Pharmacokinetics of the fetal estrogen estetrol in a multiple-rising-dose study in postmenopausal women.

OBJECTIVES: Estetrol (E4) is a natural fetal estrogen. In this open-label, multiple-rising-dose study, the pharmacokinetic effects of E4 in postmenopausal women were investigated as a secondary objective. METHODS: In total, 49 postmenopausal women were randomized to receive either 2 mg E4 or 2 mg estradiol valerate (E2V) for 28 days, or were (non-randomized) assigned to 10, 20, or 40 mg E4. The main outcome measures were: E4 plasma concentrations at trough, and on days 1 and 28; and E4 pharmacokinetic parameters AUC, Cmax and tmax on days 1 and 28. RESULTS: After oral administration, E4 showed a very fast absorption, followed by a multiphasic elimination with an initial rapid decline, gradually continuing with a slower elimination, suggesting a long terminal half-life. Steady state was reached within 2 weeks of dosing and pharmacokinetic results were generally proportional to the dose. Estetrol concentrations on day 28 were slightly higher compared to day 1, indicating some accumulation. CONCLUSION: The pharmacokinetic profile of estetrol is characterized by a very fast absorption phase, followed by an initial rapid decline, and a slow terminal elimination phase. Based on its kinetic properties, estetrol seems suitable for use as a once-daily oral drug.

Plasma estetrol as an index of fetal well-being.

Estetrol (15alpha-hydroxyestriol or E4) is considered to be a specific product of fetal liver and has been suggested as a good indicator of fetal well-being. The concentration of unconjugated estetrol (E4) was measured by rapid and specific radioimmunoassay in 1 ml of maternal plasma. E4 levels prior to the 18th week of pregnancy were often undetectable (smaller than 50 pg/ml). The mean plasma E4 level at term of 1.2 ng/ml was 7-fold higher than that observed at 24 weeks of gestation, and no diurnal variations were found. E4 levels in fetal plasma at term were 12-fold higher than those in maternal plasma and no fetal arterial venous differences were found. Umbilical vein but not maternal plasma levels of patients undergoing vaginal delivery were higher than those undergoing cesarean section (P smaller than 0.05) suggesting increased adrenal output of E4 precursors during labor. In patients with severe Rh-isoimmune disease plasma E4 levels were not helpful in assessing fetal well-being. However, in patients with chronic hypertension or pre-eclampsia, subnormal plasma E4 concentrations always preceded intrauterine fetal death. Plasma E4 appears to be a good indicator of fetal well-being in patients with hypertensive disease of pregnancy.

Studies of the diurnal pattern of plasma corticosteroids and gonadotropins in two cases of feminizing adrenal carcinoma: measurements of estrogen and corticosteroid production.

Two adult men with feminizing adrenal cortical carcinoma had measurements of their 24-h plasma corticosteroid and gonadotropin patterns as well as 24-h mean hormone levels of estradiol, estetrol, 11-desoxycortisol, DHEA-S, DHEA and testosterone. Cortisol, 11-desoxycortisol and estrogen production rates were also measured. The 24-h corticosteroid patterns showed preservation of the normal 24-h episodic and circadian patterns, albeit at higher levels. The cortisol production rates were markedly elevated despite only moderate elevation of the 24-h mean cortisol level. There were elevated plasma 11-desoxycortisol levels and a markedly elevated 11-desoxycortisol production rate in one patient and THS excretion in the other. The plasma estradiol levels, urinary excretion and production rates were markedly elevated. In addition, there was a decrease in the specific activity of estriol compared with estrone and estradiol as well as measurable levels of estetrol in both patients. These latter observations coupled with the urinary immunoassayable hCG in one patient suggest that these tumors may be functioning like trophoblastic tissue. The possibility that estetrol may serve as an additional marker for tumors of trophoblastic origin is of additional interest.

Comparison of serum unconjugated estriol and estetrol in normal and complicated pregnancies.

It has been reported that determinations of maternal serum unconjugated estriol (E3) and estetrol (E4) concentrations provide clinicians with more or less identical information on fetal status. If this is true, then theoretically the levels of E3 should be equally correlated with those of E4 in all conditions of pregnancy. To resolve this question, a study of the relationship between E3 and E4 was performed before labor in normal and complicated pregnancies. In normal pregnancy, they were highly correlated (r = .683, P less than .0001); in complicated cases, they were still correlated, but at a lower level (r = .522, P less than .003). To determine the effect of labor on this correlation, E3 and E4 levels were measured in normal subjects during labor as well as in the corresponding fetuses. The correlations between material E3 and E4, maternal and fetal E4, maternal and fetal E3, and fetal E3 and E4 were highly significant. A similar study with complicated pregnancies, however, indicated no such correlation except between fetal E3 and E4. In addition, day-to-day variability of serial measurements of E3 and E4 on an individual basis was determined in normal and diabetic subjects. The variability was qualitatively determined graphically and quantitatively determined algorithmically. The results of calculated individual variabilities indicated that the variability of E4 was less than that of E3 in most cases. It is therefore concluded that complications in pregnancy and the onset of labor have some effect on E3-E4 correlations, and that measurement of E4 has an additional advantage due to less variability.

