BACKGROUND

While advanced stages of ascending placentitis can be diagnosed by transrectal ultrasonography and clinical signs, early stages can be missed. Thus, additional tools could enhance assessment of placental health.

OBJECTIVE

To characterise peripheral dehydroepiandrosterone sulphate (DHEA-S) and testosterone concentrations in mares carrying normal pregnancies (Study 1) and compare plasma concentrations of DHEA-S, testosterone, oestradiol 17-β (oestradiol) and oestrone sulphate (OES) in mares with or without placentitis (Study 2).

METHODS

Longitudinal cohort study of healthy mares (Study 1) and controlled experiment (Study 2).

METHODS

In Study 1, mares had serum samples collected from 100 days of gestation to term. In Study 2, pregnant mares (260-280 days gestation) were assigned to a control group or a group with placentitis. Placentitis was induced via intracervical inoculation of Streptococcus equi ssp. zooepidemicus. Blood was collected at inoculation/commencement for control mares (day = 0) and daily for 12 days post inoculation (DPI) or until abortion. Steroid concentrations were determined by immunoassays. Concentrations of steroids in Study 2 were also evaluated relative to days from abortion (DFA -8 days to 0).

RESULTS

In Study 1, DHEA-S peaked by 180 days gestation, while testosterone concentrations were progressively increased from Days 100 to 180 with a plateau until ~240 days and a progressive decline until 290 days of gestation. In Study 2, concentrations of DHEA-S and testosterone were not significantly different between groups. There were significant effects of time (oestradiol P = 0.0008, OES P = 0.01) and time-by-group interactions (oestradiol P<0.001, OES P<0.0001) for oestrogen concentrations. For mares with experimental placentitis, concentrations of oestradiol were significantly reduced at -6, -2, -1 and 0 DFA, while OES concentrations were significantly reduced on the day before abortion (0 DFA).

CONCLUSIONS

Testosterone and DHEA-S were increased and varied through pregnancy. Oestrogens but not androgens decreased significantly in mares with experimentally-induced ascending placentitis.

The effect of oxytetracycline (1 g/day for five days) on the enterohepatic recycling of oestrogens and on plasma sex hormone concentrations was assessed in healthy men. Plasma oestrone (E1), oestradiol-17 beta (E2), 4-androstenedione (A), 5 alpha-dihydrotestosterone (5 alpha-DHT), total and free testosterone (T and free T), binding capacity of sex hormone binding globulin, luteinizing hormone, dehydroepiandrosterone-sulphate, urinary total E1, E2, and oestriol (E3), and oestriol-3-glucuronide (E3-3G) and faecal unconjugated and conjugated E1, E2, and E3 were measured by radioimmunoassay (RIA). Treatment with the antibiotic significantly increased the excretion of faecal conjugated oestrogens, which parallelled a decrease in urinary oestrogen excretion, especially of E3. The effect on urinary E3 could be explained almost entirely by the simultaneous decrease of urinary E3-3G concentrations. In urine and faeces the E2/E3 and E1 + E2/E3 ratios increased, probably because of the diminished reductive metabolism of oestrogens in the gut. No significant effects on plasma unconjugated oestrogen concentrations were observed. Moreover, in the present study oxytetracycline had no remarkable effect on plasma total, or free T concentrations, nor on other plasma hormones measured. Our results suggest that enterohepatic recycling and intestinal metabolism of oestrogens may be significant in men. The mechanism of action of antibiotics on oestrogen metabolism probably involves decreased hydrolysis by beta-glucuronidase of oestrogen conjugates by the intestinal contents, diminishing the reabsorption of aglycones of oestrogen conjugates and resulting in faecal loss of the steroids.

