Human Connexin 32, a gap junction protein altered in the X-linked form of Charcot-Marie-Tooth disease, is directly regulated by the transcription factor SOX10.

Mutations in SOX10, a transcription modulator crucial in the development of the enteric nervous system (ENS), melanocytes and glial cells, are found in Shah-Waardenburg syndrome (WS4), a neurocristopathy that associates intestinal aganglionosis, pigmentation defects and sensorineural deafness. Expression of MITF and RET, two genes that play important roles during melanocyte and ENS development, respectively, are controlled by SOX10. The observation that some WS4 patients present with myelination defects of the central and peripheral nervous systems correlates with the recent finding that P(0), a major component of the peripheral myelin, is another transcriptional target of SOX10. These phenotypic features suggest that SOX10 could regulate expression of other genes involved in the myelination process as well. Thus, we tested the ability of SOX10 to regulate expression of MBP, PMP22 and Connexin 32, three major proteins of the peripheral myelin. Our study shows that this factor, in synergy with EGR2, strongly activates Cx32 expression in vitro by directly binding to its promoter. In agreement with this finding, SOX10 and EGR2 mutants identified in patients with peripheral myelin defects fail to transactivate the Cx32 promoter. Moreover, we show that a mutation of the Cx32 promoter previously described in a patient with the X-linked form of Charcot-Marie-Tooth (CMTX) disease impairs SOX10 function. In addition to providing new insights into the molecular mechanisms underlying some of the peripheral myelin defects observed in CMTX disease, these results further extend the spectrum of genes that are regulated by SOX10.

The SOX10 transcription factor: evaluation as a candidate gene for central and peripheral hereditary myelin disorders.

The SOX10 transcription factor is involved in development of neural crest derivatives and fate determination in glial cells. SOX10 mutations have been found in patients with intestinal aganglionosis and depigmentation with deafness (Waardenburg-Hirschsprung). Associated neurological signs have been reported in some cases, including a patient exhibiting a central and peripheral myelin deficiency. Therefore, we screened for SOX10 mutations in a large cohort of patients with peripheral and central myelin disorders. 56 were affected by classical demyelinating Charcot-Marie-Tooth disease without identified mutations in the genes encoding PNS myelin proteins (PMP22, P0), connexin 32 and the zinc-finger transcription factor, EGR2. 88 patients with undetermined leukodystrophy were selected from a large European prospective study. Associated clinical, magnetic resonance imaging and electrophysiological signs were consistent with a defect in CNS myelination in 83 and with an active degeneration of the CNS myelin in 5. No abnormalities in the proteolipid protein gene (PLP) were found. The absence of SOX100 mutation in this large cohort of patients suggests that this gene is not frequently involved in peripheral or central inherited myelin disorders.

Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome.

Waardenburg syndrome (WS) is an autosomal dominant disorder with an incidence of 1 in 40 000 that manifests with sensorineural deafness and pigmentation defects. It is classified into four types depending on the presence or absence of additional symptoms. WS1 and WS3 are due to mutations in the PAX3 gene whereas some WS2 cases are associated with mutations in the microphthalmia-associated transcription factor (MITF) gene. The WS4 phenotype can result from mutations in the endothelin-B receptor gene (EDNRB), in the gene for its ligand, endothelin-3 (EDN3), or in the SOX10 gene. PAX3 has been shown to regulate MITF gene expression. The recent implication of SOX10 in WS4 prompted us to test whether this transcription factor, known to cooperate in vitro with PAX3, is also able to regulate expression from the MITF promoter. Here we show that SOX10, in synergy with PAX3, strongly activates MITF expression in transfection assays. Analyses revealed that PAX3 and SOX10 interact directly by binding to a proximal region of the MITF promoter containing binding sites for both factors. Moreover, SOX10 or PAX3 mutant proteins fail to transactivate this promoter, providing further evidence that the two genes act in concert to directly regulate expression of MITF. In situ hybridization experiments carried out in the dominant megacolon (DOM:) mouse, confirmed that SOX10 dysfunction impairs MITF: expression as well as melanocytic development and survival. These experiments, which demonstrate an interaction between three of the genes that are altered in WS, could explain the auditory-pigmentary symptoms of this disease.

