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Summary. Amniotic fluid concentrations of immunoreactive oestrogens and progesterone were measured at the time of caesarean section in 32 twin pregnancies; 25 women had an elective section and seven were in labour at the time of operation. No significant differences between concentrations in the amniotic fluid of the first and second twin were found in respect of conjugated and unconjugated oestrone, oestradiol, oestriol, oestetrol and unconjugated progesterone either before or during labour. It is unlikely that changes in oestrogens or progesterone in the amniotic fluid are responsible for the selective changes seen in prostaglandins and fetal adrenal steroid during labour in the first twin.

The binding affinities for the catecholestrogen metabolites of estradiol and of their methyl ethers for the rat uterine cytosol estrogen receptors were examined. Similarly the binding of the fetal estradiol metabolite, 15α-hydroxyestriol (estetrol) was also measured. All of the catecholestrogens showed binding affinities far in excess of their uterotrophic potency. This is different from estriol, the product of the alternative metabolic pathways and suggests that the direction of estradiol metabolism may have an important role in the modulation of estrogenic activity of the female sex hormone.

Several radioactive estrogens possessing one, two and three hydroxyl groups were injected orally (and in the case of estrone sulfate also intraperitoneally) into adult male rats. The rats were either intact or had ligated or cannulated bile ducts. Two unconjugated estrogen tetrols together represented 21 - 87% of the total metabolites in the intact rat. One of the tetrols was 2-hydroxyestriol (estra-1,3,5(10)-triene-2,3,16alpha,17beta-tetrol); the other may be estra-1,3,5(10)-triene-2,3,6xi,17beta-tetrol but this was not confirmed. It is concluded that poly-hydroxylated estrogens represent a very large proportion of the previously unidentified water-soluble metabolites of the estrogens in the adult male rat.

As estetrol (E4) is believed to be the steroid most likely to wholly dependent on fetal origin, we developed a radiommunoassay for unconjugated and conjugated E4 (E4-U and E4-G) and investigated plasma and urinary levels serially throughout the second half of pregnancy to establish their validity by means of monitoring or screening tests to assess fetal well-being. E4 exhibits a remarkable increase during the latter half of pregnancy. At term, the mean E4-U level in maternal peripheral plasma was 0.67 +/- 0.33 ng/ml, a five fold increase from that at 28 weeks; E4-G was 4.57 +/- 2.84 ng/ml, showing a four fold increase; and E4-G levels in maternal urine were 1.68 +/- 0.96 mg/day, showing a three fold increase from that at 28 weeks. E4-U and E4-G levels showed no diurnal change. The coefficient of the correlation between plasma E4-U and E4-G was 0.699, which is satisfactory, but no correlation was found between urinary and plasma E4 levels. A significant correlation was shown between maternal and umbilical E4-U (r=0.820) and E4-G (r=0.608). No relationships between E4 levels and birth weight were detected. Pre-eclampsia, Rh-isoimmunization and diabetes mellitus are common complications of pregnancy which may cause latent fetal distress. Prenatal fetal assessment was performed by serial daily evaluations of these E4 values. In pre-eclampsia resulting in a small full term baby, E4 levels were mostly below normal mean values or failed to show an increased pattern. In addition, the E4 levels decreased in one case of neonatal death. In Rh-isoimmunization, plasma E4-G levels were lower in the group affected severely by the hemolytic desease. In a patient with diabetes mellitus delivered of a healthy baby, E4 levels were within the range of a normal pregnancy. In order to evaluate fetal and placental reserve capacities as well as feto-placental function, the dehydroepiandrosterone sulfate (DHA-S) loading test was performed by loading selected subjects with 50 mg of DHA-S, then serially measuring the E4 in the maternal plasma and urine. Intravenous infusion of 50 mg of DHA-S was completed in 60 minutes. A rapid and sharp increase of plasma E4 was observed, reaching maximal concentrations at 120 minutes in normal pregnancies. However, urinary levels showed patterns similar to those reported for estriol (E3). In some abnormal pregnancies, no increased or delayed patterns were observed in plasma E4-G levels, while the serial levels remained within the normal range. This possibly suggests that in these pregnancies, fetal functions had been inhibited or had reached their limit. It is concluded that the simultaneous determinations of serial E4 levels accompanied by the DHA-S loading test may be of value in assessing fetal well-being and reserve capacity and may therefore improve fetal and neonatal prognosis in abnormal pregnancies.

Summary Serial measurements were made of the concentration of plasma unconjugated oestetrol in normal human pregnancy. The mean curve and limits of normal variation have been established. Plasma levels rose from just below 2 ng/ml at 20 weeks to approximately 5 ng/ml at term.

