Nestorone: a progestin with a unique pharmacological profile

Nestorone(R) (Nestorone 16-methylene-17alpha-acetoxy-19-norpregn-4-ene-3,20-dione), formerly referred to as ST 1435, is a potent progestin when given parenterally via sustained release formulations. The pharmacological profile of Nestorone was compared with that of levonorgestrel and 3-keto-desogestrel by steroid receptor binding studies and by in vivo bioassays in rats and rabbits. 3-Keto-desogestrel showed the highest binding affinity to progesterone receptors (PR) followed by Nestorone, levonorgestrel, and progesterone. The binding affinity of Nestorone to androgen receptors (AR) was 500- to 600-fold less than that of testosterone. However, both levonorgestrel and 3-keto-desogestrel showed significant binding (40 to 70% of testosterone) to AR. None of the progestins bound to estrogen receptors (ER). The progestational activity of Nestorone, levonorgestrel, and progesterone was compared using McPhail index in immature rabbits and pregnancy maintenance and ovulation inhibition tests in rats after subcutaneous (s.c.) administration. In all three tests, Nestorone was the most potent progestin. The progestational activity of Nestorone was also compared after oral and s.c. administration in rabbits. The potency of Nestorone was over 100-fold higher upon s.c. administration than via the oral route. The androgenic activity of progestins, based on the stimulation of ventral prostate (androgenic target) and levator ani (anabolic target) growth in castrated immature rats, showed good correlation with their binding affinity to AR. Nestorone showed no androgenic or anabolic activity. Nestorone did not bind to sex hormone binding globulin (SHBG), whereas both levonorgestrel and 3-keto-desogestrel showed significant binding to SHBG. The estrogenic/antiestrogenic activity of Nestorone was investigated in immature ovariectomized rats. In contrast to estradiol and levonorgestrel, Nestorone showed no uterotropic activity in ovariectomized rats. Despite significant binding to glucocorticoid receptors (GR), Nestorone showed no glucocorticoid activity in vivo. It is concluded that a strong progestational activity, combined with lack of androgenic, estrogenic, and glucocorticoid-like activities, confer special advantages to Nestorone for use in contraception and hormone replacement therapy.     

Synthesis and biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate

The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3,2 0-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches.     

Vaginal rings for contraception in lactating women

Contraceptive methods for breastfeeding women should be safe for the mother and infant and should not interfere with lactation. Progestin-only methods meet these conditions and can be used from the sixth week postpartum. Because all progestins are excreted in milk, those that are insufficiently active by the oral route are preferable to avoid any possible effect on the baby. These steroids, however, must be administered to the mother by a non-oral route. Initially, progesterone was administered subdermally to test this concept. Subsequently, a progesterone vaginal ring was developed to be used continuously for 3 to 4 months and replaced with a new device, as needed, until weaning. Clinical trials have shown a high contraceptive efficacy (over 98.5%) and safety. The gross continuation rate of this method is approximately 40% at 12 months of use, with use-related problems being the main reason for discontinuation (26.8%). Currently, a Nestorone vaginal ring is under development, delivering 50 microg of Nestorone per day. It may be used continuously for up to one year, even if weaning occurs earlier. Both of these progestin-only rings prolong lactational amenorrhea to 10 to 12 months, which represents a health benefit and convenience for many women. The registration of the progesterone vaginal ring, developed as a contraceptive method to be used exclusively during lactation, has been approved in Chile and Perú. The fact that it is a user-controlled long-term contraceptive that delivers a natural hormone makes it an attractive option for many women.     

