Twenty gravidae in whom the serum zinc concentration was less than 11.5 mumol/1 were investigated. Haemoglobin, serum vitamin B-12, serum folate, serum copper, and bone marrow smears were assessed. Thirteen showed haemoglobin concentrations below 110 g/1 and in 7 of them the cause of this anaemia was not found. Histological investigations indicated increased intramedullary cell destruction. Eight women selected at random were referred for further investigation including tests of renal and hepatic function, serum protein analyses, tests of haemolysis, and estimation of zinc and oestriol excretion in urine. The low serum zinc concentrations received no probable explanation other than zinc deficiency. Seven gravidae were treated with 90 mg Zn2+ daily as zinc sulphate by mouth during the latter part of pregnancy. Zinc excretion in urine was low and increased significantly (p less than 0.005) after one week's therapy. The serum zinc also increased (p less than 0.05). Zinc therapy gave no reticulocytosis within 8-12 days. Three women reported spontaneously an improvement in sense of taste. Five of 20 gravidae had dysmature infants. Heavy bleeding occurred at delivery in 6 cases, possibly secondary to impaired uterine contractility. Seven women who received zinc therapy had all normal deliveries, but labour was prolonged in one. No side effects of zinc therapy were noted except for nausea in one case. Further trials of zinc supplementation in larger series of women with low serum zinc concentrations during pregnancy seem to be justified.

Eight postclimacteric women were given 80 mg polyestriol phosphate by intramuscular injection in order to analyse the effect of the drug on the vaginal cytology. In four patients, whose vaginal smears showed complete atrophy before treatment, a clear but weak oestrogen effect was observed after the medication. This effect appeared after one week in three patients and after two weeks in one patient, and it lasted four weeks in three cases and two weeks in one. In four patients, whose vaginal smears showed a weak oestrogen effect before treatment, the therapy did not result in any significant change of the vaginal smear pattern.

The authors report on a preliminary study of the level of total serum oestriol in the third trimester of pregnancy by the bundle method of estimating this with "oestriol 1251 RIA Kit" (Amersham, Buchler). The criteria on which the quality of the method depends, viz, sensitivity, specificity, reliability, and accuracy, together with its practicability are satisfactory. Correlations between total serum oestriol and free serum oestriol and total urinary oestrogens are good and correspond with those given in the literature. This method therefore can be used in clinical practice. All the same, it still has to be established which parameter better reflects the feto-placental state. Is it the free serum oestriol or the total serum oestriol?

In 34 risk pregnancies with 34 hypotrophic newborns we determined during the second half of pregnancy the 24-hour urinary total oestrogens, unconjugated oestriol and total oestriol in the serum. By means of graphical evaluation of the pathological ranges in relation to the normal ranges (degree of overlapping) we investigated the relative clinical relevance. In respect of hypotrophy the ratio of dU-total oestrogens: S-unconjugated oestriol: S-total oestriol = 14:2:1. The considerable superiority of diagnosis based on urine in transverse assessment results in a statistically significant time gain of 4 weeks compared with the two serum parameters.

Plasma unconjugated oestriol (E3) and unconjugated oestradiol-17 beta (E2) were determined by radioimmunoassay. In ten normal women in their last month of pregnancy the individual fluctuation of E3 concentrations (mean of the coefficients of variation, CV) from day to day (over 5 days) was 15.6% (range 6.4--26.2%). The individual fluctuation of E2 determined in eight of these women was 16.9 (10.4--25.5) %. In six of the same women who had blood samples collected every 10 min (for 3 h) the individual fluctuation of E3 concentrations was 13.8 (7.0--25.1) %, and of E2 was 16.1 (11.3--26.0) %. The degree of fluctuation of E3 in individuals was in proportion to the mean concentration, unlike E2, suggesting that in clinical practice individual changes in E3 values would be as easy to interpret at low levels as at high levels. The finding that concentrations of plasma unconjugated E3 fluctuate in individuals no more than for E2 or than reported for total E3, and less than reported for 24-h urinary oestrogens, lends practical support to the theoretical preference for the assay of plasma unconjugated E3 to assess fetoplacental function.