Cyclofenil is a triphenylethylene derivative, similar in structure to clomiphene citrate, which is used to induce ovulation in anovulatory women. The effects of cyclofenil on a group of 10 normal cyclic and 10 oligomenorrhoeic subjects were examined in a double blind controlled cross-over study. Both groups of women were administered either cyclofenil or, following a washout cycle, a placebo in two treatment cycles. Urinary oestrone and pregnanediol excretion were measured daily and ultrasound scans performed to assess follicular development. Frequent sampling of blood was performed on day 6 to study luteinizing hormone (LH) and follicle stimulating hormone (FSH) pulsatile release. Cervical mucus changes and sperm-cervical mucus interaction were studied after identification of the LH peak. There were no significant differences between cyclofenil and placebo cycles in the following: ovulation rates, daily urinary oestrone and pregnanediol excretion, the number or size of developing follicles, LH pulsatility (parameters studied: number of peaks, pulse interval, pulse amplitude, pulse area and mean nadir LH), mean FSH level on day 6, cervical mucus and sperm-cervical mucus interaction. In view of our inability to demonstrate an effect on any parameter of endocrine function in normal and oligomenorrhoeic women, these results throw doubt on the therapeutic value of cyclofenil in its present dosage and formulation.

In an effort to investigate the structural requirements for the inhibition of the enzyme oestrone sulphatase (ES), we have previously undertaken extensive structure-activity relationship studies. Using the data from molecular modelling and structure-activity relationship determination studies, we have designed a number of compounds based upon 4-sulphamated phenyl ketones. Here, we report the results of our study into a series of these compounds as potential inhibitors of ES. The results of the study show that these compounds are potent inhibitors the possessing greater inhibitory activity than 4-methylcoumarin-7-O-sulphamate derivative (COUMATE) (a potent non-steroidal inhibitor), but are weaker than oestrone-3-sulphamate (EMATE) and the recently reported 667- and 669-COUMATE, however, they provide good lead compounds in the search for potent inhibitors of ES. Furthermore, the compounds are observed to be irreversible inhibitors. From the consideration of the structure-activity relationship of these novel compounds, we have attempted to rationalise the significance of the log P factor in the inhibition of ES and suggest that a log P requirement of approximately 3.5 aids the inhibition through the rapid expulsion of the carbon backbone from the active site. We also propose that the same factor is responsible for the hydrolysis of oestrone sulphate reaction, appearing to be an irreversible process.

Synthesis of oestrogens within breast tissues makes an important contribution to the high concentrations of oestradiol which are found in breast tumours. The activities of the enzymes involved in oestrogen synthesis, i.e. the aromatase, oestradiol dehydrogenase (E2DH) and oestrone sulphatase (E1-STS), can be stimulated by several growth factors and cytokines. As it is possible that some of these factors may be derived from cells of the immune system (macrophages and lymphocytes), the effects of basic fibroblast growth factor (bFGF) and interleukin-2 (IL-2), which are produced by these cells, on E2DH activity was examined in MCF-7 cells. Treatment of these cells with bFGF resulted in a dose-dependent increase in E2DH reductive activity whereas IL-2 was inactive at the concentration tested. To obtain further evidence that factors produced by macrophages and lymphocytes can modulate the activities of enzymes involved in oestrogen synthesis, conditioned medium was collected from these cells and found to stimulate both E1-STS and E2DH activities. In addition to understanding the control of oestrogen synthesis in breast tumours an inhibitor to block the synthesis of oestrone via the oestrone sulphatase pathway was developed. Oestrone-3-O-sulphamate (EMATE) is a potent, irreversible, inhibitor of E1-STS. A single dose of EMATE (10 mg/kg) inhibited tissue E1-STS activity in rats by more than 95% for up to 7 days, indicating that this compound may have considerable therapeutic potential for the treatment of breast cancer. Evidence is also reviewed that another steroid sulphatase, dehydroepiandrosterone sulphate sulphatase, may have a crucial role in regulating cytokine production and that this may indirectly control tumour oestrogen synthesis.

A variety of radioimmunoassays specifically characterized for use with fetal and maternal ovine plasma have been used to measure steroid hormone concentrations in small plasma samples drawn simultaneously from mother and fetus. By repeated sampling from the same animal preparation the changes over time of plasma concentrations of cortisol, pregnenolone and its sulphate, and oestrone and oestrone sulphate have been systematically studied. In animals delivering spontaneously at term the fetal plasma cortisol concentration rises between 120 and 130 days gestation and plays an important part in the maturation of several vital physiological systems, including the fetal thyroid axis. In normal term deliveries and premature deliveries induced by synthetic adrenocorticotropin (ACTH(1-24), 1 microgram h-1), the concentration of oestrone sulphate in maternal plasma increases before the fetal plasma concentration. Fetal and maternal oestrogens are probably important in the control of the low-grade tonic myometrial activity that occurs throughout gestation, in the initiation of labour and in the control of uterine blood flow. Low-grade tonic myometrial activity affects fetal oxygenation, fetal breathing and the fetal sleep state and may constitute a pathway through which the mother influences fetal development, with important physiological and possibly pathological consequences.

