The dissociation constants (Kd) and steroid specificities of oestrogen-binding species in rat granulosa cell cytosol and nuclei have been studied. Preliminary work, where diethylstilboestrol was employed as competitor in binding assays, identified the oestrogen receptor in whole ovarian tissue nuclei (Kd 0.35 +/- 0.09 nmol/l) and cytosol (Kd 0.39 +/- 0.03 nmol/l). Isolation of granulosa cells revealed that the majority of this receptor (75-96%) was present in these cells. Specificity studies on the binding of [3H]oestradiol in granulosa cell cytosol indicated the presence of an additional class of oestrogen-binding sites which were, however, not present in nuclei. Saturation analysis over an extended range of [3H]oestradiol concentrations and using unlabelled oestradiol as competitor revealed a binding species of Kd 45.8 +/- 6.9 nmol/l (capacity 16.7 pmol/mg cytosol protein) for oestradiol in addition to the cytosol oestrogen receptor of Kd 0.58 +/- 0.22 nmol/l (capacity 2.8 pmol/mg cytosol protein). The low affinity of this novel species implies that the dextran-coated charcoal techniques used in previous studies on ovarian oestrogen-binding species would cause dissociation of ligand and not allow it to be measured. The second oestrogen-binding species displayed affinity for oestradiol-17 beta, oestriol, oestrone, testosterone, 5 alpha-dihydrotestosterone, methyltrienolone, progesterone and the antioestrogens tamoxifen, nafoxidine and clomiphene citrate. The species, however, did not bind diethylstilboestrol, a characteristic shared with other low affinity cytosol oestrogen-binding species which have been reported in dog prostate, chick oviduct and male rat liver but not shared with uterine type II oestrogen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Fifty-six cases of Down syndrome were identified in a population of women who had undergone maternal serum triple marker screening [alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated oestriol (uE3) analyses]. These affected pregnancies represented all known cases present in the population of 34,368 women screened. Using a 1:270 mid-trimester Down syndrome risk to define the screen-positive group, 42 affected pregnancies were screen-positive (medians: AFP = 0.79 MOM, hCG = 2.13 MOM, uE3 = 0.62 MOM, age 34.6 years) and 14 pregnancies were screen-negative (medians: AFP = 0.82 MOM, hCG = 1.57 MOM, uE3 = 0.92 MOM, age 24.2 years). Four affected pregnancies were associated with in utero death and each of these cases was associated with relatively extreme values of AFP, hCG, and uE3, including the three highest levels of hCG in the entire series of Down syndrome pregnancies. Twenty-nine (15 screen-positive and 14 screen-negative) affected pregnancies resulted in liveborns. Down syndrome pregnancies had a significantly shorter gestational term than controls, and Down syndrome babies were also lighter than controls, even after adjustment for sex and gestational age. In affected pregnancies, a low uE3 level appeared to be associated with a greater chance of a small-for-gestational age baby. No correlations could be demonstrated between AFP or hCG levels and gestational age-adjusted term weight. Based on this small series, it would appear that uE3 may be particularly useful in detecting those Down syndrome cases associated with small-for-gestational age fetuses. A very high hCG value may indicate a higher probability of fetal death.

A set of 21 early maternal serum samples (19 first-trimester and two at 14 weeks) from pregnancies resulting in a child with Down syndrome was matched for gestation and length of storage with 63 samples from unaffected pregnancies. The concentrations of alpha-fetoprotein (AFP), unconjugated oestriol (uE3), human chorionic gonadotrophin (hCG), pregnancy-specific beta 1-glycoprotein (SP1), and placental alkaline phosphatase (PALP) were measured. The ratios of the medians for Down syndrome pregnancies compared with the medians for controls were AFP 0.71, uE3 0.67, hCG 1.43, SP1 0.79, and PALP 0.92. Although the differences between the medians for affected and unaffected pregnancies were not significant, the trends for AFP, uE3, and hCG confirm earlier findings on first-trimester samples.