Plasma lipoprotein distribution and lipid transfer activities in patients with type IIb hyperlipidemia treated with simvastatin.

The aim of the present study was to search in type IIb hyperlipidemic patients for putative concomitant effects of simvastatin on the physicochemical characteristics of low density lipoproteins (LDL) and high density lipoproteins (HDL), as well as on the activities of the cholesteryl ester transfer protein (CETP) and the phospholipid transfer protein (PLTP) that were determined in both endogenous lipoprotein-dependent and endogenous lipoprotein-independent assays. In a double-blind, randomized trial, patients received either placebo (one tablet/day; n = 12) or simvastatin (20 mg/day; n = 12) for a period of 8 weeks after a 5-week run-in period. Simvastatin, unlike placebo, reduced the lipid and apolipoprotein B contents of the most abundant LDL-1, LDL-2, and LDL-3 subfractions without inducing significant changes in the overall size distribution of LDL and HDL. Whereas simvastatin significantly increased PLTP activity in an endogenous lipoprotein-dependent assay (P < 0.01), no variation was observed in a lipoprotein-independent assay. Simvastatin significantly decreased plasma CETP activity in an endogenous lipoprotein-dependent assay (P < 0.01), and the reduction in plasma cholesteryl ester transfer rates was explained by a 16% drop in CETP mass concentration (P < 0.01). In contrast, the specific activity of CETP was unaffected by the simvastatin treatment reflecting at least in part the lack of significant alteration in plasma triglyceride-rich lipoprotein acceptors. The simvastatin-induced changes in plasma CETP mass levels correlated positively with changes in plasma CETP activity (r = 0.483, P = 0.0561), in total cholesterol levels (r = 0.769; P < 0.01), and in LDL-cholesterol levels (r = 0.736; P < 0.01). Whereas the observations suggest that simvastatin might exert concomitant beneficial effects on plasma CETP and LDL levels, neither plasma cholesteryl ester transfer activity nor plasma phospholipid transfer activity appeared as the main determinants of the LDL and HDL distribution profiles in type IIb hyperlipidemic patients.

Comparison between extra virgin olive oil and oleic acid rich sunflower oil: effects on postprandial lipemia and LDL susceptibility to oxidation.

The aim of our study was to determine whether the minor polar components of virgin olive oil could have favorable effects (1) on fasting and postprandial lipid profile and (2) on low-density lipoprotein (LDL) composition and susceptibility to oxidation in vitro. Ten normolipidic subjects were included in a crossover study (two diet periods of 3 weeks) and received either virgin olive oil (OO diet) or oleic acid rich sunflower oil. An oral fat load was performed at the end of each period. The plasma lipid levels were not significantly different after both diets in the fasting and postprandial states. A few minor variations of the LDL composition were observed only in the postprandial lipemia, and they were different after both diets. The LDL oxidation susceptibility was evaluated by the formation of conjugated dienes. With LDL isolated in the fasting state, the diene production decreased (p = 0.0573) only after the OO diet. The dienes determined at time 0 and the maximal dienes obtained during the oxidation reaction decreased (p = 0.0145 and p = 0.0184, respectively) only after the OO fat load. Nevertheless, the diene production decrease was not significant (p = 0.0848). Our results suggest a mild effect of minor components of virgin olive oil related to a decrease of LDL susceptibility to oxidation; further analyses are necessary to give clear conclusions about their role.

Expression of the SOX10 gene during human development.

SOX10, a new member of the SOX gene family, is a transcription factor defective in the Dom (Dominant megacolon) mouse and in the human Shah-Waardenburg syndrome. To help unravel its physiological role during human development, we studied SOX10 gene expression in embryonic, fetal, and adult human tissues by Northern blot and in situ hybridization. As in mice, the human SOX10 gene was essentially expressed in the neural crest derivatives that contribute to the formation of the peripheral nervous system, and in the adult central nervous system. Nevertheless, it was more widely expressed in humans than in rodents. The spatial and temporal pattern of SOX10 expression supports an important function in neural crest development.

Mutation of the Sry-related Sox10 gene in Dominant megacolon, a mouse model for human Hirschsprung disease.

