The effects of sex, menstrual cycle, and oral contraceptives on the number and activity of natural killer cells.

OBJECTIVES: The aim of this study was to study the impact of sex, the menstrual cycle, and the use of oral contraceptives (OC) on the number and activity of natural killer (NK) cells. METHODS: Both the number and the activity of NK cells were assessed per milliliter of blood, and NK activity (NKA) per NK cell and per lymphocyte was calculated. NKA was measured in each subject using a whole blood assay, which preserves the plasma and all blood cells, and using a washed blood assay, in which plasma is replaced with an artificial medium. The subjects were young (20-29 years old) women with a regular menstrual cycle (n = 39; 26 tested on both the follicular and the luteal phases), age-matched women who use OC (n = 26), and age-matched men (n = 20). RESULTS: Men showed markedly and significantly higher NKA than women with regular menstrual cycles or women using OC, who had the lowest levels of NKA. No significant differences in blood concentration of NK cell were found. Differences in NKA were of similar magnitude in the whole and washed blood assays per milliliter of blood, per NK cell, or per lymphocyte. The menstrual cycle had no significant effect on activity levels of NK cells, but during the periovulatory phase, the number of NK cells per milliliter of blood increased significantly. CONCLUSIONS: The observed differences are independent of the presence of serum factors during the in vitro assessment of NKA, but may be related to chronic exposure to sex steroids and to fluctuation in the NK cell expression of beta-adrenoceptors.

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.

Oral contraceptives, sex steroid-induced antibodies and vascular thrombosis: results from 1318 cases.

The role of antiethinyl estradiol antibodies (anti EE Ab) and associated risk factors was evaluated in 1318 cases of venous or arterial thrombosis in oral contraceptives (OC) users, and compared to 61 non-users and 124 healthy current users. Anti EE Ab were absent in non-users and present in 33% of healthy users and 72% of those with thrombosis, either arterial or venous. Age, duration of use, hyperlipidaemia and smoking were factors associated with thrombosis only in women with an arterial disease. While the two predominant factors, anti EE Ab and smoking may be risk factors in their own right, the combination of both was found in 47.7% of women with thrombosis. It is proposed that thrombosis associated with OC use may be explained by an immunological disease in which anti EE Ab and their complexes with the circulating synthetic hormones may be harmful to the vessels, as also suggested by the type of lesions already described in OC users. The determination of anti EE Ab in healthy users may identify a group at risk of thrombosis.

PIP: The significance of antibodies against ethinyl estradiol (anti-EE-Ab) and other risk factors was discussed for a series of 1318 cases of venous and arterial thrombosis in oral contraceptive users, in comparison to 61 non-users and 124 health current pill users. The cases included 264 deep vein thromboses, 159 pulmonary embolism, 37 coronary artery, 33 systemic artery, 763 cerebrovascular artery thromboses, and 10 hepatic vein thromboses collected from 88 French hospitals from 1976-1988. There were 98 cases with successive or multiple sites involved. The mean age of contraceptive users with thrombosis was 32.1, compared to 28.8 in healthy users. Duration of use was slightly longer in affected users than healthy users, but some cases were affected as early as their 1st cycle. 87.2% had no related history. The anti-EE-Ab were absent in never users, averaged 318 c./min in pill users with thrombosis, but 60 in healthy pill users. There was no correlation between anti-EE-Ab level and dose or duration of pill use. Similar anti-EE-Ab levels were found in those with venous or arterial thrombosis, but women with arterial thrombosis were older, had used pills longer, had fewer predisposing factors of surgery or labor and delivery, but more frequent incidence of hyperlipidemia, smoking, and hypertension. The most frequent associated factors with thrombosis were presence of anti-ee-Ab and smoking: 15.6% smoked, 31.1% had anti-EE-Ab, and 47.6% had both, but only 9.5% had neither factor. It is interesting that lowering the estrogen dose of oral contraceptives has decreased the frequency of venous thrombosis, but not that of arterial thrombosis or mortality, nor anti-EE-Ab levels. The vascular lesions in arterial thrombosis seen in pill users are thought to resemble those in many autoimmune diseases.

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.

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.

[Monoclonal human immunoglobulin (IgG lambda) with antiethinylestradiol activity, oral contraceptives, and arterial pulmonary thrombosis]. / Immunoglobuline humaine monoclonale (IgG lambda) anti-éthinylaestradiol, contraceptif oral et thrombose artérielle pulmonaire

In a 36-year-old woman taking an oral contraceptive containing 50 mug of ethinyloestradiol each day, a pulmonary arterial thrombosis and a monoclonal gammapathia were associated. The monoclonal IgI lambda Mai... was prepared. When purified, this IgG lambda binds ethinyloestradiol with strong affinity (Ka= 2.7 times 10(7)M-1) and also 17-beta-oestradiol with a little less affinity (Ka = 0.4 times 10(7)M-1. For those ligands each IgG lambda Mai... molecule has two sites of same affinity and specificity so that a Scatchard plot of the experimental values gives a straight line. It is likely that the antibody sites of the IgG lambda Mai... are the binding sites. These facts support the hypothesis of an immunological mechanism of the thromboembolic disease which may be induced by oral contraceptives.

PIP: An IGG lambda was purified and its binding properties analyzed from the serum of a woman who had suffered a pulmonary embolism after taking an oral contraceptive (50 mcg ethinyl estradiol and 500 mcg norethisterone) for over 2 years. The purification steps were 1) precipitation with 25% ammonium sulfate; 2) gel filtration on DEAE Sepha dex A25-Sephadex G-25 at pH 10.5 with 6 M urea; 3) repeat gel filtration, but at pH 8.6 without urea; 4) chromatography on Sepharose 4B CNBr coupled with ethinyl estradiol. The activities of the fractions were analyzed by double diffusion immunoelectrophoresis. Scatchard plots by both dialysis and by ultracentrifugation generated an association constant of 2.7 X 10 7 M -1 for ethinyl estradiol, .4 X 10 7 M -1 for 17beta-estradiol, and a valence of 2. Normal human sera had such low affinities for ethinyl estradiol that the Ka could not be calculated. Immunoelectrophoresis showed only a single protein, of about 150,000 molecular weight in polyacrylamide gel. Equilibrium dialysis against other steroids demonstrated that the IgC was specific for ethinyl estradiol, but binding was inhibited to a lesser extent by the following, in order of potency: 17beta-estradiol, progesterone, estr adiol, testosterone, and estrone. The Ka was midway between that of albuinn and the highly specific steroid binding protein. The relationships between oral contraception, this apparent monoclonal gammapathy, and the pulmonary embolism are discussed.