A total of 113 women who presented with climacteric symptoms participated in the study. They were randomly allocated to seven groups of 10-27 subjects, who received for 6 mth the following therapies, respectively: conjugated oestrogens (CE) 0.625 mg/day for 21 days + norethisterone (NET) 5 mg/day from day 12 to day 21; CE + cyproterone acetate (CPA) 12.5 mg/day from day 1 to day 10; oestradiol valerate (EV) 2 mg/day for 21 days + NET; EV + CPA; oestriol (E3) 2-4 mg/day; tibolone (ORG OD14) 2.5 mg/day; and placebo, one tablet/day. Hot flushes decreased significantly over the treatment period in all seven groups. However, E3 was less effective at the dose used than CE, EV or ORG OD 14. At the end of the 6 month treatment period histological examination revealed no changes in endometrial morphology in any of the patients treated. Indeed, the addition of a progestogen even induced regression of endometrial hyperplasia in 8 cases. No significant variation in the plasma levels of triglycerides, total cholesterol, high-density lipoprotein (HDL) or low-density lipoprotein (LDL) was observed after the second and sixth months of treatment with E3 or ORG OD 14. After 6 months, treatment with CE/EV + CPA produced a significant increase in HDL, while treatment with CE/EV + NET brought about a reduction in total cholesterol and HDL and an increase in LDL.

The metabolism of [4-(14)C]oestrone and of [6,7-(3)H(2)]oestrone sulphate was studied during cyclic perfusion and once-through perfusion of the isolated rat liver. The following results were obtained. 1. As shown by once-through perfusion, the two steroids are metabolized differently during the first passage through the organ. [4-(14)C]Oestrone was taken up by the liver and partly delivered as oestradiol-17beta and oestriol into the medium. After uptake of [6,7-(3)H(2)]oestrone sulphate, only oestrone, liberated by hydrolysis, was delivered into the medium; no oestradiol-17beta or oestriol could be detected in the medium after one passage through the organ. This indicates that intracellular oestrone, which was taken up as such, and oestrone, which derived from intracellular hydrolysis, may be metabolized in different compartments of the liver cell. 2. The results of the cyclic perfusion showed that intracellular oestrone is preferentially conjugated with glucuronic acid, and subsequently excreted into the bile. Intracellular oestrone sulphate is preferably reduced to oestradiol sulphate, thus indicating that oestrone sulphate is a better substrate for the 17beta-hydroxy steroid oxidoreductase than is oestrone. 3. Albumin-bound oestrone sulphate acts as a large reservoir, and in contrast with free oestrone is protected from enzyme attack by its strong binding to albumin. 4. Oestrone sulphate is partly converted into the hormonally active oestrone by liver tissue. This suggests that liver not only inactivates oestrogens, but also provides the organism with oestrone, which is subsequently readily taken up by other organs.

In order to elucidate the mechanism of disturbances of gonadal hormones secretion in anorexia nervosa 14 female patients were investigated. A control group also consisted of 14 women of the same age. The serum LH, progesterone, oestrogens: oestrone + oestradiol (Oe1 + Oe2), oestriol (Oe3) and testosterone were determined by radioimmunological methods. In patients with anorexia nervosa the serum testosterone and Oe3 concentrations were dramatically elevated, whereas LH, progesterone and Oe1 + Oe2 were decreased as compared with the control group. Considerable weight gain induced by cyproheptadine treatment caused a normalization of the serum testosterone and Oe3 concentrations in all the patients. A negative correlation between the testosterone level and the deficit in body weight was observed. The mechanism causing the dramatically high serum testosterone concentration in the female patients with anorexia nervosa is discussed.

An investigation into certain aspects of the interaction between oestriol and oestrogen receptors (specificity and kinetics) shows the specificity of the receptors to be very similar in various target tissues-human breast tumour and myometrium, rat myometrium, and rabbit myometrium, pituitary, and thymus-and a preferential binding of oestriol to specific oestrogen receptors to be unlikely. Competition for the oestrogen receptors in the uterus of the three species was analysed in vitro under equilibrium and non-equilibrium conditions, and oestriol and epimestrol were found to compete with oestradiol-17 beta more strongly in the latter. In rat uterus a late oestrogenic effect, the synthesis of the progesterone receptor, induced by ethinyloestradiol, is shown to be inhibited by oestriol. Oestriol emerges as a short-action agonist when administered in a single dose. It is also concluded that oestriol may prevent eostradiol-17 beta from inducing a full uterotrophic response.