The total oestrogens detected by fluorescence in 24-h urine and the concentration of total oestriol detected in serum by radioimmunoassay were estimated in 51 women during the second half of pregnancy. The statistical analysis showed a linear regression between urinary excretion and serum concentration according to the equation y = -2.89 + 2.82x (y = serum oestriol). The correlation coefficient over all pairs of estimations (N =567) was r = 0.69, which is very highly significant (P less than 0.0001). The analysis of the individual pregnancies showed that the linear correlation coefficient was significant (P less than 0.05) in 35 cases, poorly significant (0.05 less than P less than 0.1) in 3 cases and not significant (P less than 0.1) in 5 cases. The correlation could not be calculated in 8 cases because the number of estimations was too small. These values were compared empirically (normal/pathological). There was agreement in 6 cases and no agreement in 2 cases. The results show on the one hand that the radioimmunological estimation of total oestriol in serum can be used in the same way as urinary excretion of oestrogens for monitoring the feto-placental unit. On the other hand, the study confirms that a "simple" fluorimetric Kober-Ittrich method can give at least the same clinical information as a radioimmunoassay of relatively high cost. It may be concluded indirectly from the results that a considerable diurnal rhythm or substantial day-to-day fluctuation in the production of oestriol are unlikely.

Two types of oestrogen-medicated intrauterine devices (IUD) were studied in ovariectomized rhesus monkeys. An oestradiol (E2) fibre-wrapped IUD that released E2 at a rate of 3.57 micrograms/cm/day, or an oestriol (E3) fibre-wrapped IUD that releases E3 at a rate of 6.4 micrograms/cm/day, was inserted in eight animals and left in place for 4 weeks. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), E2 and E3 were determined by radioimmunoassay for 1 week before the IUD insertion, during the time the IUD was in place, and for 3 weeks after its removal. Uterine histology was performed at the time of IUD insertion and removal by light and transmission electron microscopy. Both E2 and E3 IUDs induced similar histological changes in the uterus, i.e. four- to five-fold increase in endometrial thickness, a shift of the gland/stroma ratio from 1:4 to 1:1, transformation to a marked pseudostratified epithelium with pronounced coiling of the glands, appearance of subnuclear and luminal secretion and, finally, change from spindle-dense stromal cells to plump eosinophilic cells. Oestradiol fibre-wrapped IUDs produced circulating E2 levels of 150-200 pg/ml during the entire 4 weeks. FSH and LH levels were decreased to an average of 55% and 65% from a castration baseline (P less than 0.001 and P less than 0.05, respectively). Oestriol fibre-wrapped IUDs produced circulating E3 levels of 100-250 pg/ml. However, FSH and LH levels were not altered in this group. The specific local oestrogenic effect of E3-IUDs without affecting the pituitary secretion of gonadotrophins, suggests their possible application in cases in which an exclusively oestrogenic effect at the uterine level, such as in Asherman's syndrome, is desired.

Human placenta subfractions were incubated with radioactive oestriol and 16-oxo-oestradiol. 16-oxo-oestradiol was identified from the oestriol incubation. Oestriol and 16-epi-oestriol were characterized from the incubation while 16-epi-oestriol were characterized from the incubation with 16-oxo-oestradiol. The 16alpha-dehydrogenase has been located in the soluble fraction (105 000 g supernatant) of the human placenta.

Oestrogen replacement therapy relieves many post-menopausal symptoms and has been successfully employed clinically for this purpose for more than four decades. Recently the alleged relationship between oestrogens and cancer has stimulated a re-evaluation of an old oestrogen preparation, oestriol (E3). The dosages of E3 employed appear to vary considerably, and the need was felt to establish the dosage on a scientific basis. Accordingly in the study reported here E3 was administered in various dosages (2, 4, 6, and 8 mg/d) to 52 symptomatic post-menopausal women as oestrogen replacement therapy for a six-month period. Assays of follicle-stimulating hormone (FSH), luteinizing hormone (LH), oestrone (E1) and oestradiol (E2) were performed before and during therapy and vaginal cytology, cervical mucus and endometrial studies were performed during the period of administration. The clinical effectiveness of E3 was found to be directly related to dosage. E3 did not induce endometrial proliferation and proved a poor suppressor of FSH and LH. The ability of oestriol to relieve vasomotor instability and to improve vaginal maturation without inducing notable side effects is sufficient reason for it to be included in the management of the post-menopausal syndrome.

