A rodent model of human dose-equivalent progestin-only implantable contraception.

BACKGROUND: Long-acting, reversible contraceptives (LARC; progestin only) are an increasingly common hormonal contraceptive choice in reproductive aged women looking to suppress ovarian function and menstrual cyclicity. The overall objective was to develop and validate a rodent model of implanted etonogestrel (ENG) LARC, at body size equivalent doses to the average dose received by women during each of the first 3 years of ENG subdermal rod LARC use. METHODS: Intact, virgin, female Sprague-Dawley rats (16-wk-old) were randomized to 1 of 4 groups (n = 8/group) of ENG LARC (high-0.30µg/d, medium-0.17µg/d, low-0.09µg/d, placebo-0.00µg/d) via a slow-release pellet implanted subcutaneously. Animals were monitored for 21 days before and 29 days following pellet implantation using vaginal smears, ultrasound biomicroscopy (UBM), saphenous blood draws, food consumption, and body weights. Data were analyzed by chi-square, non-parametric, univariate, and repeated measures 2-way ANOVA. RESULTS: Prior to pellet implantation there was no difference in time spent in estrus cycle phases among the treatment groups (p > 0.30). Following pellet implantation there was a dose-dependent impact on the time spent in diestrus and estrus (p < 0.05), with the high dose group spending more days in diestrus and fewer days in estrus. Prior to pellet insertion there was not an association between treatment group and estrus cycle classification (p = 0.57) but following pellet implantation there was a dose-dependent association with cycle classification (p < 0.02). Measurements from the UBM (ovarian volume, follicle count, corpora lutea count) indicate an alteration of ovarian function following pellet implantation. CONCLUSION: Assessment of estrus cyclicity indicated a dose-response relationship in the shift to a larger number of acyclic rats and longer in duration spent in the diestrus phase. Therefore, each dose in this model mimics some of the changes observed in the ovaries of women using ENG LARC and provides an opportunity for investigating the impacts on non-reproductive tissues in the future.

Contraceptive efficacy of sperm agglutinating factor from Staphylococcus warneri, isolated from the cervix of a woman with inexplicable infertility.

BACKGROUND: Voluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility. METHODS: Genomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-α sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg++-dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice. RESULTS: The 80 kDa rSAF showed complete sperm agglutination, inhibited its Mg2+-ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests. CONCLUSION: The Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.

Differential metabolism of clinically-relevant progestogens in cell lines and tissue: Implications for biological mechanisms.

Steroid hormones regulate a variety of physiological processes, including reproductive function, and are widely used in hormonal therapy. Synthetic progestogens, or progestins, were designed to mimic progesterone (P4) for use in contraception and hormonal replacement therapy in women. Medroxyprogesterone acetate (MPA) and norethisterone (NET) are the most widely used injectable contraceptives in the developing world, while other progestins such as levonorgestrel (LNG), etonogestrel (ETG) and nestorone (NES) are used in or being developed for other forms of contraception. As concerns remain about the most appropriate choice of progestin and dosage, and the associated side-effects, the mechanisms and biological effects of progestins are frequently investigated in various in vitro mammalian cell line and tissue models. However, whether progestogens are differentially metabolised in different cell types in vivo or in vitro is unknown. For nine mammalian cell lines commonly used to investigate progestogen mechanisms of action, we developed and validated an ultra-high performance supercritical fluid chromatography-tandem mass spectrometry (UHPSFC-MS/MS) protocol for simultaneously quantifying the metabolism of the above-mentioned steroids. We show for the first time that, while 50-100% of P4 was metabolised within 24 h in all cell lines, the metabolism of the progestins is progestin- and cell line-specific. We also show that MPA and NET are significantly metabolised in human cervical tissue, but to a lesser extent than P4. Taken together, our findings suggest that differential progestogen metabolism may play a role in cell-specific therapeutic and side-effects. Relative affinities for binding to steroid receptors as well as potencies, efficacies and biocharacters for transcriptional activity of progestins, relative to P4, are most frequently determined using some of the cell lines investigated. Our results, however, suggest that differential metabolism of progestins and P4 may confound these results. In particular, metabolism may under-estimate the receptor-mediated intrinsic in vitro binding and dose-response values and predicted endogenous physiological effects of P4.