Pre-eclampsia and eclampsia, along with its complications such as prematurity, intrauterine growth retardation, perinatal asphyxia and abruptio placenta, continues to be one of the major causes of maternal and fetal morbidity and mortality. Due to its morbid course, it is necessary to identify those at risk for the illness and take precautions. However, the lack of knowledge about the aetiology makes it difficult to assess risk factors. We studied the role of the serum androgens in the pathogenesis of pre-eclampsia. Ninety severe pre-eclamptic, 10 mild pre-eclamptic and 50 normotensive pregnant women were studied. The patient and control groups' blood total testosterone, free oestriol (FE3), FAI (free androgene index), sex hormone binding globulin (SHBG) and dehydroepiandrostenedione sulphate (DHEA-S) values were compared. The sex, weight, APGAR scores and hospitalisation in the neonatal intensive care unit of the babies were also evaluated. No statistically significant difference in the total testosterone and free oestriol values was found between the groups. SHBG was found to be higher in the pre-eclamptic group, whereas free testosterone and DHEA-S were higher in the normotensive group. Compared to the severe pre-eclamptic group, no difference was found in the total testosterone levels, whereas free testosterone levels were significantly higher in the mild pre-eclamptic group. We reached the opinion that androgens do not play a clinically significant role in the pathogenesis of pre-eclampsia.

The plasma levels of oestrone (Oe1), 17 beta-oestradiol (Oe2), oestriol (Oe3), testosterone (T), 5 alpha-dehydrotestosterone (DHT) and androstenedione (A) were assayed by RIA in plasma obtained from peripheral venous blood. The hormones are isolated from the plasma extract, first by Sephadex LH-20 column chromatography (Oe2/Oe3/Oe1, T, DHT, A) and after Oe1, T, DHT, A by TLC on silica gel 60 F254. The accuracy, reproducibility and sensitivity of the method make it satisfactory for clinical studies.

Eight bilaterally oophorectomized women were given a depot injection of 200 mg DHEA-enanthate to study the effect on endocrine and lipid metabolism. A decrease in sex-hormone binding globulin (SHBG) and an increase in androstenedione was found 14 and 30 days after the injection. No changes could be detected in LH, FSH, oestrone, oestradiol or oestriol. Testosterone showed a tendency towards an increase. As compared to pre-treatment values, plasma lipids were unaltered after 30 days. A decrease in high density lipoproteins (HDL), cholesterol and in very low density lipoproteins (VLDL), free cholesterol, total cholesterol and phospholipids were seen in the lipid composition of the lipoproteins on day 30. These findings are in agreement with previous data reported after the administration of drugs with androgen-like effects. The relative fatty acid composition of plasma lecithin revealed only minor changes while the fatty acid composition of cholesterol esters indicated a decreased portion of essential fatty acids. These results suggest, in agreement with previous studies, an impaired endogenous cholesterol formation in the liver. The results from the analysis of the fatty acid composition of lecithin and cholesterol esters might indicate a decreased percentage of exogenous (dietary) cholesterol ester in plasma.

1. To investigate the hypothesis that disruption of glucuronidation of endogenous compounds by drugs represents a potential mechanism for pathogenesis of adverse drug reactions, the effects of a range of tertiary amine and amide drugs (many with effects on sex hormone function) on steroid hormone and xenobiotic UDP-glucuronosyltransferase activities in human and rabbit liver microsomes were studied in vitro. 2. Chlorpromazine, amitriptyline, imipramine, promethazine and cyproheptadine were consistently the most potent inhibitors of the glucuronidation of testosterone, androsterone, oestriol and 1-naphthol, the steroid activities being more susceptible to inhibition (up to 90%). 3. Carbamazepine, diphenhydramine, sulphadimethoxine, dimenhydrinate and (+/-)-chlorpheniramine had little effect on the UDPGT activities measured. 4. The structural features within this group of compounds required for inhibitory potency were the presence of a rigid tricyclic ring (e.g. phenothiazine) and either a dimethylaminopropyl or a methylpiperidine side-chain. 5. The implications of these data for involvement of disruption of the normal cellular function of glucuronidation in the pathogenesis of frequently observed adverse side-effects associated with these compounds are discussed.

