results suggest that the interactions between oral contraceptive hormones and ascorbic acid may be of clinical importance only in the case of borderline intake of the vitamin.