Progestin-only contraceptive rings

Several progestin-only long acting contraceptives are currently available in the form of implants or injectables. Vaginal rings are another contraceptive option in the final stages of development. These steroid-containing polymer rings are placed in the vagina, providing relatively constant drug release, thus allowing for lower effective doses. Vaginal rings have the advantage of being user-controlled and non-provider dependent, and their use is non-coital related. The first clinical study with medroxyprogesterone acetate vaginal rings was published in 1970. Since then numerous clinical trials testing different steroids and doses have followed. A large Phase III multicenter clinical trial with a levonorgestrel ring, releasing 20 microg/day, was coordinated and sponsored by WHO. The cumulative one-year pregnancy rate was 4. 5%. The principal reasons for discontinuation were menstrual disturbances (17.2%), followed by frequent expulsion of the ring (7. 1%), and vaginal symptoms (6.0%). The finding of erythematous lesions in the vagina in some women has led to the development of a more flexible device. Collaboration with industry should facilitate the manufacture of a redesigned levonorgestrel ring with a higher release rate. The Population Council is also developing a vaginal ring containing Nestorone for 6 months of continuous use. Ovulation inhibition was achieved in over 97% of the segments studied, with rings releasing either 50, 75, or 100 microg/day. No pregnancies occurred in women using the low-dose ring, while one pregnancy each occurred in the intermediate- and high-dose ring groups for a 6-month cumulative pregnancy rate of 0.0, 1.9, and 2.1%. Bleeding irregularities were common. Nestorone is orally inactive; therefore this ring is also excellent for use in lactating women.     

Conception and pharmacodynamic profile of drospirenone

Progesterone is more than a progestin. Beyond functions in cycle and pregnancy, progesterone binds with high affinity to the mineralocorticoid receptor (MR) acting as an antagonist, with obvious significance for electrolyte homeostasis, an array of MR-related functions in the circulation as well as in the CNS. Progesterone induces natriuresis at physiological concentrations. Lack of antimineralocorticoid activity with conventional progestins may account for sodium and water retention, minor elevation of blood pressure and "pill hypertension" in susceptible women on oral contraceptives. Ethinylestradiol (EE) contributes to this problem by distinct activation of the renin-angiotensin-aldosterone (RAAS) system. Drospirenone (DRSP: 6beta,7beta,15beta,16beta-dimethylene-3-oxo 17alpha-pregn-4-ene-21,17 carbolactone) is the first synthetic progestin with antialdosterone activity. DRSP and progesterone bind to PR in uterine (affinity of both is about 30% of R5020) and MR in kidney cytosol (affinity about 230 and 100% of aldosterone, respectively). Intrauterine administration of DRSP in silastic tubes induced maximum local progestational effects in rabbits. At systemic subcutaneous (s.c.) administration (McPhail-assay) full endometrial transformation was obtained at 1mg per animal per day. At 1-3mg DRSP per animal per day subcutaneously, pregnancy maintenance after ovariectomy, antiovulatory activity, and antimineralocorticoid activity were seen in the respective assays in rats. The latter activity indicates about eight-fold higher potency than spironolactone. DRSP decreased blood pressure in male hypertensive rats, whereas an increase was noted under conventional progestins. DRSP also prevented hypertension and fetal growth retardation in pregnant rats after L-NAME, an inhibitor of nitric oxide synthase. DRSP has antiandrogenic activity. Feminizing effects were recorded during sexual differentiation in male fetuses at high doses. Powerful antiandrogenic effects were also seen in gonad intact and testosterone substituted castrated male rats. The antiandrogenic potency of DRSP is superior to that of spironolactone but below that of cyproterone acetate. Endometrial transformation, inhibition of ovulation, and antimineralocorticoid, i.e. natriuretic effects and mild antiandrogenic effects were recorded at the same range of oral doses (0.5-4 mg per day) in humans. Combined with EE (3 mg DRSP+30 microg EE), DRSP provides effective inhibition of ovulation and cycle control. Body weight compared to conventional oral contraceptives was reduced. DRSP (3 mg per day+15, 20, or 30 microg ethinyl estradiol per day) prevented the mild increase of blood pressure seen under a conventional levonorgestrel-containing contraceptive and even tended to reduce pretreatment blood pressure. Studies on modulation (i.e. inhibition) of glucocorticoid effects at the MR in the CNS remain an unexplored and interesting area for research.     