Fourteen recently ovariectomized women received 4 mg of oestradiol valerate therapy daily for 6 mth. Serum oestrogens were measured by radioimmunoassay at monthly intervals. The results of therapy produced a 25-fold increase in serum oestrone (E1) concentration. Circulating oestradiol (E2) increased 5-to 6-fold. No accumulation of oestrogens was observed as a consequence of treatment. Serum concentration of oestriol (E3) showed no elevation. One month after the completion of therapy the E1 and E2 concentrations had returned to pre-treatment levels. Although therapy of high-dose oestradiol valerate was well-tolerated, it is not recommended because of the non-physiological high serum concentrations of oestrone and high serum level of oestradiol.

The tremendous interpatient variations that occur for oestrogen excretion during pregnancy are largely overcome by expressing results in terms of relative concentrations (i.e., as percent of total oestrogens excreted). Specimens of urine from 28 pregnant patients with a variety of complications were assayed for their oestrogen content by an "oestrogen profile" technique and the results thus expressed were compared with those from 20 normal pregnancies. Statistically significant changes are shown in the group of patients in the "above" normal range (normal range = normal mean +/- 3 X SE) for five oestrogens (oestrone, 2-hydroxyoestrone, 2-methoxyoestrone, 2-hydroxyoestradiol, and 2-hydroxyoestriol) and "below" the normal range for one oestrogen (oestriol). When the means of the oestrogen values for the different groups of patients were examined, two things became obvious; firstly, only patients with hypertension showed these changes, and secondly, two additional oestrogens (16-hydroxyoestrone and 16-epioestriol) were excreted in large amounts by the normotensive "high risk" patients. A comparison of the two methods of expressing oestrogen assay results is made quantitatively (as milligrams per day) versus relatively (as percent of total oestrogens excreted), and advantages and disadvantages of each are discussed.

Oestrone, oestradiol-17 beta and oestriol were measured in plasma samples from non-pregnant and pregnant African elephants shot in the wild. Enzymic hydrolysis of plasma showed that approximately 90 and 96% of the total (i.e. conjugated plus unconjugated) concentrations of oestrone and oestradiol-17 beta, respectively were represented by conjugated hormones. Unconjugated oestrogens remained low (less than 50 pg ml) in all samples, with no distinction between non-pregnant and pregnant animals. Levels of total oestrone during pregnancy varied between 160 and 594 pg/ml but were not significantly different from non-pregnant values. Total oestradiol-17 beta concentrations were significantly elevated during pregnancy (P less than 0 X 01) and, despite considerable individual variation (193-1428 pg/ml), were consistently higher than non-pregnant values after 6 months of gestation. The elevated levels of oestradiol-17 beta resulted in a reversal of the total oestradiol-17 beta: oestrone concentration ratio at about 6 months of pregnancy. Concentrations of total oestriol did not exceed 103 pg/ml. An indirect method of measurement indicated that oestradiol-17 beta sulphate was probably the most abundant circulating oestrogen during pregnancy in the African elephant.

The chemistry of oestrogens, and the metabolism of the oestrogens during pregnancy is considered. On this basis, appropriate methods of oestrogen determination have been devised for the recognition of foetuses at risk; the diagnostic significance of these methods is discussed. A method is regarded as suitable if the results can be quickly available, and give a reliable representation of the rate of oestriol production by the foeto-placental unit. Following a review of the development of eostrogen determinations, one method for the photometric measurement of total urinary oestrogens and one method for the radio-immunological determination of serum oestriol are discussed in detail. The advantages and disadvantages of studies on urine and serum are summarized in a table. Finally, problems in the clinical evaluation of the results of oestriol determinations are discussed.