Control of endometriosis-associated pain with etonogestrel-releasing contraceptive implant and 52-mg levonorgestrel-releasing intrauterine system: randomized clinical trial.

OBJECTIVE: To assess the efficacy of an etonogestrel (ENG)-releasing contraceptive implant or the 52-mg levonorgestrel-releasing intrauterine system (LNG-IUS) in the control of endometriosis-associated pelvic pain. DESIGN: Noninferiority randomized clinical trial in which women with endometriosis were assigned to use an ENG implant (experimental treatment) or an LNG-IUS (active comparator). Monthly follow-up visits were conducted up to 6 months. SETTING: University teaching hospital. PATIENT(S): One hundred three women, with endometriosis-associated chronic pelvic pain, dysmenorrhea, or both for more than 6 months. In cases of deep endometriosis, vaginal ultrasonography and magnetic resonance imaging were used as additional diagnostic tools. INTERVENTION(S): The ENG implant or the LNG-IUS were inserted within the first 5 days of the menstrual cycle. MAIN OUTCOME MEASURE(S): Daily scores of noncyclic pelvic pain and dysmenorrhea were evaluated using a daily visual analogue scale. Health-related quality of life was evaluated using the Endometriosis Health Profile-30 questionnaire at baseline and up to 6 months. Bleeding patterns were assessed daily from a menstrual calendar. RESULT(S): Both contraceptives improved significantly the mean visual analogue scale endometriosis-associated pelvic pain and dysmenorrhea, without significant differences between treatment group profiles. Health-related quality of life improved significantly in all domains of the core and modular segments of the Endometriosis Health Profile-30 questionnaire, with no difference between both treatment groups. The most common bleeding patterns at 180 days of follow-up were amenorrhea and infrequent bleeding and infrequent bleeding and spotting among ENG implant and LNG-IUS users, respectively. CONCLUSION(S): In this noninferiority study both contraceptives improved significantly pelvic pain, dysmenorrhea, and health-related quality of life in endometriosis. CLINICAL TRIAL REGISTRATION NUMBER: Clinicaltrials.gov under number NCT02480647.

Nestorone® as a Novel Progestin for Nonoral Contraception: Structure-Activity Relationships and Brain Metabolism Studies.

Nestorone® (NES) is a potent nonandrogenic progestin being developed for contraception. NES is a synthetic progestin that may possess neuroprotective and myelin regenerative potential as added health benefits. In receptor transactivation experiments, NES displayed greater potency than progesterone to transactivate the human progesterone receptor (PR). This was confirmed by docking experiments where NES adopts the same docking position within the PR ligand-binding domain (LBD) as progesterone and forms additional stabilizing contacts between 17α-acetoxy and 16-methylene groups and PR LBD, supporting its higher potency than progesterone. The analog 13-ethyl NES also establishes similar contacts as NES with Met909, leading to comparable potency as NES. In contrast, NES is not stabilized within the human androgen receptor LBD, leading to negligible androgen receptor transactivation. Because progesterone acts in the brain by both PR binding and indirectly via binding of the metabolite allopregnanolone to γ-aminobutyric acid type A receptor (GABAAR), we investigated if NES is metabolized to 3α, 5α-tetrahydronestorone (3α, 5α-THNES) in the brain and if this metabolite could interact with GABAAR. In female mice, low concentrations of reduced NES metabolites were identified by gas chromatography/mass spectrometry in both plasma and brain. Electrophysiological studies showed that 3α, 5α-THNES exhibited only limited activity to enhance GABAAR-evoked responses with WSS-1 cells and did not modulate synaptic GABAARs of mouse cortical neurons. Thus, the inability of reduced metabolite of NES (3α, 5α-THNES) to activate GABAAR suggests that the neuroprotective and myelin regenerative effects of NES are mediated via PR binding and not via its interaction with the GABAAR.

The other side of progestins: effects in the brain.