The use of progesterone antagonists and progesterone receptor modulators in contraception

Progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) have contraceptive potential by suppressing follicular development, delaying the surge of luteinizing hormone (LH), retarding endometrial maturation, and promoting endometrial bleeding. Mifepristone, in daily doses of 2-10 mg, blocks the LH surge and ovulation. Many of the studies were conducted in women not at risk of pregnancy, and thus the contraceptive efficacy is not yet known. Nevertheless, there is evidence that daily doses of 2 or 5 mg of mifepristone have contraceptive potential. Because of anovulation, there may be an unopposed estrogen effect on the endometrium, although this risk may be mitigated by the noncompetitive anti-estrogenic activity exhibited by both PAs and PRMs. Low doses of PAs and PRMs, which do not affect ovulation, retard endometrial maturation, indicating that the endometrium is exquisitely sensitive to these compounds. This raises the prospect of endometrial contraception, i.e. prevention of endometrial maturation without disturbing ovulation or producing alterations in bleeding patterns. This approach works well in monkeys but was not found to be very promising when given to women not using contraception. On the other hand, 200 mg mifepristone administered 48 h after the LH surge, which has minimal or no effect on ovulation and bleeding patterns, is an effective contraceptive; yet, it is not a practical approach to contraception. Late luteal phase administration of mifepristone produces menstrual bleeding. However, when mifepristone was administered every month at the end of the cycle either alone or together with prostaglandins, it was not very effective in preventing pregnancy. In contrast, a mifepristone-prostaglandin combination has been shown to be a very effective treatment for occasional menstrual regulation, with vaginal bleeding induced in 98% of pregnant women, with menses delay of 11 days or less. Mifepristone is an excellent agent for emergency contraception when used within 120 h of unprotected intercourse. It is also possible that PAs and PRMs may be used to reduce the occurrence of bleeding irregularities induced by progestin-only contraceptive methods. Both classes of progesterone receptor ligands may also have contraceptive efficacy by having a pharmacological effect on the embryo or altering tubal transport or other aspects of tubal physiology.     

Current Concepts in Conception Control

The progressive increase in world population has become a most urgent global problem in recent years. Man has, however, been interested in controlling his reproductivity at the family level for many centuries. Historical aspects of this saga are reviewed.The modern era of conception control was ushered in by Makepeace et al. in 1937 when ovulation inhibition by progesterone was demonstrated. Confirmation of this by Pincus and associates, and development of the potent oral progestational agents, the 19-norsteroids, have made efficient reliable contraception a reality.Experience with one of these agents (Ortho-Novum, 2 mg.) in 115 patients through 805 cycles is presented. Conception control was 100% effective at this dosage. Side effects were minimal. Only three of the women discontinued the tablets because of these effects. All but five in this group of 115 preferred the oral contraceptives to methods previously employed.     

Progesterone and progestins: applications in gynecology

Achievements obtained in infertility treatments over the past two decades have sparked interest in optimizing progesterone administration. Although progesterone is absorbed orally when ingested in micronized form, bioavailability is poor because of extensive liver metabolism. This explains why full predecidual transformation of the endometrium cannot be achieved with oral progesterone and is therefore ineffective for luteal support in in vitro fertilization (IVF). Progesterone administered non-orally can duplicate the endometrial changes normally seen in the menstrual cycle in women whose ovaries are inactive. Similar results have been reported with intramuscular (i.m.) injections and vaginal administration, although tissue levels are higher in the latter case. The recent development of a controlled and sustained release vaginal progesterone gel, Crinone(R) 8%, has made the vaginal route clinically practical by limiting the number of necessary applications to 1 per day. This regimen has been found at least as effective as intramuscular (i.m.) injections in women whose ovaries are inactive (donor egg IVF) and for luteal support in regular IVF. Hence, painful daily i.m. injections of progesterone in oil become unnecessary. The possibility of reducing the number of daily applications of vaginal progesterone to 1 per day, made possible by the sustained release gel Crinone, has opened new possibilities for long-term treatments, as in hormone replacement therapy (HRT). The low incidence of systemic side effects with use of the vaginal progesterone gel used for HRT in amenorrheic women, contrasts with findings related to use of synthetic progestins. Preliminary data suggest that vaginal progesterone can be instrumental in enhancing the notoriously poor long-term compliance of HRT.     