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder caused by reduced activity of 7-dehydrocholesterol (7DHC) reductase, resulting in a decreased level of cholesterol and increased concentrations of 7DHC and 8DHC in body fluids and tissues. Ten pregnancies at 25% risk of SLOS underwent prenatal testing. Diagnostic studies included DHCR7 mutation analysis in chorionic villus samples, amniotic fluid sterol analysis and serial measurements of oestriol (E3), pregnanetriol (PT), 7-dehydropregnanetriol (7DHPT) and 8-dehydroesteriol (8DHE3) concentrations in maternal urine samples obtained between 9 and 20 weeks of gestation. All tests were diagnostic and revealed nine unaffected foetuses (two normal homozygotes and seven DHCR7 heterozygotes) and one affected foetus. In the affected pregnancy, 7DHC and 8DHC in amniotic fluid were 9.87 and 3.7 microg/ml, respectively [reference range (RR) 0.0026 +/- 0.0015 microg/ml and not detectable, respectively] and maternal urinary steroid analyses showed increased ratios of 7DHPT/PT and 8DHE3/E3 of 0.74 and 1.7, respectively (RR 0-0.0147 and 0-0.019). In the heterozygous foetuses, 7DHPT/PT and 8DHE3/E3 ratios did not exceed those found in 48 normal controls. This is the first series of prenatal diagnostic testing for SLOS where non-invasive biochemical testing was performed in tandem with invasive diagnostic testing. We conclude that steroid measurements in maternal urine are a reliable means of prenatal diagnosis for SLOS.

At menopause, several abnormalities in oestrogen metabolism have been reported, which may increase the likelihood of cancer development in the breast or uterus following oestrone or oestradiol-17 beta supplementation. Occult hypothyroidism reduces the rate of oestrogen inactivation by C2 hydroxylation, and 15-20% of women have low rates of C16 hydroxylation to oestriol. Reduced sex hormone binding globulin concentration occurs in association with obesity, thereby increasing the biologically active unbound fraction of oestradiol in plasma. Since oestriol undergoes minimal metabolism after absorption, does not bind to sex hormone binding globulin, and has an anti-oestradiol action by decreasing the duration of nuclear binding of oestradiol-receptor proteins, it is less likely to induce proliferative changes in target organs of cancer-prone women than oestrone or oestradiol. Intermittent non-conjugated oestriol treatment has demonstrated the most significant anti-mammary carcinogenic activity of 22 tested compounds as well as anti-uterotropic activity in intact female Sprague Dawley rats fed either of two dissimilar carcinogens (7, 12 dimethylbenz(a) anthracene, procarbazine) and followed for their natural life span. The protective effect was specific for mammary carcinomas only and has been decreased in rats with a 20% increase in growth curves. Clinical experience thus far with oral oestriol therapy of post-menopausal women has indicated little hazard of cancer development.

To increase the sample-handling capacity for an induction of ovulation programme, direct urinary radioimmunoassays (RIA) for three steroid glucuronides, pregnanediol-3-glucuronide (Pd-3-G), oestrone-3-glucuronide (E1-3-G), and oestriol-16 alpha-glucuronide (E3-16-G), were established. Results obtained for urinary Pd-3-G measured by direct RIA show an excellent correlation (r = 0.98, N = 46) with those for urinary pregnanediol measured by gas-liquid chromatography. Estrone-3-glucuronide (E1-3-G) and estriol-16 alpha-glucuronide (E3-16-G) values, measured by direct RIA, closely paralleled the total urinary oestrogen measured fluorimetrically. Ovarian response to ovulation induction therapy can be monitored by observing the changes in the levels of E1-3-G in urine. Pre-ovulatory levels of urinary E3-16-G were found to be too low for use in this regard. Direct RIA for E1-3-G and Pd-3-G are recommended as reliable indices of ovarian function in the monitoring of patients receiving treatment for the induction of ovulation.