Serum E1, E2 and E3 concentrations and E2/E1 ratio were measured after vaginal application of conjugated oestrogens, micronized 17 beta-oestradiol and oestriol. 2.4 mg of conjugated oestrogens caused a prompt elevation in the serum E1 concentration; the E2 level changed only slightly. After vaginal application of 2 mg micronized 17 beta-oestradiol the main serum oestrogen is E2 and the conversion of E2 to E1, as in oral administration, does not occur. A significant elevation in the serum E3 concentration was noted 2 h after the vaginal application of 0.5 mg oestriol. The E2/E1 ratio changed little after the application of conjugated oestrogens but increased considerably after the vaginal administration of 2 mg micronized 17 beta-oestradiol.

In ovariectomized rats oestradiol and oestriol administered sc for 5 days increased basal plasma prolactin in a dose related manner to similar maximal values. The data fitted log dose-response curves. Oestradiol was about 12 times more potent than oestriol. These dose response relationships were retained on thyrotrophin releasing hormone (TRH) administration (50 ng iv, blood sampled 5 min post TRH). In addition, significantly higher maximal response values for both oestradiol and oestriol were now seen. The relative potency also increased in oestradiols favour. We subsequently examined the effect of co-administering oestriol with oestradiol on plasma secretory responses to a single dose of oestradiol in young male rats: Oestriol (30 micrograms) potentiated increases in basal plasma prolactin, TRH stimulated prolactin secretion and radioimmunoassayable pituitary prolactin in rats in response to a single sc injection of oestradiol (10 micrograms). Tamoxifen, administered once also potentiated these responses to a single sc injection of oestradiol. However, if the tamoxifen was administered sequentially i.e. 48 and 24 h before or 48 and 24 h before and simultaneous with the oestradiol the potentiation was completely reversed.

Changes in the relative sensitivity of reaction oestrone, oestradiol, oestriol, and their monoglucuronides due to changes in reaction temperature and the acid a nd quinol concentrations of Kober's reagent have been investigated by means of an automated system. Similar investigations have been carried out with a number of standardized pregnancy urines. All measurements have been made with reference to an oestriol-16(beta-D-glucuronide)standard. These changes have been examined with trichloracetic acid and p-nitrophenol in Ittrich's reagent. The importance of the final acid concentration has also been examined. As a result of these investigations a modified scheme is suggested which gives maximal sensitivity to oestriol conjugates and minimal sensitivity to oestradiol conjugates, and may be calibrated by using an oestriol conjugate as a primary standard. The effect of varying the concentration of trichloracetic acid and p-nitrophenol in Ittrich's reagent has been examined under these modified conditions. More than 200 urines have been assayed by this method and the results compared with those obtained by the present automated procedure. The effect of dilution on many of these specimens has also been examined.

Synopsis Using high performance liquid chromatography the presence of natural oestrogen traces in aqueous placenta extracts of human origin has been demonstrated. This study, corroborated by results obtained by colorimetric dosage and by gaseous phase chromatography, reveals the preponderance of oestriol, which represents 80% of all the oestrogens. In all the extracts studied, oestriol concentration was not more than 100 degrees per litre of extract. This quantity of oestrogen contained in human placenta extracts of the Filatov type, even in wide cutaneous applications, cannot influence the subject's hormonal balance.

Serum total oestriol concentration was estimated by radioimmunoassay in 370 healthy pregnant women during the 27th and 41st week of gestation. The rise of the serum oestriol was noted from 230,4 +/- 128,2 nmol/L in the 27th week to the peak value of 732,8 +/- 322,4 nmol/L obtained in the 39th week. The changes in the urine total oestrogens measured by immunochemical method were similar but the increase was more uniform and stable. Low correlation (r = 0,25) was found between serum total oestriol and total oestrogens in urine. The results comparison of total oestrogens from urine determined by immunochemical method and obtained by radioimmunoassay has shown excellent and significant accordance (r = 0,98 p less than 0,01).