The spontaneous mouse mutant Dominant megacolon (Dom) is a valuable model for the study of human congenital megacolon (Hirschsprung disease). Here we report that the defect in the Dom mouse is caused by mutation of the gene encoding the Sry-related transcription factor Sox10. This assignment is based on (i) colocalization of the Sox10 gene with the Dom mutation on chromosome 15; (ii) altered Sox10 expression in the gut and in neural-crest derived structures of cranial ganglia of Dom mice; (iii) presence of a frameshift in the Sox10 coding region, and (iv) functional inactivation of the resulting truncated protein. These results identify the transcriptional regulator Sox10 as an essential factor in mouse neural crest development and as a further candidate gene for human Hirschsprung disease, especially in cases where it is associated with features of Waardenburg syndrome.

Lp(a) levels and antiestrogen antibodies in women with and without thrombosis in the course of oral contraception.

Several reports have shown that lipoprotein(a) is associated with ischemic diseases. Two characteristics might explain this association. Firstly, Lp(a) is an LDL-like lipoprotein which may be implicated in the atherosclerotic process and secondly, Lp(a) possesses an additional apolipoprotein(a) whose structure is close to that of plasminogen and might confer to the molecule prothrombotic properties. It seemed of interest to see whether Lp(a) was a risk factor in oral contraceptive users with thrombotic complications, a group of young women with presumably little or no atherosclerosis. Three groups of women were compared: 25 of them served as controls and did not use oral contraceptives (OC) (group 1); 25 women were healthy current users of OC (group 2); 35 women suffered thrombotic complications in the course of OC (group 3). Mean levels of Lp(a), estimated by RID, were not found to be significantly different in the 3 groups: 19 +/- 18, 20 +/- 23 and 16 +/- 22 mg/dl, respectively. Levels above 30 mg/dl were similarly distributed. Among the other risk factors studied, antiestrogen antibodies were absent in group 1, present in 24% of group 2 and 71.4% of group 3 (P < 0.01). Serum cholesterol levels were similar in the 3 groups: 209 +/- 33, 220 +/- 41, 213 +/- 45 mg/dl respectively. Mean serum triglyceride levels were higher in group 2 than in group 1 (61 +/- 18 and 83 +/- 32, P < 0.01), and higher in group 3 than in group 2 (116 +/- 66 and 83 +/- 32, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperhomocyst(e)inemia, anti-estrogen antibodies and other risk factors for thrombosis in women on oral contraceptives.

Hyperhomocyst(e)inemia was shown to be associated with vascular occlusion in atherosclerotic patients. We have conducted a study to determine if hyperhomocyst(e)inemia was also related to the vascular events observed in women on oral contraceptives, presumably having little or no atherosclerosis. Two hundred women receiving oral contraceptives were included in the study: 100 were healthy controls and 100 had documented vascular occlusion. Determination of serum homocyst(e)ine and anti-estrogen antibody levels wore performed under blind conditions. They were evaluated in logistic regression models in which age and smoking were also included. Women with vascular occlusion had higher levels of homocyst(e)ine (P less than 0.001) and of anti-estrogen antibodies (P less than 0.001) when compared to controls. They were also older (P less than 0.001) and more frequently smokers (P less than 0.05). The above mentioned variables were, in isolation, independent predictors of vascular occlusion. Moreover, a model assessing those variables and their interactions indicated that the levels of anti-estrogen antibodies and smoking increased the predictability in older women, as well as the levels of age-adjusted homocyst(e)ine. The study suggests that the above factors can identify women at risk and that determination of anti-estrogen antibodies and homocyst(e)ine levels may help to detect women predisposed to vascular occlusions when taking oral contraceptives.

