Blood changes in sex steroid hormone users. Circulating immune complexes induced by estrogens and progestogens and their relation to vascular thrombosis.

Oral contraceptives (OC) have been shown to induce in some women antiethinylestradiol antibodies which may be detected as circulating immune complexes by precipitation in ammonium sulphate at 25% saturation (CIC.AS). A reevaluation of the presence of CIC.AS in 644 women either receiving sex steroid hormones or not was made, and the respective role of estrogens and progestogens investigated, together with the influence of the dose. The study confirmed that CIC.AS levels were significantly different in controls (442 +/- 246 micrograms/ml serum), healthy gonadal hormone users (754 +/- 700 micrograms) and users with thrombosis (1331 +/- 1099 micrograms/ml). These results indicated that: 1. CIC.AS could be induced by synthetic estrogens as well as progestogens, but not by non-synthetic hormones; 2. the induction of CIC.AS seemed poorly dose-related, and 3. was not correlated with the duration of use; 4. in reactive women, high CIC.AS levels occurred as soon as 3 weeks after the beginning of synthetic gonadal hormones use, persisted throughout treatment and decreased slowly when discontinued; 5. in women with thrombosis CIC.AS were more frequently detected (64.7%) than in healthy users (32.2%) P less than 0.001. The importance of the immunologic changes as a risk factor in thrombosis in OC users was evaluated in comparison with other predisposing factors and tobacco smoking.

PIP: Oral contraceptives (OCs) have been shown to induce antiethinyl estradiol antibodies in some women which may be detected as circulating immune complexes by precipitation in ammonium sulphate at 25% saturation (CIC.AS). A reevaluation of the presence of CIC.AS in 644 women either receiving sex steroid hormones or not was made, and the respective role of estrogens and progestogens investigated, together with the influence of the dose. The study confirmed that CIC.AS levels were significantly different in controls (442 +or- 246 mcg/ml serum), healthy gonadal hormone users (754 +or- 700 mcg) and users with thrombosis (1331 +or- 1099 mcg/ml). These results indicated that: 1) CIC.AS could be induced by synthetic estrogens as well as progestogens but not by nonsynthetic hormones; 2) the induction of CIC.AS seemed poorly dose-related; and 3) the induction was not correlated with the duration of use; 4) high CIC.AS levels occurred as soon as 3 weeks after the beginning of synthetic gonadal hormone use in reactive women and persisted throughout treatment and decreased slowly when discontinued; and 5) CIC.AS was detected more frequently (64.7%) in women with thrombosis than in healthy users (32.2%), P0.001. The importance of the immunologic changes as a risk factor in thrombosis in OC users was evaluated in comparison with other predisposing factors and tobacco smoking.

Enzyme immunoassay for nomegestrol acetate in human plasma.

Currently available chromatographic assays of the progestative drug nomegestrol acetate in human plasma are not suitable for monitoring drug kinetics more than 24 h after clinical dosage. A specific and sensitive enzyme immunoassay was therefore developed. A 3(O-carboxymethyl)oxime derivative of nomegestrol acetate was synthesized and coupled to bovine serum albumin in order to raise polyclonal antibodies in rabbits. The enzymatic tracer was obtained by coupling the 3(O-carboxymethyl)oxime derivative to acetylcholinesterase (E.C.3.1.1.7.). HPLC fractionation of human plasma samples followed by enzyme immunoassay revealed the presence of cross-reacting metabolites. An automated procedure of metabolite separation was developed using silica bonded with diol groups (Diol Bakerbond column). This procedure ensured assay specificity. The quantification limit in human plasma was 0.1 ng/ml. Mean repeatability (intra-assay variation) and reproducibility (inter-assay variation) were 9 and 15%, respectively. The enzyme immunoassay allowed monitoring of the kinetics of nomegestrol acetate 144 h after oral administration of a single 5 mg dose. Values for human samples were in excellent agreement with those assayable by HPLC followed by u.v. detection.

Phytohemagglutin-induced lymphocyte transformation in oral contraceptive users.

Phytohemagglutin (PHA)-induced lymphocyte transformation (PILT) was determined in 217 women taking oral contraceptives and 203 control women by means of the uptake of 3H-thymidine into DNA of lymphocytes cultured in heterologous serum. Depressed PILT responses were observed in oral contraceptive users as compared with age-matched controls, and the magnitude of depression correlated with the duration of oral contraception and was inversely related to the clinical progestagenic potency of the component steroids. An additional group of 21 women, tested within 1 year (mean 3 months) of cessation of oral contraception, showed persistent depression of PILT responses. Suppression of lymphocyte transformation in autologous as compared with homologous, normal serum suggests that serum inhibitory factors amy be important. We found no evidence for a direct suppressive in vitro effect of synthetic estrogens and gestagens. The prevalence of autoantibodies in oral contraceptive users was similar to that in control subjects.

Production of antisera against contraceptive steroids.

A four step synthesis of 6-(0-carboxymethyl) oximinoethynylestradiol is reported. This compound, 6-(0-carboxymethyl) oximinomestranol, the 3-(0-carboxymethyl) oximes of norethindrone and norgestrel and the 3-hemisuccinate of ethynylestradiol were synthesized and conjugated with bovine serum albumin. Rabbits were immunized at 3 dose levels of haptene (20, 66 and 200 nmoles) and eight weeks later with a booster containing 66 nmoles of haptene. The antibody titer and association constant of responding rabbits was nearly independent of dose although most antibody production occurred after the booster injection. Antibodies to mestranol crossreacted more than 100 percent with ethynylestradiol and to a small extent with norethindrone and norgestrel.