The Velhas River is the most polluted river in the state of Minas Gerais, south-eastern Brazil. Due to its historical and environmental relevance, the aim of this study was to evaluate the effects of oestrogenic endocrine disruptors on the reproduction of the lambari Astyanax rivularis, a small-sized species found in headwaters of the São Francisco River basin. Quarterly field samplings were carried out during a reproductive cycle in three streams of the upper Velhas River: S1 (reference site) and S2 and S3 (sites contaminated by untreated sewage). The main oestrogenic compounds were evaluated in water using HPLC/MS. Molecular, histological and reproductive biomarkers were assessed in liver and gonad. The results showed higher average concentrations of oestradiol (>200ng/l) in S2 and S3, oestrone (>250ng/l) in S2 as well as oestriol (>200ng/l), bisphenol A (>190ng/l), and nonylphenol (>600ng/l) in S3 compared to S1 (<70ng/l for all compounds). In S2 and S3, there was an increase in the proportion of females, higher ELISA levels of vitellogenin (Vtg) and proteins of the zona radiata (Zrp) in liver males. Insulin-like growth factor (IGF-I) levels were lower in S2 males, which also had a smaller body size, a smaller seminiferous tubule diameter, a higher proportion of spermatogonia, and lower proportion of spermatozoa in relation to S1. Histopathological analyses detected an increase in yolk deficient oocytes and over-ripening in the contaminated sites, and these alterations were associated to a reduction of hepatic Vtg levels and a delay in spawning, respectively. Intersex specimens with perinucleolar follicles in a multifocal distribution in the testis were detected in S2 and S3. These results indicate that chronic exposure to oestrogenic compounds induced endocrine disruption that may affect wild populations of A. rivularis in the Velhas River.

The binding of oestradiol to a nuclear fraction extracted from human breast carcinomatous tissue was demonstrated. The material, which was extracted with KCl, sedimented at 3--4S and bound oestradiol with high affinity (dissociation constant approximately 2 X 10(-10) mol/l). Oestriol, diethylstilboestrol and 5 alpha-dihydrotestosterone (100-fold excesses) competed with [3H]oestradiol for the binding sites (binding inhibited by 89 +/- 8 (S.D.), 92 +/- 6 and 57 +/- 8% respectively), whereas progesterone and cortisol (100-fold excesses) did not (binding suppressed by 5 +/- 5 and 2 +/- 3% respectively). Similar competition patterns were found for cytoplasmic material which bound oestradiol. The binding occurred at 4 degrees C and was therefore considered to be a measure of the amount of binding material unoccupied by endogenous oestrogen, Unoccupied binding sites for oestradiol in the nucleus and cytoplasm were measured in 35 samples of breast carcinomatous tissue using sucrose gradient centrifugation. In 17 out of 35 tumorus, unoccupied nuclear and cytoplasmic 8S and 4S binding sites could be detected. Three out of 35 tumours contained unoccupied nuclear binding sites and 4S cytoplasmic binding sites. Nuclear binding sites only were found in two out of 35 tumours. Unoccupied nuclear binding sites were not detected in 13 out of 35 tumours and ten of these tumours also did not contain unoccupied cytoplasmic binding sites.

In 100 normal singleton pregnancies and in 44 patients with pregnancy hypertension (pre-eclampsia) linear regression analyses demonstrated highly significant positive correlations between birth weights and late third trimester maternal serum unconjugated oestriol (O3) levels. Correlation coefficients increased, though not significantly, after standardising birth weights according to maternal size, parity and sex of infant. Pregnancy hypertension was classified according to the duration of clinical signs. In 23 patients with short duration pregnancy hypertension (onset 14 days or less before delivery) neither maternal age nor birth weight differed from normal controls. Mid-pregnancy weights were greater and unconjugated O3 levels lower and only one infant was growth retarded. Twenty one patients with long duration pregnancy hypertension (onset more than 14 days before delivery) were found on average to be heavier at mid-pregnancy and older than those in whom the condition was short-lived. Unconjugated O3 levels in patients with long duration hypertension were significantly below those in the short duration group. One third of infants born following long duration hypertension were growth retarded.