This open-label, randomized study in 36 healthy postmenopausal women, investigated the pharmacokinetics of Fem7, (an oestradiol matrix-type patch), at doses of 25, 50, 75 and 100 microg/24 h, applied once weekly. Maximum plasma concentrations of oestradiol and oestrone were observed 14-20 h after patch application, remaining within the therapeutic range until removal, and returning to baseline within 12 h thereafter. Plasma oestradiol concentrations increased in a dose-dependent manner for all four doses, and plasma oestrone increased for the three highest doses. Fem7 treatment was well tolerated at all four doses and no severe adverse reactions were reported. Fem7 is a convenient and well-tolerated form of hormone-replacement therapy that delivers oestradiol in a consistent and predictable manner.

The endocrine and therapeutic effects of the luteinising hormone-releasing hormone (LHRH) analogue, Zoladex, have been assessed in 28 postmenopausal women with advanced breast cancer. Fourteen had responded to previous hormone therapy and 14 had received no previous hormone therapy. Zoladex treatment resulted in 2 partial responses and 2 patients with stable disease for more than 6 months in the former group, and 1 partial response and 2 with stable disease for more than 6 months in the latter group. Toxicity was minimal. All responses occurred in soft tissue disease. Peripheral oestradiol levels fell after 1 month of Zoladex from 33 pmol/l (+/- 20 SD) to 22 pmol/l (+/- 11 SD) (p less than 0.005) and both responders and nonresponders showed similar changes in oestradiol. Oestrone levels did not change significantly. Six out of 7 patients who received tamoxifen after progression of disease on Zoladex, showed a response. These results suggest that Zoladex acts indirectly via changes in peripheral hormone levels rather than directly on LHRH receptors on the tumour.

Glucuronidation of oestrone and of oestradiol in microsomal fractions was markedly and significantly stimulated by UDP-N-acetylglucosamine and by ultrasonication: Triton X-100 also stimulated. This is consistent with compartmentation of UDP-glucuronyl-transferase. Stimulation by UDP-N-acetylglucosamine may be physiologically significant.

The effect of adding oestriol or oestriol succinate to oestradiol in the treatment of climacteric postmenopausal patients was examined in a cross-over trial. Plasma concentrations of oestrone (E1), oestradiol (E2), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured by radioimmunoassay after collection over a day after each medication. The subjective effect of each therapy was evaluated by the patient herself. The combination of 1 mg oestriol with 2 mg oestradiol did not change the daily profiles of plasma E1, E2, LH and FSH in three out of four patients, compared with only 2 mg of oestradiol. The other combination, 8 mg of oestriol succinate with 1 mg oestradiol, similarly did not change the daily plasma profiles of oestrogens or gonadotrophins, compared with only 1 mg of oestradiol. Neither oestriol succinate changed the clinical response during the treatment of these climacteric patients.

A randomised, single-blind comparative study was carried out in 9 ovariectomized women to evaluate the kinetics of single doses of three different steroid combinations: 0.150 mg desogestrel + 2.0 mg micronized 17 beta-oestradiol, 0.150 mg desogestrel + 0.500 mg 17 beta-oestradiol cyclo-octyl acetate and 0.150 mg desogestrel + 1.0 mg 17 beta-oestradiol decanoate. Serum levels of 17 beta-oestradiol and oestrone were measured, as well as the excretion of 17 beta-oestradiol and its metabolites (oestrone and oestriol) in urine. In relation to the doses given, higher peak serum concentrations of 17 beta-oestradiol were obtained after the two fat soluble analogues, while the AUCs were similar to that after micronised 17 beta-oestradiol. However, there was more extensive conversion of the micronised 17 beta-oestradiol preparation into oestrone compared to 17 beta-oestradiol cyclo-octyl acetate and 17 beta-oestradiol decanoate. The oestrone/17 beta-oestradiol serum concentration ratio was approximately 2.6 before tablet intake and remained essentially unchanged after intake of 17 beta-oestradiol cyclo-octyl acetate and 17 beta-oestradiol decanoate. After micronized 17 beta-oestradiol however, there was a 2-3-fold increase in the ratio at Cmax and slower elimination of 17 beta-oestradiol from plasma, which may be due to the fact that high serum oestrone levels may serve as a reservoir, since both a metabolite and also a precursor of 17 beta-oestradiol. The urinary excretion of 17 beta-oestradiol, oestrone and oestriol was highest after oral administration of micronized 17 beta-oestradiol compared to 17 beta-oestradiol cyclo-octyl acetate and 17 beta-oestradiol decanoate.(ABSTRACT TRUNCATED AT 250 WORDS)

