The effects of germ cells prepared from adult rats and of media conditioned by some of these germ cells have been studied in vitro on both ABP and oestradiol-17 beta secretion by immature rat Sertoli cells. Addition of the germ cells to the Sertoli cell cultures resulted in both a dose-dependent increase of ABP secretion and a dose-dependent inhibition of oestradiol production. These effects were suppressed after removal of germ cells by hypotonic treatment. Furthermore, spent media of highly viable germ cells (SMGC), but not spent media of an epithelial cell line, mimicked the effects of germ cells themselves on ABP and oestradiol levels after FSH or dbcAMP stimulation. These effects were reversible when SMGC were replaced by fresh media and did not result from a change in the conversion of oestradiol to oestrone. SMGC effects were unaltered by heating at 60 degrees C for 30 min, by freezing and thawing and non dialysable (MW greater than 10,000). However, heating at 100 degrees C for 3 min and treatment by trypsin, suppressed the SMGC effects. This indicates that the stimulation of ABP and inhibition of oestradiol levels by germ cells, in vitro, could be mediated by factor(s) of proteinaceous nature.

Plasma levels of oestradiol (Oe2), oestrone (Oe1) oestrone sulphate (Oe1S), androstenedione, testosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulphate (DHEAS), sex hormone-binding globulin (SHBG) and the gonadotrophins (FSH and LH) were determined in 20 postmenopausal women with breast cancer treated with the anti-oestrogen droloxifene (3-hydroxytamoxifen). Plasma oestrogens were measured before and after 3, 6 and 12 months of therapy. The other hormones were measured before and after 6 months of therapy. Droloxifene treatment had no significant influence on plasma levels of Oe2. Plasma levels of Oe1 and Oe1S increased during treatment (mean increase of 11.9-15.9% and 24.5-69.4% respectively after different time-intervals on treatment). The Oe1S/Oe1 and Oe1S/Oe2 ratios increased by mean values of 13.8-45.2% and 25.9-52.4% respectively. Plasma SHBG increased significantly by a mean value of 73.9%, while FSH and LH fell non-significantly by 19.7% and 20.4% respectively. Plasma levels of testosterone, androstenedione, DHEA and DHEAS all increased during treatment, but none of these alterations were of statistical significance. While the influence of droloxifene on plasma SHBG resembled that which is seen during treatment with tamoxifen, its influence on plasma oestrogens and the gonadotrophins seems to be different. Possible explanations of such differences and the clinical implications of alterations in plasma hormones during treatment with droloxifene are discussed.

Endogenous oestrogens play a crucial role in endometrial cancer pathogenesis, with most endometrial cancer risk factors causing an increase in oestrogens. Adipose tissue, where androgens are converted to oestrogens by the enzyme aromatase, is an important source of endogenous oestrogen production in the postmenopausal woman. The peroxisome proliferator-activated receptor-gamma (PPARgamma), a key transcriptional regulator of adipogenesis, may also play a role in the regulation of aromatase expression in adipose tissue. We hypothesized that the functional PPARgamma ProAla polymorphism may alter aromatase expression, ultimately affecting endometrial cancer susceptibility. We genotyped the PPARgamma ProAla polymorphism in a study of invasive endometrial cancer cases (n = 222) and matched controls (n = 666) nested within the Nurses' Health Study Cohort. We found little or no evidence of an association between the Ala allele of the PPARgamma codon 12 polymorphism and endometrial cancer risk (adjusted odds ratio = 1.18, 95% confidence interval = 0.80-1.76). Furthermore, we found no association with the PPARgamma ProAla polymorphism and the ratio of oestrone to androstenedione or oestradiol to testosterone plasma hormone levels, measures of aromatase activity. Consistent with previous findings for breast cancer, these results suggest that the PPARgamma ProAla polymorphism does not play a major role in mediating circulating oestrogen levels or endometrial cancer susceptibility.

The possibility that medroxyprogesterone acetate (MPA) is clinically effective at least in part by its suppression of adrenal steroidogenesis and a resultant reduction of circulating oestrogen levels was investigated in 49 postmenopausal patients with advanced breast cancer. Thirty-one patients were treated with low dose MPA (100 mg three times daily) and 16 patients with high dose MPA (250 mg four times daily). Plasma levels of androstenedione, testosterone, oestrone and oestradiol were all significantly reduced during treatment, with the suppression being most marked for the 17 beta hydroxysteroids, testosterone and oestradiol. The fall in oestradiol levels was to about 50% of pretreatment levels, but a concomitant fall in SHBG levels to less than 25% of baseline probably resulted in the fall in free, biologically active oestradiol being only to about 70-80% of pretreatment. It is unlikely that this is a major determinant of the activity of MPA in breast cancer.