Transdermal application of steroid hormones for contraception

The concept of transdermal delivery (TD) for steroid application has nowadays been largely accepted for hormone replacement therapy in the menopause. It is only recently that the same concept has been envisaged for contraception. The skin can be penetrated by both estrogens and progestins, provided they are delivered in an appropriate solvent. About 10% of the total dose applied topically is actually absorbed. The transdermal delivery systems (TDS) presently available are either of the reservoir type (membrane-moderated system) or of the matrix dispersion type where the drug is dispersed into a polymer matrix. Estradiol (E₂) is the most appropriate steroid for TD and can be combined with progestins to ensure a contraceptive effect. Only potent progestins should be used to achieve effective plasma levels with low doses in order to maintain an acceptable small surface of TDS. TDS changed weekly and delivering both E₂ and levonorgestrel (L-NG) at daily dosages of 38.4 (±7.5) and 28.8 (±7.2) μg/10 cm² per day respectively, showed ovulation suppression. Another progestin derived from norprogesterone (ST 1435) has been shown to penetrate the skin when suspended in acetylated lanolin or dissolved in a hydroalcoholic gel and to ensure ovulation suppression at a dose of 2 mg per day in a small number of cycles. These preliminary data demonstrate the feasibility of suppressing ovulation in women by transdermal absorption of steroids. Using TDS for contraception implies that such systems should be perfectly adhesive, well tolerated locally and achieve nearly 100% efficacy. These targets are very challenging, however, the potential advantages are so high that the concept deserves further development.     

Determination of ovulation time in bitches based on teasing, vaginal cytology, and elisa for progesterone

The estrous cycle of 16 mature mongrel female dogs was monitored to evaluate the accuracy of teasing, vaginal cytology and quantitative ELISA progesterone assay to determine ovulation. The dogs were presented to male, and blood samples and vaginal swabs were taken daily during proestrus and estrus. Selected serum samples collected during estrus were assayed for endogenous LH by radioimmunoassay (RIA). Plasma samples collected during proestrus and estrus were assayed for progesterone with a commercially avialable ELISA kit. Ovulation was considered to take place 48 h after the preovulatory LH peak. Vaginal cytology smears were stained with Wright's stain and evaluated for the percentage of superficial squamous cells. Day 1 of diestrus (Day 1) was defined as a drop of 20% or more in the total number of superficial cells. Two standard curves (linear and best fitted curves) commonly used with ELISA were compared together and with the RIA progesterone assay. Ovulation was estimated to occur when progesterone concentration was 4.9 +/- 1.0ng/ml (mean +/- SD, n = 15), with a range of 3.4 to 6.6 ng/ml. Based on vaginal cytology, ovulation took place 6.9 +/- 1.6 d (n = 15) after 80% of the squamous cells were superficial and 6.8 +/- 1.4 d (n = 16) before Day 1. Ovulation took place 2.1 +/- 3.9 d (n=11) after the first day of standing estrus and 8.8 +/- 1.5 d (n = 10) before the last day of receptivity. The two standard curves were found parallel to each other and to the RIA progesterone assay. Based on the results of the present study, ELISA progesterone assay and determination of the first day of estrus by vaginal cytology are reliable methods for predicting ovulation, whereas the last day of receptivity as determined by teasing and Day 1 as determined by vaginal cytology are reliable methods to retrospectively estimate ovulation time.     

Mifepristone: a novel estrogen-free daily contraceptive pill

When the first synthetic progesterone antagonist (mifepristone) was synthesized over 20 years ago, it was clear that it had a potential as an antifertility agent. Research into the use of antiprogestogens for contraception have concentrated on three general approaches: (1) inhibition of ovulation, (2) inhibition of implantation and (3) disruption of implantation or "menstrual induction". The effect of mifepristone on the ovarian and endometrial cycle depends on dose, timing and frequency of administration. Doses of 10 mg per day or more suppress follicular development and estradiol levels. Ovulatory cycles are maintained in the dose of less than 2 mg although there is increased variability in cycle length. The endometrium shows some minor asynchronous changes, although these are not sufficient to prevent pregnancy. We have chosen to investigate daily doses between 2 and 5 mg which inhibit ovulation and menstruation in over 90% of cycles while still maintaining follicular development and levels of estradiol within the range found during the follicular phase. The endometrium shows proliferative or cystic changes lined by a layer of inactive glandular epithelium set in densely packed stroma. There is, however, an absence of proliferative activity as reflected by a reduced mitotic index and Ki67 staining. These unusual histological appearances are associated with downregulation of PR but a massive upregulation of AR in particularly glandular epithelium. The antiproliferative effect of mifepristone is reassuring suggesting that the risk of atypical hyperplasia due to the effect of prolonged exposure to estrogen unopposed by progesterone is low. In a pilot study, there were no pregnancies in 200 months of exposure in 50 women who used this method as their sole method of contraception. Daily mifepristone could provide a novel contraceptive method which should be devoid of the risks associated with estrogen containing combined oral contraceptive (COC), e.g. venous thromboembolism. The health benefits of avoiding the morbidity associated with menstruation are considerable. Recent surveys suggest that amenorrhoea would be popular with many women.     