Ozone oxidation is proven to be an effective solution for the degradation of selected oestrogenic active substances detected in secondary wastewaters such as beta-oestradiol, oestrone, oestriol, 17-alpha-ethinyloestradiol, mestranol, daidzeine, beta-sitosterol, bisphenol A, norethisterone, 4-tert-octylphenol and 4-iso-nonylphenol, up to their limit of detection. The matrix-effect of wastewater was investigated performing ozone experiments under batch mode and continuous mode using drinking water and a wastewater issued from a local plant both spiked with the non-detected substances. The results obtained indicate that the wastewater matrix greatly affects the kinetics of ozone reaction with these substances but does not really change the related reactivity scale. The ozone dose corresponding to the full conversion of target EDCs consequently increases as their oxidation takes place competing with reactions of background pollutants represented by the COD and DOC content. However, a usual dose close to 12 mg/L was found sufficient to provide high degradation yields for all substances studied while 35% of COD was removed. This is a contribution to the numerous current works focused on technologies able to improve the quality of water discharged from wastewater treatment plants, both considering conventional parameters and emerging contaminants.

OBJECTIVE

Hyperprolactinaemic amenorrhoea is associated with disturbances of pulsatile gonadotrophin secretion. The underlying mechanism remains unclear and the aim of this study was to investigate the 24-hour secretory pattern of gonadotrophins in women with hyperprolactinaemic amenorrhoea. The effect of opioid blockade using naloxone infusion on LH secretory pattern was also studied.

METHODS

The secretory patterns of LH, FSH, PRL and their responses to naloxone infusion were studied by serial blood samples collected at 10-minute intervals for 24 hours. On the following day, naloxone was infused at a dose of 1.6 mg per hour for 4 hours.

METHODS

Eight women with hyperprolactinaemic amenorrhoea, two women hyperprolactinaemic but with normal ovarian cycles, and nine control subjects in the early follicular phase of menstrual cycle.

METHODS

Concentrations of LH, FSH and PRL were measured in plasma samples obtained at 10-minute intervals for 24 hours. In one woman, concentrations of urinary oestrone glucuronide were measured daily during treatment with pulsatile GnRH.

RESULTS

The number of LH pulses per 24 hours was significantly fewer in women with hyperprolactinaemic amenorrhoea than in those with hyperprolactinaemia with normal cycles or control subjects (mean +/- SEM 4.5 +/- 2.4 vs 13.5 +/- 2.5 vs 17.3 +/- 0.8, P < 0.001). The magnitude of each episode of secretion was significantly higher in the hyperprolactinaemic amenorrhoeic women (P < 0.05) so the overall mean concentrations of LH throughout the 24-hour period was similar in the three groups (5.2 +/- 1.1, 4.8 +/- 0.8 and 5.2 +/- 0.4 U/l respectively). In women with hyperprolactinaemic amenorrhoea there was no significant change in the pattern of LH secretion during sleep in contrast to the control women in whom there was a slowing in the LH pulse frequency during the night. There was no significant change in the mean concentrations of LH, FSH and PRL during the naloxone infusion. There were also no significant changes in the LH pulse frequency in response to naloxone infusion when compared with an equivalent period of time in the previous 24 hours. In one hyperprolactinaemic amenorrhoeic woman, follicular development, ovulation and pregnancy were induced when gonadotrophin releasing hormone (GnRH) was infused in a pulsatile manner at a dose of 5 micrograms every 90 minutes.