PIP: The methods of synthesis of the 6-(0-carboxymethyl) oxime derivative of 6-oxoethynylestradiol-17beta, the 3-succinyl derivative of ethiny estradiol-17beta and the 3-carboxymethyl oxime derivative of norethindrone and norgestrel as well as the synthesis of the corresponding bovine serum albumin (BSA) conjugates required for the specific antisera for mestranol, ethinyl estradiol, norethindrone, and norgestrel in rabbits are described. The synthesis of 6-(0-carboxymethyl) oxime derivative involves a 4-step synthesis and a chromatographic purification which is an improvement over past methods. Conjugation to BSA was by the carbodiimide method. Response of immunization was measured in terms of titer, association constant, and cross-reactivity as a function of time after immunization with 3 dose levels of each antigen. Titer response was independent of dose for the original injection. Ethiny estradiol-3-conjugates had very low titers.

Absence of antisteroid antibodies in oral contraceptive users presenting with vascular events.

Previous reports speculated that vascular events could be related to the development of antibodies against synthetic steroids contained in oral contraceptives or other hormonal treatments. This study describes original immunoassays designed to detect antisynthetic steroid antibodies. In a first step, the assays were characterized and validated using animal-raised antisteroid antibodies. In a second step, a population of 88 oral contraceptive users, 47 of them having developed a vascular thrombosis during synthetic steroid use and 41 serving as healthy control users, were tested. Detection of antibodies against ethinylestradiol, levonorgestrel, norethisterone, cyproterone acetate, and gestodene showed that the values obtained in normal oral contraceptive users as well as thrombosis patients are very low, and show no statistically significant difference between the two groups tested. Taken together, these data indicate that the "immunological hypothesis" related to antisteroid antibodies is unlikely to explain the pathogenesis of vascular events in oral contraceptive users.

[Bovine serum albumin conjugates of the new progestagen dienogest, synthesis and immunogenic properties]. / Rinderserumalbuminkonjugate des neuen Progestagens Dienogest--Syntheseund immunogene Eigenschaften.

In order to develop a radioimmunoassay for the new progestagen dienogest (STS 557, 17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one), bovine serum albumin (BSA) conjugates of STS 557-3-carboxymethyloxime and of STS 557-11-hemisuccinate were synthesized as antigens for the production of antisera. It was proved that an excess of isobutylchlorocarbonate in the coupling reaction using the "mixed anhydride method" results in an acylation of free NH2-groups in the BSA. By the immunization of rabbits with the STS 557-antigen-antisera of high specificity and affinity to STS 557 were produced. Endogenous steroids show no cross reaction with the STS 557-antisera. Steroids with a 17 alpha-CH2CN-group, being obtained by chemical synthesis or microbial transformation, compete with STS 557 for the binding positions of the antibodies to a different extent.

Medroxyprogesterone acetate enhances in vivo and in vitro antibody production.

In the present study we examine the effects of medroxyprogesterone acetate (MPA) on the specific antibody secretion to T-dependent antigens. Our results show that the in vivo administration of MPA to mice, 7 or 90 days before immunization with sheep red blood cells (SRBC), significantly enhanced both, primary and secondary antibody responses, without affecting delayed-type hypersensitivity (DTH). These effects could be counteracted by the anti-progestin onapristone or ZK 98299 (ZK) suggesting that MPA interacted with progesterone (PRG) receptors to increase B-cell response. To better understand the mechanisms involved in MPA activity we carried out cultures of splenocytes, bone marrow cells or lymph node cells from immunized mice in the presence of MPA, and evaluated the amount of antibody release to supernatants. We found that low doses of MPA (10(-9) M and 10(-10) M) significantly enhanced the in vitro production of specific immunoglobulin G (IgG) antibodies, an effect that appears to involve the interaction of the progestin with PRG receptors, as judged by the inhibition of MPA effects with ZK (10(-8) M) or RU486 (10(-9) M). These receptors were detected by flow cytometry analysis in a proportion of T lymphocytes. Because MPA did not increase the number of immunoglobulin-secreting cells, our findings suggest that MPA enhanced the capacity of individual cells to produce specific immunoglobulin.

Biotin-labelled and photoactivatable aldosterone and progesterone derivatives as ligands for affinity chromatography, fluorescence immunoassays and photoaffinity labelling.

New derivatives of progesterone and aldosterone were synthesized and functionally tested with commercially available antibodies. The covalent labelling of antibodies specific for aldosterone and progesterone was detected by SDS/PAGE analysis and subsequent autoradiography after using 3-(O-carboxymethyl)-oximino-(3-[125I]iodo-4-azidosalicylamidobu tylamine) derivatives of aldosterone and progesterone, respectively, as photoactivatable radioligands. Labelling was not observed in the presence of an excess of the unlabelled steroid. Aldosterone was labelled with biotin and used as a tracer in a time-resolved fluorescence immunoassay. The nonradioactive tracer is highly selective for its antibody-binding site, with almost no detectable cross-reactivity for other steroids. Biotin-labelled progesterone was immobilized by avidin-agarose and used for affinity chromatography. This yielded a more than 20-fold enrichment of an anti-progesterone polyclonal antibody. These results demonstrate that derivatives of steroids are particularly useful for the development of nonradioactive assays for the determination of natural steroids and may be also useful for the detection of specific binding sites in biological material such as plasma membranes.