A pilot study of the endogenous steroid concentrations in human breast tumours was performed. The technique of high-resolution molecular-ion monitoring during combined g.l.c.-mass spectrometry was used to determine oestrone, oestradiol-17beta and oestriol in concentrations above 1ng/g wet wt. of tissue, and dehydroepiandrosterone, testosterone, androsterone (3alpha-hydroxy-5alpha-androstan-17-one) and 3beta-hydroxy-5alpha-androstan-17-one in concentrations exceeding 5ng/g, in extracts of five primary breast tumours.

Transporters are carrier proteins that may influence pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs. The development of validated in vitro transporter models is imperative to support regulatory submissions of drug candidates. This study is focused on utilizing human embryonic kidney (HEK) 293 cell cultures genetically transfected with the human organic anion transporting polypeptides (OATP) 1B1 transporter to identify substrates and inhibitors in drug development. The kinetics of OATP1B1-mediated uptake of [(3)H]-oestradiol 17beta-glucuronide and inhibition of uptake by rifamycin SV were used to determine K(m), V(max), and IC(50) values over a range of passage numbers to investigate accuracy and precision. The mean K(m) and V(max) values were found to be 6.3 +/- 1.2 microM and 460 +/- 96 pmol min(-1) mg(-1), respectively. The mean IC(50) value for rifamycin SV was 0.23 +/- 0.07 microM on uptake of 1 microM [(3)H]-oestradiol 17beta-glucuronide. These data were similar to previously reported values (accuracy greater than 82%), reproducible (precision less than 29%) and exhibited low standard deviations (SDs) obviating the need to study test compounds on more than one occasion. [(3)H]-oestrone 3-sulfate and [(3)H]-pravastatin exhibited concentration-dependent OATP1B1 uptake, and statistically significant differences were observed at each concentration between uptake rates of HEK293-OATP1B1 and HEK293-MOCK cells (uptake ratios greater than or equal to 3). Propranolol showed no positive uptake ratio. Bezafibrate and gemfibrozil exhibited concentration-dependent inhibition of OATP1B1-mediated uptake of [(3)H]-oestradiol 17beta-glucuronide with mean IC(50) values of 16 and 27 microM, respectively. Based on the validation results, acceptance criteria to identify a test compound as a substrate and/or inhibitor using these specific cell lines were determined. These validated OATP1B1 assays were robust, reproducible, and suitable for routine in vitro evaluation of candidate drugs.

In a 23-year old primipara who was in her 34th week of pregnancy, serum oestriol could not be identified, with no other abnormal clinical and endocrinological findings, which is why the authors suspected placental sulphatase deficiency. The placental sulphatase activity was tested by the intravenous application of dehydroepiandrosterone sulphate, and compared with the data obtained from a control patient with normal endocrinological findings. There was a slight increase in DHEA from 7.5 ng/ml to 18.0 ng/ml, whereas the serum values of oestrone and oestradiol did not change. In contrast, the control patient showed an excessive increase of DHEA from 5.5 ng/ml to 56.8 ng/ml. Oestrone and oestradiol also reacted with a strong increase of activity from 12.7 ng/ml to 42.7 ng/ml or 25.2 ng/ml to 61.2 ng/ml, respectively. A placental sulphatase deficiency with absence of hydrolysis of the applied DHEAS must be assumed as the reason for the deficient or absent reaction of DHEA and the oestrogens. The patient gave birth to a healthy boy in the 41st week of pregnancy, delivery being performed by caesarian section. Clinically normal course of pregnancy with lowered oestriol values, delivery by caesarian section in the case of primiparae, as well as male sex of the newborn, are criteria of placental sulphatase deficiency. These criteria correspond to the few observations published so far.

Peripheral plasma from five postmenopausal women was analyzed for oestrone and oestradiol during 48 hours after administration of 1.25 mg Premarin (conjugated equine oestrogen) orally and vaginally. Oestrone and oestradiol were separated on columns of Sephadex LH 20 before radioimmunoassay. Administration of 1.25 mg of Premarin produced a marked increase in plasma oestrone levels and 24 hours after treatment the oestrone levels were still above follicular phase levels. The plasma pattern of oestradiol was similar but the elevation was less pronounced. The biological activity of Premarin as indicated by depressed gonadotrophin levels was similar with the intravaginal and oral routes of administration.