Contraception with progestogens and progesterone during lactation

The growth and development of breastfed infants whose mothers used the contraceptive implants Norplant® containing levonorgestrel and the injectable containing norethisterone enanthate were studied. Each group comprised of 120 women who initiated the use during the 5th to 7th week postpartum and were compared with a similar number of IUD using mothers. The breastfeeding performance did not differ between groups. The infants of the three groups performed similarly as regards their physical growth and health as well as the time of acquisition of the various milestones of psychomental development. A vaginal ring releasing 10 mg of the “natural” progesterone per 24 h was tested in breastfeeding mothers. The continuous use of the ring produced a serum level of progesterone around 4 ng/ml. This was effective in augmenting lactational infertility even through the later phases of breastfeeding when such an effect starts to wane off. The use of the ring proved to be acceptable and had no ill-effect on breastfeeding or infant growth or health. Using the natural progesterone as a contraceptive adds a new measure of safety, since the amount of the steroid secreted in the mother's milk will not be effectively absorbed from the infant's gut. These studies suggest the possibility of using two new methods for breastfeeding mothers; Norplant and the progesterone vaginal contraceptive ring. These can be initiated early postpartum, whenever this is considered needed.     

Progestin implants

Progestin implants for contraception are highly effective, safe, and the most convenient choice for many women. Progestin implants currently on the market, preparing for launch, or under investigation are reviewed here. Their basic galenic and pharmacokinetic features, as well as their contraceptive effectiveness, are described. The first progestin-only contraceptive implant placed on the market was Norplant, a multiunit system. Since then, several single- and double-rod implants have been developed, each using one of four different progestins: levonorgestrel, etonogestrel, Nestorone and nomegestrol acetate. Jadelle is similar to Norplant but consists of only two, rather than six, Silastic rods to simplify insertion and removal; nevertheless, levonorgesterel serum levels are identical, and performance is the same for both systems. The single implant systems reviewed here are: Implanon with a 3-year duration; Nestorone implants for breast feeding and non-breast feeding women lasting up to 2 years; and Uniplant, which is effective for 1 year. The advantages and disadvantages of progestin implants, the importance of counseling for increasing user satisfaction, and the future outlook for this contraceptive method are also discussed.     

Use of an immediate, qualitative progesterone assay for determination of day of ovulation in an equine embryo transfer program

An immediate, qualitative enzyme-linked immunosorbent assay (ELISA) for progesterone was evaluated for use in determining the day of ovulation in an equine embryo transfer program. Plasma samples were collected from 27 mares from the third day of estrus to the second day of diestrus for 50 cycles. Ovulation was detected by ultrasound examination per rectum. Plasma progesterone concentrations were estimated using the qualitative assay to detect the time of the rise in progesterone after ovulation. Qualitative scores were compared to progesterone concentrations for the same samples as measured by a quantitative ELISA; the correlation between the two methods, expressed as a contingency coefficient, was 0.56. The accuracy of determining day of ovulation using qualitative progesterone results was compared to that achieved using the quantitative assay or detection of the first day of diestrus by teasing. Accuracy in determining day of ovulation +/- 1 d using the three methods was qualitative, 36/50 (72%); quantitative, 44/50 (88%); and teasing, 43/50 (86%). There was a significant difference in accuracy between the qualitative and quantitative progesterone assays (P<0.05).     