Progestins are a broad class of progestational agents widely differing in their chemical structures and pharmacological properties. Despite emerging data suggest that progestins, besides their action as endometrial protection, can also have multiple nonreproductive functions, much remains to be discovered regarding the actions exerted by these molecules in the nervous system. Here, we report the role exerted by different progestins, currently used for contraception or in postmenopausal hormone replacement therapies, in regulating cognitive functions as well as social behavior and mood. We provide evidence that the effects and mechanisms underlying their actions are still confusing due to the use of different estrogens and progestins as well as different doses, duration of exposure, route of administration, baseline hormonal status and age of treated women. We also discuss the emerging issue concerning the relevant increase of these substances in the environment, able to deeply affect aquatic wildlife as well as to exert a possible influence in humans, which may be exposed to these compounds via contaminated drinking water and seafood. Finally, we report literature data showing the neurobiological action of progestins and in particular their importance during neurodegenerative events. This is extremely interesting, since some of the progestins currently used in clinical practice exert neuroprotective and anti-inflammatory effects in the nervous system, opening new promising opportunities for the use of these molecules as therapeutic agents for trauma and neurodegenerative disorders.

Controlling levonorgestrel binding and release in a multi-purpose prevention technology vaginal ring device.

Despite a long history of incorporating steroids into silicone elastomers for drug delivery applications, little is presently known about the propensity for irreversible drug binding in these systems. In this study, the ability of the contraceptive progestin levonorgestrel to bind chemically with hydrosilane groups in addition-cure silicone elastomers has been thoroughly investigated. Cure time, cure temperature, levonorgestrel particle size, initial levonorgestrel loading and silicone elastomer type were demonstrated to be key parameters impacting the extent of levonorgestrel binding, each through their influence on the solubility of levonorgestrel in the silicone elastomer. Understanding and overcoming this levonorgestrel binding phenomenon is critical for the ongoing development of a number of drug delivery products, including a multi-purpose technology vaginal ring device offering simultaneous release of levonorgestrel and dapivirine - a lead candidate antiretroviral microbicide - for combination HIV prevention and hormonal contraception.

Development of controlled release systems over the past 50years in the area of contraception.

The field of controlled release has contributed significantly to female reproductive health and in particular the prevention of unintended pregnancy. For at least 50years, there have been significant advances in controlled release dosage forms used for contraception. These advances have been driven by the need to provide women a wide array of products that address adherence problems noted with oral contraceptives. The first long-acting injectable product (Depo-Provera®) was approved in the US in 1959. Since then, there has been an emphasis on development of long-acting reversible contraceptives. These products include implants, intrauterine systems, and vaginal rings. A shorter acting contraceptive option is the transdermal patch. Despite these advances there are still a large number of unplanned pregnancies around the world. New controlled release technologies will be needed to continue providing women safe and easy to use contraceptive products.

[Contemporary possibilities of non-oral estrogen-progestin contraception]. / Soucasné moznosti non-orální estrogen-progestinové antikoncepce.

Besides the classic combined oral contraception (pill), there are alternative medical forms as transdermal contraception system, vaginal ring, intramuscular injections, and percutaneous gel, respectively. The mechanism of contraceptive effect is the same as well as they are the contraindications. The advantage of non-oral application is the elimination of the first-pass effect that enables reduction of doses, diminishing of undesirable side-effects and medical interactions, and only small effect on enzymatic systems associated with cytochrome P450. As the main advantage, comfort and better compliance of patients who have problems with daily use of tablets, is considered.

Effect of progestins on immunity: medroxyprogesterone but not norethisterone or levonorgestrel suppresses the function of T cells and pDCs.

OBJECTIVES: The potential effect of hormonal contraception on HIV-1 acquisition and transmission represents an important public health issue. Several observational studies have suggested an association between the use of hormonal contraception, in particular injectable depot medroxyprogesterone acetate (DMPA), and an increased risk of HIV-1 acquisition and transmission. We and others have previously demonstrated that DMPA acts as a potent inhibitor of innate and adaptive immune mechanisms. The study presented here addresses the immunomodulatory properties of several common progestins with a potential to replace DMPA. STUDY DESIGN: To identify safe alternatives to DMPA, we tested the effect of commonly used progestins on the function of human primary T cells and plasmacytoid dendritic cells (pDCs) obtained from the blood of healthy premenopausal women. RESULTS: Medroxyprogesterone acetate (MPA) inhibited the activation of T cells and pDCs in response to T cell receptor- and Toll-like receptor-mediated activation at physiological concentrations. Etonogestrel exerted a partial suppressive activity at high concentrations. In sharp contrast, norethisterone (NET) and levonorgestrel (LNG) did not exhibit detectable immunosuppressive activity. CONCLUSION: Evidence indicating the immunosuppressive properties of DMPA strongly suggests that DMPA should be discontinued and replaced with other forms of long-term contraception. Since NET and LNG do not exert immunosuppressive properties at physiological concentrations, these progestins should be considered as alternative contraceptives for women at high risk for HIV-1 infection. IMPLICATIONS: The presented data suggest that, at physiological levels, the progestins NET and LNG do not suppress cytokine production by immune cells and should be considered as alternatives to DMPA; however, more in vivo testing is needed to confirm this data.