PIP: This study was conducted to test the hypothesis that hyperhomocyst(e)inemia may be an additional risk factor for vascular occlusions in women taking oral contraceptives (OCs). A total of 200 women who were regular users of OC tablets containing 30 or 50 mcg ethinyl estradiol were studied: 100 were controls and 100 had documented vascular occlusion. Serum levels of homocyst(e)ine and anti-estrogen antibody were determined under blind conditions. These were evaluated in logistic regression models in which age and smoking were also included. Women with vascular occlusion had higher levels of homocyst(e)ine and anti-estrogen antibodies when compared to controls. They were also older and frequent smokers. The variables investigated--namely, anti-estrogen antibody, age-adjusted log, transformed homocyst(e)ine, age, and smoking--were independent predictors of vascular occlusions when considered in isolation. Moreover, a model assessing these variables and their interactions, indicated that the levels of anti-estrogen antibodies and smoking increased the predictability in older women, as well as the levels of age-adjusted log and homocyst(e)ine. The study suggests that these variables can identify women at risk, and the determination of anti-estrogen antibody and homocyst(e)ine levels may help to detect women predisposed to vascular occlusions when taking OCs.

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.

Residual vascular risk of discontinued oral contraception. Role of antibodies to synthetic sex hormones.

Recent epidemiological data indicate that the risk of thromboembolic disease associated with oral contraception (OC) may persist after discontinuation of the drug. It was demonstrated on the other hand that antibodies to sex steroid hormones which develop in OC users, were significantly correlated with the incidence of thrombosis. It is well known that antibodies may persist years after the antigenic stimulation. So it was of interest to see if the eventual occurrence of thrombosis in ex-users might be correlated with the presence of anti-sex steroid antibodies remaining after stopping OC. Thirty-eight women with thrombosis on OC and positive antibody levels, who were required to stop the pill, were followed for periods ranging from 1 to 10 years. No disappearance of anti-ethinyl-estradiol antibodies (anti-EE ab) was observed except for 2 cases. On the other hand, 109 patients with thrombosis either current- (50), past- (29), or never-users (30) of OC were compared to 102 controls of similar groups. Results indicate that the levels of anti-EE ab, and the percentage of women who had anti-EE ab, were similar in those who experienced thrombosis either in the course of OC or after discontinuation. A significant difference was observed between both cases who were current- or ex-users and their controls.

[Vascular complications of oral contraception. In whom and how to prevent them?]. / Complications vasculaires de la contraception orale. Chez qui et comment les prévenir?

Oral contraception entails an increased risk of arterial and venous thrombosis which can only be prevented by detecting women at risk. The relative importance of various predisposing or precipitating factors was evaluated by comparing 3 groups of women: 50 oral contraceptive (OC) users with thrombosis; 50 healthy OC users and 30 controls who had never used OC's. The factors investigated were: duration of use and dose of oestrogens, age, blood pressure, serum lipid levels and tobacco smoking. In addition, all women were tested for the presence of anti-ethinylestradiol antibodies (anti-EE ab) which we had previously shown to be induced by OC's in a number of women. Our results indicated that the most frequently encountered risk factor associated with vascular thrombosis was the presence of anti-EE ab and that the risk was further increased by smoking in women with these antibodies.

Evaluation of risk factors associated with vascular thrombosis in women on oral contraceptives. Possible role of anti-sex steroid hormone antibodies.

Women on oral contraceptives have an increased risk of thrombosis. The prevention of the vascular complications relies on the detection of women who are at risk. In order to find out which characteristics correlate with the occurrence of the vascular disease, 3 groups of women were compared; 50 oral contraceptive users with thrombosis, 50 healthy users, and 30 controls. Apart from the modality of oral contraception (duration of use, dose of estrogens), the following parameters were tested as possible risk factors: age, serum lipid levels, tobacco smoking, and especially presence of antiethinylestradiol antibodies (anti-EE ab) which we had previously shown to be induced by oral contraceptives in a number of women. Results indicated that the major risk factor associated with vascular thrombosis was the presence of anti-EE ab. Furthermore, the risk was highly increased by the association of tobacco smoking to anti-EE ab.

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.

Oral contraception, circulating immune complexes, antiethinylestradiol antibodies, and thrombosis.