Molecular and pharmacological properties of a potent and selective novel nonsteroidal progesterone receptor agonist tanaproget

Progesterone receptor (PR) agonists have several important applications in women's health, such as in oral contraception and post-menopausal hormone therapy. Currently, all PR agonists used clinically are steroids. Because of their interactions with other steroid receptors, steroid-metabolizing enzymes, or other steroid-signaling pathways, these drugs can pose significant side effects in some women. Efforts to discover novel nonsteroidal PR agonists with improved biological properties led to the discovery of tanaproget (TNPR). TNPR binds to the PR from various species with a higher relative affinity than reference steroidal progestins. In T47D cells, TNPR induces alkaline phosphatase activity with an EC(50) value of 0.1 nm, comparable with potent steroidal progestins such as medroxyprogesterone acetate (MPA) and trimegestone (TMG), albeit with a reduced efficacy ( approximately 60%). In a mammalian two-hybrid assay to measure PR agonist-induced interaction between steroid receptor co-activator-1 and PR, TNPR showed similar potency (EC(50) value of 0.02 nm) and efficacy to MPA and TMG. Importantly, in key animal models such as the rat ovulation inhibition assay, TNPR demonstrates full efficacy and an enhanced progestational potency (30-fold) when compared with MPA and TMG. Furthermore, TNPR has relatively weak interactions with other steroid receptors and binding proteins and little effect on cytochrome P450 metabolic pathways. Finally, the three-dimensional crystal structure of the PR ligand binding domain with TNPR has been delineated to demonstrate how this nonsteroidal ligand achieves its high binding affinity. Therefore, TNPR is a structurally novel and very selective PR agonist with an improved preclinical pharmacological profile.     

Clinical and Laboratory Assessment of a New Progestational Agent

Studies of the effects of megestrol (6-methyl, 6-dehydro, 17α-acetoxy progesterone) on human subjects indicated that this drug is a potent, orally effective progestational agent. The progestational effect was enhanced by estrogen. When combined with ethinyl estradiol satisfactory control of menstrual function and inhibition of ovulation were achieved. Increasing doses of this combination were effective in four patients with endometriosis. Side effects of the drug were minimal. No toxic effects on adrenal, hepatic or hematopoietic function were noted.     

Effects of hormonal treatments on reproductive performance and embryo production

Developments in the use of drugs to improve reproduction and embryo production have focused on estrus and ovulation synchronization protocols and embryonic survival. Protocols for synchronization of ovulation eliminate the need for detection of estrus and allow timed insemination of all cows enrolled. Various estrogenic, progestational, GnRH and PGF2 alpha-like drugs are used to synchronize follicle development, CL regression and induction of ovulation. Strategies are discussed to optimize such programs to maximize herd pregnancy rates. Use of bovine Somatotrophin (bST) in combination with the Ovsynch protocol resulted in increased pregnancy rates, indicating possible effects on oocyte and embryonic development. Treatment of embryo donor cows with bST reduced the proportion of unfertilized oocytes and increased the number of transferable embryos. Furthermore, bST increased pregnancy rate when given to the recipient. Sub-luteal plasma progesterone concentrations after insemination have been associated with lower pregnancy rates. Injection of hCG on day 5 post-insemination resulted in induction of an accessory CL, increased plasma progesterone concentrations and increased conception rates. Strategies involving the use of sustained GnRH agonists to enhance CL development and alter follicular development are considered for future programs to enhance pregnancy rates.     

The relationship between vaginal mucous impedance and serum concentrations of estradiol and progesterone throughout the sheep estrous cycle