CONCLUSIONS

The suppression of normal ovarian cycles in women with hyperprolactinaemic amenorrhoea is due to a significant reduction in frequency of LH (GnRH) secretion which is not due to an increase in hypothalamic opioid activity. As normal ovarian cycles can occur or be induced by exogenous GnRH in hyperprolactinaemia, it is unlikely that a high level of prolactin by itself inhibits follicular development and ovulation.

Unconjugated oestrone and unconjugated oestradiol-17 beta were measured in 24 h urine specimens from twenty normal men. The concentrations of non-protein-bound oestrone and non-protein-bound oestradiol-17 beta, in samples of blood obtained from these subjects during the time in which the urine was collected, were measured by equilibrium dialysis (using a correction for plasma dilution). The mean excretion of unconjugated oestrone (0.44+/- 0.36 nmol/24 h, mean +/- SD) was significantly greater (Pless than 0.01) than that of unconjugated oestradiol-17 beta (0.20 +/- 0.13 nmol/24 h). The concentrations of non-protein-bound oestrone and non-protein-bound oestradiol-17 beta in plasma were 5.52 +/- 2.69pmol/1 and 2.42 +/- 0.73 pmol/1, respectively. There was no correlation between the quantity of unconjugated oestrone excreted and the concentration of non-protein-bound oestrone in plasma (r=0.27) nor between the quantity of unconjugated oestradiol-17 beta in the urine and the concentration of non-protein-bound oestradiol-17 beta in plasma (r=0.05). Therefore in normal men, estimation of unconjugated oestrone or oestradiol-17 beta in urine provides no guide to the concentration of the corresponding non-protein-bound oestrogen in plasma.

Progesterone, oestradiol and oestrone were measured in plasma from four captive muskoxen during three consecutive pregnancies (1983-1984, 1984-1985 and 1985-1986). Jugular blood samples were collected weekly (1983) or on an alternating 3:4 day schedule (1984-1986) during the first 12-15 weeks and last 6-10 weeks of pregnancy. Sampling during mid-pregnancy was at intervals of 2 weeks (1983 and 1985) or 1 week (1986). Duration of gestation was about 34 weeks (235 +/- 4 (SD) days (n = 10), range 230-242 days). Progesterone remained at concentrations similar to those found during the luteal phase of the oestrous cycle for the first 10-12 weeks (mean +/- SEM 1.6 +/- 0.1 ng ml-1) after which it rose to a peak (mean 5.5 +/- 0.65 ng ml-1) between weeks 12 and 20. In all ten pregnancies progesterone concentrations declined dramatically between weeks 20 and 22 to luteal phase values where they remained until parturition. The decline was accompanied by an increase in oestradiol and oestrone concentrations which reached mean peak values of 199.23 +/- 87.23 pg ml-1 and 980.48 +/- 203.91 pg ml-1, respectively. Corpora lutea collected from wild muskoxen between 45 and 80 days gestation all showed histological evidence of regression, while corpora lutea from mid-gestation (112-125 days) were in advanced stages of involution. Repeated ovarian ultrasonography of captive muskoxen during the first 100 days of pregnancy confirmed these findings. The unusual, early regression of the corpus luteum of pregnancy indicates that progesterone and oestrogen of mid- and late pregnancy are probably of placental origin.(ABSTRACT TRUNCATED AT 250 WORDS)

The duration of the oestrous cycle and day of ovulation were recorded in six mares which were used for concurrent assay of plasma levels of sex steroids and pituitary LH concentration. Peak of progesterone were reached during dioestrus and those of oestradiol and androstenedione occurred 2 days before ovulation and were in decline on the day of ovulation. Plasma oestrone remained constant (between 9 and 12 pg/ml) throughout the cycle. Plasma LH rose to a maximum near to the time of ovulation, and thereafter decreased gradually until mid-dioestrus. The pattern of secretion is compared with that reported in other domesticated animals.