Association between caffeine intake and bone mass among young women: potential effect modification by depot medroxyprogesterone acetate use.

UNLABELLED: This study assessed associations between habitual caffeine intake and bone mass among young women. Analyses of the entire study population revealed no significant associations, while analyses restricted to women using depot medroxyprogesterone acetate (DMPA) showed modest inverse associations between caffeine intake and bone mineral content (BMC). INTRODUCTION: Some previous investigations among postmenopausal women suggest an inverse relationship between caffeine intake and bone mass, yet studies of this association among young women are few. METHODS: The association between habitual caffeine intake and bone mass was evaluated prospectively in a population-based cohort of 625 females, aged 14 to 40 years, adjusting for relevant biological and lifestyle factors. Caffeinated beverage intake was self-reported, and bone mineral content (BMC) and bone mineral density (BMD) were measured at baseline and every 6 months throughout a 24-month follow-up period using dual-energy x-ray absorptiometry. RESULTS: Cross-sectional analyses revealed no significant differences in mean BMC or BMD at baseline. Mean percentage and absolute changes in BMC and BMD were not associated with caffeine use. Repeated measures analyses similarly showed no significant association between caffeine intake at baseline and mean BMC or BMD measured during follow-up. However, among women using depot medroxyprogesterone acetate (DMPA), modest inverse associations between caffeine and BMC (but not BMD) were detected. CONCLUSIONS: Our data suggest that heavy habitual consumption of caffeinated beverages does not adversely impact bone mass among young women in general. Greater caffeine intake may be associated with lower BMC among DMPA users.

Contraception for women with epilepsy.

Antiepileptic drugs that induce hepatic enzyme activity may alter the metabolism of most hormonal methods of contraception, and this affects the contraceptive regime. This effect should be considered in the choice of both the treatment of the epilepsy and the choice of contraceptive method. This review considers these interactions and offers advice about their management.

The cytokine receptor gp130 and its soluble form are under hormonal control in human endometrium and decidua.

The transmembrane protein gp130 plays a central role in cytokine action as a signal transducing receptor subunit common to all interleukin-6 type cytokines. Endometrial tissue obtained from women with a normal menstrual cycle and decidua obtained from women in the first or second trimester of pregnancy were assessed for gp130 by western blotting, immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) analysis. By immunoblotting, two forms of gp130 were detected: one-the soluble form-of approximately 100 kDa and a larger membrane-bound form of approximately 150 kDa. The latter became clearly visible in the mid to late secretory phase and was more pronounced in decidual tissue of second trimester compared to first trimester. Immunohistochemically, gp130 was located in glandular epithelial cells during the mid to late secretory phase, whereas staining in the proliferative phase was rather weak. In first and second trimester decidua, glandular cells were also positively stained. In addition, the invading trophoblast cells were gp130 positive. Soluble gp130 release was measured in the supernatants from primary endometrial and decidual cell cultures by ELISA and reached maximum values in cell cultures without addition of hormones. In cultured endometrial epithelial cells obtained during the proliferative phase of the cycle, the soluble gp130 release increased significantly under combined estradiol/progesterone supplementation which mimics the secretory phase conditions compared to estradiol supplementation alone. In cultured epithelial cells derived from decidual tissue of first trimester of pregnancy, similar effects of hormonal regulation were observed. Our results suggest that the balance between soluble gp130 and its membrane-bound form may play an important role in regulating cytokine action necessary for blastocyst implantation and for further interaction between the decidualized endometrium and the invading trophoblast.