Mean serum concentrations of oestradiol-17beta, oestrone, and oestrone sulphate in postmenopausal women were the same when measured up to six hours after treatment with either piperazine oestrone sulphate 1.5 mg or oestradiol valerate 2 mg. Maximum concentrations of oestradiol were less than those of oestrone, but oestrone sulphate reached concentrations about 30 times higher than those of oestrone. The rapid conversion of oestradiol valerate to oestrone and oestrone sulphate does not support the suggestion that in menopausal women oestradiol is less likely to be associated with a risk of endometrial carcinoma than oestrone sulphate, since the two preparations appear to become identical after ingestion.

Oestrogens protect neurons against excitatory amino acid-induced toxicity; however data on their interaction with particular subtype of glutamate receptors are sparse. Therefore in the present study we investigated oestrogen effects on kainate neurotoxicity in primary cortical neurons. The data showed that both oestradiol-17 beta and oestrone (100 nM and 200 nM) reduced kainate toxicity by ca. 40%. Since tamoxifen only partly inhibited the above effects, we suggest that both genomic and non-genomic mechanisms are involved in the anti-kainate action of oestrogens.

The concentration of oestrone sulphate in peripheral plasma from postmenopausal women was investigated using a method which involved extraction of the conjugate, which was then hydrolysed with acid, and determination (by radioimmunoassay) of the purified oestrone fraction obtained. The concentration of unconjugated oestrone in the same plasma samples was also measured. Postmenopausal women had concentrations of oestrone sulphate in plasma (1.1 +/- 0.36 nmol/l, mean +/- SD, n = 39) similar to those found in women in the follicular phase of the menstrual cycle and less than those found in males (3.2 +/- 0.61 nmol/l, n = 21). The mean ratio of the concentration of oestrone sulphate to that of oestrone in plasma from postmenopausal women (7.9 +/- 3.3) was significantly lower (P less than 0.001, t test) than the mean ratio in men (19.8 +/- 3.8). Treatment with conjugated oestrogens, oestradiol in a cream, oestradiol valerate or ethinyl oestradiol, increased the concentration of oestrone sulphate in the peripheral circulation. In contrast, chronic corticosteroid therapy reduced the level of oestrone sulphate (0.5 +/- 0.11 nmol/l, n = 10) but this was partly restored (to 0.7 +/- 0.13 nmol/l) by concomitant oral dehydroepiandrosterone. Ingestion of piperazine oestrone sulphate (Harmogen, 1.5 mg) by three fasting postmenopausal women was followed 4 h later by oestrone sulphate concentrations five to ten times those found at midcycle.

Steroid sulphates such as oestrone sulphate (OE1S) and dehydroepiandrosterone sulphate (DHEAS) have been suggested to be of biological importance in different disease states such as breast cancer and atherosclerosis. Previous studies have shown that drugs such as aminoglutethimide and rifampicin that induce P450-dependent mixed-function oxygenases selectively suppress plasma OE1S levels. The aim of this study was to evaluate the influence of treatment with carbamazepine, an antiepileptic drug known to stimulate mixed-function oxygenases, on plasma levels of OE1S and DHEAS. We measured plasma OE1S and DHEAS together with other plasma oestrogens and androgens in male epileptic patients before and during carbamazepine monotherapy. Patients treated with valproate monotherapy acted as a control group. Treatment with carbamazepine decreased plasma OE1S levels from a mean value of 810.8 to 411.6 pmol/l (mean suppression to 50.7% of pretreatment levels, P < 0.001). Similarly, the ratio of OE1S to OE1 fell to 59.9% of pretreatment levels (P < 0.001)). DHEAS decreased from a mean level of 4.9 mumol/l before treatment to 3.0 mumol/l during carbamazepine therapy (mean reduction to 62.7% of pretreatment levels (P < 0.001), while the ratio of DHEAS to DHEA fell to 63.0% of pretreatment values (P < 0.01). No significant change in plasma levels of the other oestrogens or androgens measured was observed. Treatment with valproate caused a slight decrease in FSH levels (P < 0.05), but no change in any of the other hormones measured was observed. Studies are warranted to evaluate the possible effects of long-term treatment with carbamazepine on the risk of developing endocrine-sensitive tumours and cardiovascular disease and also the possible effects of alterations in plasma DHEAS on epileptic activity.

Plasma oestrone sulphate (E(1)S) concentration and doppler ultrasound as methods of pregnancy diagnosis in sows were compared. Using either method, pregnancy was accurately detected (test sensitivity>> 94% for pregnant sows). E(1)S was a better predictor of nonpregnant animals (test specificity 78 vs 66%, respectively; P < 0.01) and could be used at least 1 wk earlier than doppler ultrasound (24 to 30 d vs 35 d postservice, respectively). E(1)S concentration was not an accurate predictor of litter size.