The levels of immunoreactive oestrone, oestradiol-17 beta and oestriol in plasma and urine were measured during early, mid- and late pregnancy in the marmoset monkey. In plasma, unconjugated oestrone remained less than 2% of total (conjugated plus unconjugated) oestrone throughout gestation, whereas unconjugated oestradiol-17 beta increased from 3% of the total value in early and mid-pregnancy to 35% in late pregnancy. The reversal in the unconjugated oestrone: oestradiol-17 beta concentration ratio from early (12:1) to late (0 . 15:1) pregnancy occurred despite the continuing predominance of oestrone in terms of total hormone. Total oestriol was measurable but in relatively low concentrations. Oestradiol conjugate was the predominant urinary oestrogen metabolite measured at each stage of pregnancy. The pattern of urinary oestrone and oestradiol-17 beta reflected plasma levels of total hormone, rather than unconjugated hormone, showing no further increase after mid-pregnancy. In contrast, oestriol increased throughout pregnancy and to a proportionately greater extent than oestrone or oestradiol-17 beta, but at lower absolute levels. High-pressure liquid chromatography of urine extract indicated the presence of considerable amounts of oestrogen immunoreactivity not accounted for by oestrone, oestradiol-17 beta and oestriol and with a retention time similar to that of 16 alpha-hydroxyoestrone. Gas chromatography and mass spectroscopy provided further evidence to suggest that 16 alpha-hydroxyoestrone is an abundant urinary oestrogen metabolite during pregnancy in the marmoset monkey.

The separations achieved when mixtures of both free and conjugated oestrogens from a variety of sources are chromatographed on columns of Sephadex gel are reviewed. The molecular identities of oestrogen conjugates which have been separated from human urine by these methods are listed in Table 1. Tables 2 and 3 contain the key experimental details for a total of 26 separations of oestrogen mixtures, abstracted from the total of 20 papers which were published during the period 1961-82. Table 4 details corresponding experimental data for the separation of free oestriol (in human blood) achieved by methods involving a combined Sephadex gel and immunochemical procedure abstracted from a further two papers. A careful analysis of the separation data given in the tables leads to the initial conclusion that the elution profile depends on the expected chromatographic variables for gel filtration chromatography, namely, type of Sephadex gel, length of column, nature and amount of sample applied and the sensitivity of detection methods. However, the separation achieved by the Sephadex columns is also shown to be critically dependent on the column temperature and the pH and chemical composition of the eluent and wash solvents. These latter effects, together with the realization that the molecular weights of the oestrogens being separated are very similar, leads to the conclusion that the separations summarized in Tables 2 and 3 are all being achieved by an absorption process. This being the case, it is suggested that the time-consuming methods of gel filtration chromatography need not be used. Confirmation of this proposal is afforded by a discussion of a recent paper in which the rapid separation of the oestrogens from other components in the biological matrix (urine) was achieved by an adsorption procedure. It is suggested that in the future, separations of oestrogens in biological materials may be most rapidly achieved in combining this type of adsorption procedure with HPLC.

Although oestriol measurements are well established for the assessment of 'at risk' pregnancies, there are a number of other oestrogens, excreted during pregnancy, which contain additional hydroxyl groups and might be more sensitive indicators of the condition of mother or fetus. Some of these result from the action of hydroxylases possibly present only in the fetus and others from maternal hydroxylations. We review the evidence for the biosynthesis of these polar oestrogens, summarise methods of measurement, and compare values obtained in normal and pathological pregnancies. There is as yet insufficient evidence to enable their potential value to be confirmed.

An automated assay suitable for estimating urinary oestrogens in pregnant women has been investigated. Fluorimetry was found to have considerable advantages over colorimetry. The fluorimetric assay was simpler, more precise, more sensitive, and eliminated the need for correction for non-specific chromogens; in the assay of oestriol in pregnant women there was no need for correction for non-specific fluorescence. Spectrofluorimetric and photometric analyses, recoveries, and reproducibility show that the method offers a robust means of providing values for urinary oestrogen in pregnant women on a scale of up to 100 tests a day, the time of the assay being one and a half hours.