Sequential determination of serum human placental lactogen, estriol, and estetrol for assessment of fetal morbidity.

Serial measurements were made of the concentrations of maternal serum human placental lactogen (hPL) (300 determinations), estriol (E3) (460 determinations), and estetrol (E4) (275 determinations) in normal human pregnancy during the third trimester period. Simultaneous determinations of serum hPL, E3, and E4 were also made sequentially on blood samples from 6 diabetic and 5 toxemic pregnant women to ascertain the relative usefulness of these parameters as indicators of fetal welfare. In uncomplicated diabetic patients controlled with insulin, all parameters increased with gestational age. In three pregnancies complicated by severe toxemia in which fetal distress progressed to intrauterine fetal death, both serum E3 and E4 levels decreased progressively, but E4 concentration started to decrease at least 1 day earlier than E3 prior to fetal death. In other women, the E4 levels appeared to drop or decrease significantly whereas the E3 levels remained almost unchanged. Daily hPL levels remained low in chronic fetal distress and, therefore, appeared to be of minimal value for predicting either intrauterine death or acute fetal distress. Therefore, serum E4 measurement seems to provide a more sensitive and reliable indicator of fetal morbidity than the measurement of serum E3 during toxemic pregnancies.

The applications of steroid hormone radioimmunoassays to clinical obstetrics.

This is a selective survey of recent publications dealing with theoretical and established applications of steroid hormone radioimmunoassay procedures applied to clinical obstetric investigation. This subject is reviewed in three sections: first, basic principles of steroid hormone radioimmunoassay methodology; second, steroidogenesis in normal pregnancy and its relation to patterns of steroid hormones measured in maternal and fetal circulations; third, applied steroid radioimmunoassay technology as it is being used in established and potential clinical applications. It is concluded that steroid hormone radioimmunoassay procedures have been and will continue to be a highly productive technology applied to clinical obstetric investigation.

A radioimmunoassay of plasma unconjugated and conjugated estetrol.

A radioimmunoassay for the measurement of both unconjugated and conjugaged estetrol in plasma has been developed. The antiserum obtained after 6 months of immunization with 6-oxoestetrol-6-(O-carboxy-methyl)oxim-BSA was used at a final dilution of 1:90,000 and showed almost no cross reaction with other steroids except for estriol at 1.24%. Esterol-glucosiduronate was synthesized by incubating with adrenalectomized rat liver homogenate and uridine diphosphoglucuronic acid. Then, plasma estetrol-glucosiduronate was measured in the same manner for unconjugated estetrol after hydrolysis with beta-glucuronidase. Sephadex LH-20 column chromatography (7X110 mm, benzene:methanol, 85:15) was employed for accurate assessment. The sensitivity was 10 pg and the smallest amount measurable was 40 pg/sample. The method bland was consistently negligible. The intra and inter assay precision was 11.8% and 14.2% for unconjugated estetrol and that for estetrol-glucosiduronate was 13.5% and 17.1%.

Radioimmunoassay of 15alpha-hydroxyestriol (estetrol) in pregnancy serum.

A more specific antisera against 15alpha-hydroxyestriol (estetrol) was produced in rabbits by immunization with 6-oxoestetrol-6-carboxy-methyloximino-bovine serum albumin. The synthesis of estetrol tetraacetate was greatly improved. Characterization of estetrol antisera revealed that it is more specific than those formerly obtained. A simple method of extraction of estetrol with alcohol from serum with high recovery is described. A radioimmunoassay method for pregnancy serum is characterized in regard to its specificity, accuracy, sensitivity and precision. The method is able to measure as little as 50 pg/tube or 167 pg/ml of serum. The intraassay coefficient of variation is 7.8% and that for interassay including personnel variation is 14.2%. Accuracy of the method, as determined by the recovery of non-radioactive estetrol (1 ng/ml) added to non-pregnancy serum, is found to be 89 +/- 8 (SD)%, CV = 8.7%, without correction for experimental losses. One hundred twelve pregnancy sera were analysed; the values obtained from normal subjects in the third trimester ranged from 240-950 pg/ml. The values obtained by this method are somewhat lower than those reported previously, presumably due to the higher specificity of the antibody.