Four possible monoglucuronides of estetrol (estra-1,3,5(10)-triene-3,15 alpha, 16 alpha, 17 beta-tetraol) have been prepared from appropriately protected estetrol by the Koenigs-Knorr reaction employing cadmium carbonate as a catalyst. Condensation of methyl acetobromoglucuronate with estetrol 15,16,17-triacetate provided the 3-glucuronide acetate-methyl ester in a satisfactory yield. Introduction of the glucuronyl residue into C-17 was similarly attained by the use of estetrol 3-benzoate 15,16-acetonide. When estetrol 3,17-diacetate and acetobromosugar were stirred in anhydrous toluene in the presence of cadmium salt, the reaction occurred at C-16 and C-15 yielding two isomeric monoglucuronide derivatives in a ratio of ca. 5 to 2. Removal of the protecting groups in the four glucuronide acetate-methyl esters gave the desired estetrol glucuronides, respectively. These synthetic substrates underwent readily enzymatic hydrolysis with beef-liver beta-glucuronidase to afford estetrol.

A radioimmunoassay (RIA) for the measurement of both unconjugated and total serum estetrol has been developed, using an antiserum to an E4-3-conjugate and a 125I-radioiodinated E

The estrogenic effects on this immature rat uterus of estetrol 11,3 ,5( 10)-estratriene-3 ,1 So, 16o, 1 713-tetrol] (E4), a major metabolite of placental estrogens in the human fetus, and of tamoxifen (TAM), an antiestrogen, were compared with those of estradiol (E2) and estriol (E3). The compounds were injected s.c. daily, for 3 days, into 22- or 23-day-old Sprague-Dawley rats at a dose of 50 �g/100 g BW of E4 or TAM, and 1 pg/100 g BW of E2 or E3. Tissue was collected 24 to 27 h after the third injection. Both E4 and TAM increased uterine cytosol progesterone receptor (PR) content and PR/DNA. Progesterone receptor levels per mg DNA were 1.7, 2.8 and 5.1 pmol in the control, E4 and TAM groups, respectively. Both compounds showed estrogenic activity in raising uterine wet weight and content of total and soluble (cytosol) protein. Tamoxifen augmented DNA content but the effect of E4 on this parameter was statistically significant in only one of two series of experiments. Specific induction of soluble protein, assessed by the ratio cytosoh/total protein, was noted after E2 or E3 injection, but not after treatment with E4 or TAM. Evaluation of morphologic changes in luminal and glandular epithehium by light and electron microscopy indicated enhanced synthetic and secretory activity after administration of all four compounds. Estradiol and TAM changed the shape of luminal epithelial cells from cuboidal to tall columnar, developed prominent Golgi systems and rough endoplasmic reticulum, and promoted the formation of secretory granules. Similar but less pronounced ultrastructural changes were observed after E3 or E4 treatment: the height of the luminal columnar epithehium was lower, the microvilli were shorter and the secretory granules were absent. The ultrastructural changes in glandular epithelium paralleled those found in the luminal epithehium. Cell degeneration was observed in luminal and glandular epithelium after E3 or E4 treatment and, to a small degree, after TAM treatment. Estradiol produced this effect in glandular but not in luminal epithelium. The phenolic steroids, but not TAM, increased the number of uterine eosinophils significantly. TAM raised the endometrial/myometrial ratio of cross-sectional areas significantly, whereas E

3H-15α-Hydroxyestrone-3-sulfate-N-acetylglucosaminide (15α-OHE1 SGNAc) was injected via the T-tube into the duodenum of 2 subjects (JG and MC) with biliary drainage, and biliary and urinary metabolites were determined. Within 48 hr 33% (JG) and 20% (MC) of the injected dose was found in the urine, while 36% (JG) and 56% (MC) was excreted in the bile. Separation of metabolites according to conjugate type was effected by chromatography on alumina and Sephadex G-10. Individual conjugates were purified by Celite partition chromatography. Each metabolite was assayed by treatment with appropriate enzymes and subsequent purification of the estrogen by Celite chromatography and isotope dilution techniques. About 30% of the urinary and 75% of the biliary radioactivity was identified. About 65% (JG) and 62% (MC) of the biliary tritium consisted of sulfo-N-acetyl-glucosaminides (SGNAc) of 15α-hydroxyestrone (15α-OHE1) and 15α-hydroxyestradiol (15α-OHE2). An additional 2% (JG) and 12% (MC) consisted of N-ace...