LDL susceptibility to copper-induced oxidation after administration of a single dose of free or esterified beta-cryptoxanthin.

BACKGROUND: The oxidative modification of LDL is believed to be an initial step in atherosclerosis. Thus, antioxidative substances such as carotenoids may have a role in the prevention of coronary heart disease. We examined the susceptibility of LDL to Cu2+ oxidation in young adults before and after a single dose of beta-cryptoxanthin. METHODS: 1.3 mg of beta-cryptoxanthin was administered to 12 apparently healthy young volunteers. Six of the volunteers received esters, the other six free beta-cryptoxanthin. The plasma concentration of beta-cryptoxanthin and the susceptibility of LDL to copper-induced oxidation ex vivo in terms of the duration of lag time were measured before and 12 h after beta-cryptoxanthin ingestion. RESULTS: A single dose of beta-cryptoxanthin significantly increased the mean plasma beta-cryptoxanthin concentration and the mean cholesterol adjusted beta-cryptoxanthin concentration by 117 and 133%, respectively. No effect on the length of lag time was assessed. However, in LDL isolated from plasma 12 h after beta-cryptoxanthin administration the lengths of lag time correlated significantly with the plasma beta-cryptoxanthin concentration and with the cholesterol adjusted beta-cryptoxanthin levels. The lag time did not differ significantly between volunteers who received esters and those who received the same dosage as free beta-cryptoxanthin. At both measuring points, smokers, male volunteers and women using oral contraceptives tended to exhibit lower beta-cryptoxanthin concentrations and lower cholesterol adjusted beta-cryptoxanthin concentrations as well as increased LDL oxidizability compared to nonsmokers and women not using oral contraceptives. CONCLUSION: A single dose of beta-cryptoxanthin does not enhance the duration of LDL lag time ex vivo in healthy young subjects.

Membrane heme as a host factor in reducing effectiveness of dihydroartemisinin.

Plasmodium falciparum infecting alpha-thalassemic erythrocytes are resistant to artemisinin and its derivatives. Binding of the drug to hemoglobin H resulting in drug inactivation was previously demonstrated. We now show that an additional host factor, membrane heme, significantly accounted for decreased antimalarial activity of artemisinin. The antimalarial activity of dihydroartemisinin in the presence of normal and thalassemic erythrocyte membranes showed a correlation with the heme content of the membrane (r(2)=0.466, P<0.01). The correlation was more clearly seen when the drug effectiveness was correlated with the heme content of alpha-thalassemic membrane (r(2)=0.636, P<0.01). However, the drug effectiveness showed no correlation to ferrozine-reactive (free or non-heme) iron content (r(2)=0.0001, P>0.05). alpha-Thalassemic erythrocytes contained higher amounts of membrane heme (11.04+/-8.96 nmol/mg membrane protein) than those from normal and beta-thalassemia/HbE erythrocytes (2.68+/-1.28 and 3.98+/-3.98 nmol/mg membrane protein, respectively, P<0.01). Loss of drug effectiveness was also correlated with increment of heme content in membrane prepared from normal erythrocytes treated with phenylhydrazine. It is concluded that heme in both normal and thalassemic erythrocyte membranes is an important factor in drug inactivation.

Effects of levonorgestrel implant upon TSH and LH levels in male infants during lactation.

OBJECTIVES: The objective of the study is to evaluate the effects of levonorgestrel transferred through breast milk on thyroid stimulating hormone (TSH) and luteinizing hormone (LH) levels in full breast-fed infants. METHODS: Forty healthy postpartum women and their male newborns were recruited for the study. Women were randomly allocated to two study groups: Group 1, IUD users and group 2, Norplant users. Blood and milk samples were collected on the day of IUD or Norplant insertion and 3 and 6 months later for TSH and levonorgestrel measurements. RESULTS: The results disclosed a significant decrease in TSH levels, and a negative relationship between LNG levels and TSH concentration in breast feeding infants at 3 months after implant insertion. The lowest TSH levels were observed at 6 months in the women from group 2. CONCLUSIONS: The overall data indicate that the LNG levels transferred to fully breast-fed infants through breast milk from Norplant users significantly modified their TSH levels.

Assessment of the oestrogenic activity of the contraceptive progestin levonorgestrel and its non-phenolic metabolites.

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.