The objective of this experiment was to assess the relationship between electrical resistance of the vaginal mucosa and serum concentrations of estradiol (E2) and progesterone (P4) during the estrous cycle in ewes. Vaginal impedance was recorded daily using a 2-electrode impedometer in 10 nonprolific Western white-faced and 7 prolific Finn ewes, during the mid-breeding season (October to December). Transrectal ultrasonography of ovaries was performed once a day to confirm ovulation and monitor follicle growth (follicles > or =3 mm in diameter) and development of corpora lutea (CL). Jugular blood samples were collected daily for radioimmunoassay (RIA) of estradiol and progesterone. In all ewes, a decline in vaginal impedance (to <40 ohms) was closely associated with the onset of behavioral estrus. In both breeds of sheep, there was no significant correlation between daily serum concentrations of estradiol and vaginal impedance throughout the estrous cycle. Daily serum concentrations of progesterone and the E2:P4 ratio were correlated with vaginal impedance during the period of luteolysis and follicular phase in both breeds (Western white-faced ewes: r = 0.62, P = 0.0002 and r = -0.56, P = 0.0002; Finn ewes: r = 0.61, P = 0.001 and r = -0.45, P = 0.03, respectively) and early in the cycle (Days 0 to 2, Day 0 = day of ovulation) in white-faced ewes (r = 0.61, P = 0.0003 and r = -0.36, P = 0.052, respectively) but not during the remaining portion of the luteal phase in either breed. In conclusion, vaginal mucous impedance appears to be primarily controlled by progesterone, but it also changes in response to shifts in the E2:P4 ratio when progesterone concentrations are low. Impedometric characteristics of the vaginal mucosa in cyclic ewes are an indicator of serum concentrations of progesterone and E2:P4 ratios during the terminal stage of the estrous cycle.     

Progesterone and estradiol in biodegradable microspheres for control of estrus and ovulation in mares

Progesterone and estradiol 17-beta in poly (DL-lactide) microspheres were used to control estrus and ovulation in mares after luteolysis was induced by prostaglandin F(2)infinity. Mares were given a single intramuscular injection of biodegradable poly (DL-lactide) microspheres, 1 day following prostaglandin treatment, containing no hormones (control), 0.625 g progesterone and 50 mg estradiol (low dose), 1.25 g progesterone and 100 mg estradiol (medium dose), or 1.875 g progesterone and 150 mg estradiol (high dose; n=15 mares per group). Mares treated with the low dose had significantly longer intervals (P<0.05) to estrus and ovulation than the control mares; however, low dose mares had shorter intervals (P<0.05) to estrus than high dose mares and shorter intervals to ovulation than medium and high dose mares. Regression analysis indicated that the medium dose was sufficient for maximizing interval to ovulation while the high dose maximized interval to estrus. All groups of mares exhibited similar (P>0.05) post-treatment estrus lengths. A clinical response scoring system based on synchrony of both estrus and ovulation within a treatment group was also used to measure the effectiveness of treatments on control of estrus and ovulation. Clinical response scores did not differ (P>0.05) among treatment groups. Mares were randomly assigned for insemination at the beginning of the first post-treatment estrus. Rates for embryo recovery performed by uterine lavage 7 days post-ovulation did not differ (P>0.05) among groups. Concentrations of serum progesterone increased in mares receiving progesterone and estradiol microspheres. At 10 to 14 days post-injection of microspheres, progesterone concentrations were higher (P<0.05) and remained above 1 ng/ml in the mares receiving the high dose. Progesterone concentrations were also higher (P<0.05) on Days -3 to -1 (Day 0 = day of post-treatment ovulation) in mares receiving the high dose when compared to control mares. Gonadotropin concentrations were suppressed (P<0.05) in the medium and high dose groups.     

Relationship between peri-oestrus progesterone levels and time of ovulation by echography in pigs and influence of the interval between ovulation and artificial insemination (AI) on litter size

Two methods for the determination of ovulation were compared to one ultrasonography performed 5 times a day. Time of ovulation by echography was 40 +/- 5.8 h (mean +/- SD) after the onset of oestrus. Preovulatory LH rise (two blood samples per day) began near the onset of oestrus but, in our conditions, this parameter could not be used to predict ovulation. The basal level of progesterone (two blood samples per day) was determined with a non-linear model, the timing when progesterone rose more than one SD (0.3 ng x mL(-1)) coincided with the timing of ovulation determined by echography (R2 = 0.98). This method was efficient and was used in a field trial to measure the consequences of the variability of the interval between Al and ovulation on litter size. The interval between Al and ovulation had an effect on litter size; litter size decreased by one piglet when this interval increased by 10h.