Rapid and simultaneous measurement of estrone, estradiol, estriol and estetrol in serum by high performance liquid chromatography with electrochemical detection.

A high performance liquid chromatographic (HPLC) method with electrochemical detection (ECD) was developed for the simultaneous measurement of estrone, estradiol, estriol and estetrol in serum. These hormones were extracted with diethylether, chromatographed on an silica-octadecyl silane (ODS) column with an eluent of phosphate buffer solution-acetonitrile-methanol (volume ratio 152:85:40), and detected by ECD at +1.0V vs. Ag/AgCl. In comparisons between the values measured by this method and radioimmunoassay, significant correlations were noted for estrone (r = 0.759, p less than 0.01), estradiol (r = 0.816, p less than 0.001) and estriol (r = 0.830, p less than 0.001). In clinical applications of this method, differences between cases of the normal and the anencephalic pregnancy in the thirty-eighth week of gestation were distinct not only in the individual estrogen, but also in the profile analysis of estrogens. With this method, all 4 serum estrogens above approximately 500 pg/ml could be measured within 2 h, and the method seemed to be clinically applicable.

Unconjugated estetrol in normal pregnancy.

The Authors studied the levels of Estetrol (15 alpha-hydroxyestriol) in the amniotic fluid, in maternal and foetal plasma, by the RIA method, in near-term pregnancies. Higher concentrations of this steroid were found in the foetal plasma and in amniotic fluid than in the maternal plasma. These data, even though of little clinical importance, confirm the foetal origin of this compound and suggest further studies, especially in the amniotic compartment.

What are the most suitable fetoplacental tests in the monitoring of the third trimester of pregnancy?

The case-series of the Institute of Obstetrics and Gynaecology were examined to evaluate the suitability of urinary estriol, total plasma estriol, unconjugated plasma estriol, unconjugated plasma estetrol, plasma placental lactogen, plasma S.P.-1 glycoprotein, plasma alphafetoprotein and biparietal diameter in correctly forecasting the perinatal risk, when performed after the 25th week of pregnancy. In high-risk pregnancies, according to our results, S.P.-1 glycoprotein and urinary estriol are the most sensitive tests, while S.P.-1 glycoprotein, placental lactogen and biparietal diameter are found to have the highest predictive value. The repetition of the considered tests increases their sensitivity, but not their predictive value. In pregnancy mass screening the most suitable tests, on the basis of the "relative risk" are S.P.-1 glycoprotein (or even placental lactogen), estriol and biparietal diameter. For the last one a single measurement seems to be enough during the third trimester.

Estetrol and utero-placental flow after progesterone load.

On the basis of recent demonstration in animals of the effect of some hormones on uteroplacental flow, the Authors examined the response of plasmatic Estetrol (15 alpha-hydroxy-estriol) after the administration of progesterone to pregnant women with low Estrogen values. The increase of this compound was related to an improvement of placental function, probably dependent on an increase of available O2, and therefore on uterine blood flow. This can justify a progesterone treatment in such pregnancies.

Daily variation of serum unconjugated estriol and estetrol in normal pregnancy.

Serum unconjugated estriol and estetrol were assayed daily in seven nondiabetic, uncomplicated third-trimester pregnancies to define the daily variation of these compounds. When compared to the mean of the three preceding days' values, or to the highest mean of three consecutive daily values previously obtained in a pregnancy, daily estriol and estetrol values fell greater than or equal to 40% on 1.2 and 0% of occasions, respectively. Isolated estriol values represented falls of greater than or equal to 40% from previously obtained single estriol values on 2% of occasions, and no isolated estetrol values fell greater than or equal to 40% from any other isolated values obtained in a given pregnancy. These results define the stability of daily serum estriol and estetrol in late-gestation normal pregnancy, although they emphasize the large variability encountered when comparing isolated estriol values.

Plasma levels of unconjugated estetrol and estriol and of total estriol in normal human pregnancy.

The course of normal pregnancy in 54 patients was monitored by weekly assays of Unconjugated Estriol (E3U), Total Estriol (E3T) and Unconjugated Estetrol (E4U). These subjects were divided into two groups: one of those patients who delivered a fetus with a weight above the 50th percentile and the other of those who delivered a fetus with a weight below the 50th percentile. No significant difference was found between these two groups and it is not therefore possible to have information regarding the weight of the fetus, starting from the weekly values of these hormones. Analogous variations of the three hormones were found as pregnancy progresses. However, the rate of increase for E4U was higher than for E3T and E3U.