Circulating immune complexes were detected in women on oral contraceptives (OC) by a simple antigen nonspecific method using precipitation of serum in 25% saturated ammonium sulfate (CIC-AS). A significant correlation was found between the presence of CIC-AS and the OC vascular risk. A radioimmunoassay with tritiated ethinylestradiol indicated that CIC-AS contained antiethinylestradiol antibodies (anti-EE Ab) in a number of OC users, but indicated also that 1) anti-EE Ab may be found in cases with no detectable CIC-AS, 2) CIC-AS containing no anti-EE Ab are found in nonusers, and 3) even in OC users the CIC-AS may contain antibodies to other ligands than EE. The study demonstrated also that, in OC users with a vascular complication, anti-EE Ab were more frequently detected (78% of cases) than CIC-AS (60%). Moreover, among OC users with CIC-AS, anti-EE Ab were found in 95% of women with a vascular complication and only 37% of current healthy users. The detection of anti-EE Ab appears to be more predictive, with regard to the vascular risk of OC, than the detection of CIC-AS.

[Automatic assay of circulating immune complexes induced by oral contraceptives (author's transl)]. / Dépistage automatique des complexes immuns circulants induits par les hormones contraceptives.

PIP: The authors describe an automatic method to assay circulating immune complexes induced by OC (oral contraception). The method is simple, quick, and cheap; it consists of detecting the immune complexes precipitating by ammonium sulphate at 25% saturation, as in the manual method. In the automatic method the procedure is done on an Auto Analyzer apparatus, which allows direct reading of the floculation, and does not require washing of the precipitate. Results obtained for 164 sera tested automatically were compared with results obtained by the manual method; the correlation was very significant. The same method can be used for screening other immune complexes.^ieng

Vascular thrombosis and oral contraceptives - a risk correlated study.

An immunological mechanism of the vascular complications related to oral contraceptives (O.C.) was suggested by the demonstration of circulating anti-ethinylestradiol antibodies in several cases. According to the characteristics of the specific immunoglobulins (Ig), a test based upon Ig precipitability in 25% saturated ammonium sulfate was developed. This test was applied to several groups of women on O.C. with and without thrombosis, and to a control group. Results indicated: (1) that the test was positive in nearly 100% of women with a vascular thrombosis on O.C.; and (2) that the test could detect among healthy contraceptive users a group of women who may be considered as at risk. The role of adjuvant factors is considered.

[Detection of pill-induced antiethinylestradiol antibodies (author's transl)]. / Détection des anticorps anti-éthinyl-oestradiol induits par les contracetifs oraux.

The synthetic hormones contained in contraceptive pills were shown to induce antiethinylestradiol (EE) antibodies in some women. These antibodies can be detected by the presence of circulating immune complexes (CIC) which are precipitated from serum in 25 p. cent saturated ammonium sulphate. A method of analysis of the anti-EE antibodies is described, in which binding of tritiated EE is measured with and without addition of unlabeled EE in excess. This method allows to identify specific reversible binding on saturable sites. Because of the large excess of non specific sites, the whole serum was enriched in antibodies before the binding measurement, either by previous separation of CIC, or by an affinity chromatography on a EE column. Results of this method confirmed the presence in a number of women on oral contraceptives, of immunoglobulins which were able to bind EE reversibly. The antibodies were found in CIC. in the absence of CIC, they were occasionally found in serum after an affinity chromatography on a EE column. This method is felt to allow a more accurate detection of women immunoreactive to the pill.

PIP: OC (oral contraception) use provokes in some women the production of EE (ethinyl estradiol) antibodies which circulate in the blood as immune complexes. These CIC (circulating immune complexes) precipitate when the serum is brought to 25% saturation in ammonium sulphate. The localization of CICs in OC users has allowed the detection of reactive women, and has shown that vascular thrombosis happens almost exclusively in reactive users. The method of detection described above is the one commonly used, being simple and practical; however, it can be at times erroneous. The authors of this article describe a new method of analysis of EE antibodies which allows to detect the presence of proteins able to fix EE in a reversible way in OC users. In this method binding of tritiated EE is measured with and without addition of unlabeled excess EE. Because of the large excess of nonspecific sites, the whole serum was enriched in antibodies before the binding measurements, either by previous separation of CIC, or by an affinity chromatography on an EE column. The method confirms the presence of immunoglobulins which were able to bind EE reversibly in a number of OC users. The antibodies were found in CICs; in the absence of CICs they are sometimes found in the serum after affinity chromatography on an EE column. This method allows a more accurate detection of women immunoreactive to the pill.