A cytosolic oestrogen receptor from baboon endometria was detected and partially characterized. The apparent dissociation constant for oestradiol was 1.5 x 10(-10)--4 x 10(-10) mol/l. Steroids that competed with the [3H]oestradiol binding to the receptor were oestradiol and ethynyloestradiol greater than oestriol greater than oestrone; progesterone, testosterone and corticosterone were not competitors. The [3H]oestradiol-receptor complexes migrated as a 3-3.5S peak during sucrose density-gradient centrifugation when endometrial samples were taken during either the proliferative or the secretory phase. A 7S peak was observed for samples taken at the period of ovulation. A [3H]oestradiol exchange technique was used to detect changes in the receptor concentration during the menstrual cycle. This concentration which was high during the early follicular phase fell sharply before the ovulatory peak of ovarian oestrogens. It remained at a base level during the early secretory phase and then rose during the last days of the cycle to the same concentration as that measured at the beginning of the cycle.

The maturation value (MV), cervical mucus parameters (ferning, Spinnbarkeit), oestrone (E1), oestradiol (E2), oestriol (E3), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), thyrotropin (TSH), growth hormone (GH), sex hormone binding globulin (SHBG), corticosteroid binding globulin (CBG) and thyroxin-binding globulin (TBG) were determined in 11 post-menopausal women presenting with vaginal atrophy prior to, and following, treatment with Ovestin vaginal cream containing 0.5 mg/day of E3 for 8 wk. In 6 of the patients E3 was measured during frequent plasma sampling on days 1, 21 and 56; in the same patients and on the same days TRH-stimulated PRL, TSH and GH levels were estimated. While the therapy induced a sharp rise in the MV, there was a moderate effect on ferning/Spinnbarkeit. Baseline E3 rose from undetectable levels to a mean value of 86.8 pmol/l at day 21. E3 levels achieved during frequent plasma sampling were higher on day 1 than on days 21 and 56 - a decline of the areas under the response curves being significant (P2-sided = 0.03). There was a slight suppression of FSH and LH. No changes in the circulating levels of E1, E2, SHBG, CBG, TBG, PRL, TSH and GH were seen. TRH-stimulated PRL, TSH and GH levels remained unaffected. Clinical effect was excellent and no untoward effects were reported.

The levels of oestrone sulphate in plasma of pregnant cows was followed from 30 days of pregnancy until parturition. The Swedish Jersey Breed (SJB) showed significantly lower levels of oestrone sulphate between 101 and 200 days of pregnancy than either the Swedish Red and White (SRB) or the Swedish Lowland Breed (SLB). No significant difference was noted between SRB and SLB. On days 141-160 of gestation the oestrone sulphate values were still below the 10 nmol/l level for the SJB while they were above this level for the SRB and the SLB, and the difference was significant. In the SJB, levels above 10 nmol/l were reached on days 161-180 of gestation. In the second part of this study the levels of oestrone sulphate were measured around parturition in SRB cows. At parturition, the levels of oestrone sulphate rose to peak values of 79.9 +/- 5.2 nmol/l and then decreased to 6.6 +/- 0.5 nmol/l on the day after calving. In one cow peak values of 66.0 nmol/l were reached 2 days prior to parturition, and subsequently dropped to 7.0 nmol/l at parturition. This cow had retained foetal membranes. A possible relationship between low oestrone sulphate levels prior to parturition and retained foetal membranes is discussed.

Oestrone sulphate concentrations were measured by radioimmunoassay in milk samples obtained weekly during pregnancy from Jersey and Friesian cows, with each breed grazed at two different stocking rates. Mean milk yields differed significantly (P<0.05) between the four herds, while mean percentage milk fat and protein values differed significantly (P<0.05) between the two breeds. In all four herds, oestrone sulphate concentrations in milk rose progressively during pregnancy from a mean value of approximately 80-100 pg/ml at 60-80 days of pregnancy to a plateau value of approximately 1 ng/ml at 181-200 days. In non-pregnant cows, oestrone sulphate concentrations in milk ranged from non-detectable to 110 pg/ml, with a mean +/- s.e.m. value of 59 +/- 4 pg/ml. There was considerable variation in milk oestrone sulphate concentrations between cows in each herd, and oestrone sulphate concentrations could also fluctuate markedly within cows from week to week. Despite this variation, the concentration of oestrone sulphate in 98% of milk samples obtained after 120 days of pregnancy was greater than the highest concentration found in milk from non-pregnant cows. Measurement of oestrone sulphate concentrations in milk samples taken at least 120 days after mating or insemination may provide an alternative, non-invasive means of determining or confirming pregnancy in New Zealand dairy cows.