An antiserum raised against oestrone-3-glucuronide-BSA was used for the direct radioimmunoassay of oestrone sulphate in pig serum. The method has a minimum sensitivity of 0.2 ng/ml and is potentially an accurate pregnancy test in pigs. Serum oestrone sulphate concentrations were less than or equal to 0.5 ng/ml in non-pregnant cyclic sows and greater than 0.5 ng/ml in pregnant sows bled 26-29 days after service.

A radioimmunoassay for oestrone sulphate in unextracted samples of milk has been developed. The assay was validated by comparison with a method involving hydrolysis and extraction. The direct assay was used to measure oestrone sulphate in milk samples taken at weekly intervals throughout pregnancy in a commercial dairy herd. Concentrations of oestrone sulphate increased approximately 100 days after insemination and were maintained throughout the remainder of pregnancy in the range of 1.85-3.70 nmol/l.

An oestrogen-sensitive rat mammary tumour (OES HR1) has been grown in normal female rats and in female and male rats supplemented with oestrone. In some rats, after the tumour was established, both exogenous and endogenous sources of oestrogen were removed--a treatment which inhibited further growth of the tumour. The proliferative characteristics of the tumours were measured by injecting the rats with deoxybromouridine (BrdU) 4 h before removing the tumour. Extracted nuclei were reacted with anti-BrdU and the labelling index and DNA content measured by flow cytometry. A correlation between the number of (diploid) host cells present and the number of (aneuploid) tumour cells in S-phase of the cell cycle was observed. This result suggests that there are paracrine interactions between tumour and host cells. We also observed that, on oestrogen ablation, the labelling index was significantly reduced while the percentage of cells in S-phase changed far less. The demonstration that there are cells in S-phase which are not proliferating highlights a possible problem with the measurement of proliferation in human tumours from a DNA histogram.

OBJECTIVE

What are the characteristics of, and how variable are, individual normal menstrual cycle profiles of excretion rates for the urinary metabolites oestrone glucuronide (E1G) and pregnanediol glucuronide (PdG)?

CONCLUSIONS

There is a continuum of menstrual cycle profiles that differ from standard textbook profiles but which can be understood simply in terms of growth, atresia and ovulation of ovarian follicles.

BACKGROUND

Point-of-care assays with the Ovarian Monitor pre-coated assay tubes, using urine samples diluted to a constant volume per unit time, give laboratory accurate clinical data for individual menstrual cycles. Lay operators can perform the point-of-care assay system at home to achieve reliable and reproducible results, which can be used for natural family planning.

METHODS

This prospective study involved 62 women, with normal menstrual cycles, recruited from three centres: Palmerston North, New Zealand, Sydney, Australia and Santiago, Chile. The study lasted 3 years.

METHODS

Women collected daily urine samples and determined their E1G and PdG rates with a pre-coated enzyme assay system known as the Ovarian Monitor. For two cycles, the assays were repeated in a study centre and the results were averaged to give 113 individual menstrual cycles for analysis. The cycles were displayed individually in a proprietary database program.

RESULTS

The individual normal hormonal profiles were more complex than the classic composite curves for 40% of the cycles. Of 113 ostensibly normal cycles, only 91 were potentially fertile and 22 had some luteal phase defect. The oestrone glucuronide and PdG excretion rates were reliable and informative in the non-invasive elucidation of ovulation and ovarian function for both simple and complex profiles. Daily monitoring revealed the variability of normal menstrual cycle profiles. The LH peaks were variable and ambiguous markers for ovulation.

CONCLUSIONS

The study consisted of cycles only from women with regular cycles of 20-40 days duration. All the women were intending to avoid a pregnancy during the study thus the limits of the fertile window were not tested.

CONCLUSIONS

The principles established in this study should apply to cycles of any length. All peaks in oestrone glucuronide excretion should be tested by concurrent measurements of PdG, which gives a positive indication of the fate of the follicle it represents. The Ovarian Monitor provides a useful addition for practitioners of natural family planning.

BACKGROUND

Financial support for this study was obtained from the UNDP/UNFPA/World Bank/WHO Special Programme of Research, Development and Research Training in Human Reproduction (HRP). D.G.C. is currently employed by and holds stock in Manawatu Diagnostics Ltd, a company in the development phase of a potentially competing product. The remaining authors have nothing to declare.