An association between various abnormal mid-trimester maternal serum analyte values and adverse perinatal outcome has been reported. From an original sample of 14,857 women, we observed five women who were 'screen-positive' for both neural tube defects [maternal serum alpha-fetoprotein (MSAFP)>> or = 2.5 multiples of the median] and Down syndrome [risk>> or = 1/274 using MSAFP, maternal serum unconjugated oestriol (MSuE3), maternal serum human chorionic gonadotropin (MShCG), and maternal age]. The four patients who elected to undergo amniocentesis all demonstrated both normal karyotype and normal amniotic fluid AFP levels. All five cases were associated with intrauterine growth retardation (IUGR) and abnormal pregnancy outcomes. Two cases exhibiting severe IUGR on ultrasound examination were terminated at 19.1 and 21.2 weeks, respectively; the former also exhibited fetal calcifications and positive maternal serology for toxoplasmosis. In another case, fetal demise occurred at 36 weeks' gestation in a patient who had been treated for syphilis in the second trimester. Neither infection was confirmed in fetal tissue studies. Though resulting in live births, the remaining two cases required operative deliveries; emergency Caesarean sections for fetal distress were performed at 38 and 32 weeks, respectively, the latter case being associated with severe pre-eclampsia. We conclude that elevated mid-trimester MSAFP levels concurrent with maternal serum analyte values associated with increased risk for fetal Down syndrome may presage a poor perinatal outcome, particularly IUGR and possibly congenital infection.

We previously reported that postmenopausal obese women exhibit increased levels of circulating adipocyte fatty acid binding protein (A-FABP), which is associated with breast cancer (BC) development. In postmenopause, increased oestrogen levels are reported to be associated with increased BC risk. Herein, we assessed if oestrogens, including oestrone (E1), oestradiol (E2) and oestriol (E3), are associated with A-FABP in the obesity-related BC development. We collected 249 serum samples from women with or without BC and measured serum levels of E1, E2, E3 and A-FABP. Considering all subjects, E1 and E2 but not E3 levels were significantly higher in pre- than in postmenopause individuals. E3 and E1 levels were higher in non-obese than in obese women. When samples were separated by BC status, E2 levels were significantly higher, while E1 and E3 levels were significantly lower in postmenopausal obese than non-obese women without BC. These differences based on body mass index (BMI) were not observed among women with BC. E3 levels were higher in obese women with BC than those without. A-FABP levels were significantly higher in postmenopausal obese women regardless of BC status. In addition, A-FABP was not associated with E1, E2 or E3. Altogether, our data suggest that A-FABP is independently regulated by obesity and menopausal status compared to oestrogens, thus playing a unique role in the development of BC.

In the present work, the dose-response relationship of highly purified porcine relaxin has been examined on broadening of the pubic ligament in mice. Using the method of Steinetz et al. with 7 days of oestriol priming, higher sensitivity of the pubic ligament was attained in mice with an original weight of 10 g than in those with a weight of 20 g. S-shaped curves were obtained; by increasing the relaxin doses after maximal effect had been reached a decreased action was observed. With presomen priming, a relaxin effect was established after only 2 days instead of the usual 8 days but the action was markedly less than in the Steinetz test.

In a prospective, double-blind, randomized, cross-over trial, the effects of oral oestradiol, 2 mg daily, on the endometrial histology, frequency and severity of vaginal bleeding, and the symptomatic and psychological status of postmenopausal women were compared with those of oral oestradiol, 2 mg daily, plus oestriol, 1 mg daily. Both therapies were prescribed for 3 months on a cyclical basis. The addition of oestriol to oestradiol did not modify the endometrial response. The prevalence of proliferative/hyperplastic endometrium (64%: 9 of 14 biopsies) was similar after both treatments and there were no significant differences in either the frequency or heaviness of vaginal bleeding. Both therapies significantly reduced hot flushes, night sweats and vaginal dryness: no significant differences in effect on the symptomatic and psychological status were recorded. The addition of 1 mg of oestriol to 2 mg of oestradiol did not confer any benefit and the value of such an addition is challenged.