To elucidate the main metabolic pathways which lead to the foeto-placental biosynthesis of estetrol (I), we investigated the 15 alpha- and 16 alpha-hydroxylations of potential precursors of this estrogen in the human fetal liver. We determined the 15 alpha- and 16 alpha-hydroxylation capacity of the fetal liver for each precursor by GC-MS. The results suggest that estetrol is derived only from estradiol sulfate (II) and DHEA sulfate (III). 15 alpha-Hydroxy-androstenedione (IV) can no longer be regarded as a good precursor of estetrol. The phenolic pathway appears to be a more likely route than the neutral pathway, even when derived from DHEA sulfate.

The uterotrophic activities of the catechol metabolites of estradiol 2-hydroxyestrone, 2-methoxyestrone and 2-hydroxyestradiol were measured under conditions of continuous administration of sc implanted paraffin pellets. The activity of these estrogens was compared to that of estradiol-17beta and its other principal metabolites estrone, estriol and 15alpha-hydroxyestriol (estetrol). The major catechol estrogens, 2-hydroxyestrone and 2-methoxyestrone, and the pregnancy metabolite, 15alpha-hydroxyestriol, exhibited no uterotrophic activity. The minor catecholestrogen, 2-hydroxyestradiol, showed some activity whose character was different from that exhibited by implants of estradiol, estrone and estriol all of which were equipotent uterotrophic agents. Implants of 2-hydroxyestrone in the presence of estradiol or estriol pellets did not diminish the response to the latter indicating that the 2-hydroxyestrone is not antiestrogenic under these conditions. It is concluded that the direction of estradiol metabolism can have a profound influence on the expression of peripheral hormonal activity with hydroxylation at C-2 terminating and hydroxylation at C-16 extending it.

Gas chromatographic-mass spectrometric analysis of steroid extracts of pregnancy urine resulted in the identification of four estetrols. Three of these compounds were epimeric 15-hydroxyestriois, the major compound of the group being 15α-hydroxyestriol. The fourth estetrol was characterized as 18-hydroxyestriol, a compound not previously identified in pregnancy urine. The stereochemical configuration of the hydroxyl groups of these steroids was not determined.

A more specific antisera against 15α-hydroxyestriol (estetrol) was produced in rabbits by immunization with 6-oxoestetrol-6-carboxymethyloximino-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 intraasaay 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.

Simultaneous determinations of unconjugated estradiol (E2), estriol (E3) and 15 alpha-hydroxyestriol (E4) levels in maternal serum were studied serially to ascertain the significance of these estrogens in the feto-placental unit. The samples of serum were collected serially from 25 normal and 44 abnormal pregnancies. In normal pregnancy, these estrogen levels increased throughout pregnancy, especially E3 and E4 nearing the term. In 15 cases of IUGR pregnancy (including 4 cases of perinatal death), E2 levels were mostly low (less than M -- S.D.), E3 was within normal limits (M +/- S.D.) or low, and E4 was either high (greater than M + S.D.) or relatively low, and normal. In 9 cases of twin pregnancy, most E2 levels were within normal limits, while E3 and E4 were remarkedly high. The results signified that E2 indicated placental function, that E3 indicated placental and fetal function, and that E4 indicated fetal function.

The synthesis of 16 alpha-3H androgens and estrogens is described. 1-(3H)-Acetic acid in the presence of zinc dust reacts with 16 alpha-bromo-17-ketosteroids to produce 16 alpha-3H-17-ketosteroids. This chemical reaction was used to prepare 16 alpha-3H-dehydroepiandrosterone (I) and 16 alpha-3H-estrone acetate (XI) from 16 alpha-bromo-dehydroepiandrosterone (X) and from 16 alpha-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16 alpha-3H-androstenedione (II) and 16 alpha-3H-estradiol-17 beta (VII). 16 alpha-3H-Estrone (VI) was obtained by the chemical hydrolysis of 16 alpha-3H-estrone acetate. The label distribution as determined by microbiological 16 alpha-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16 alpha position. These substrates can be used for measuring the 16 alpha hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).

We have investigated the variability of four hormonal parameters used for the endocrine monitoring of the third trimester of pregnancy (E3, E4, HPL, SP1). We have observed no circadian rhythm of Estriol, Estetrol, HPL, and SP1. The within-a-day and the day-to-day variations are much greater for E4 than for E3 and for HPL than for SP1. The standard deviations were great for the steroids, especially for E4 and much less for the protein hormones, especially SP1. This implies that it is necessary to have serial assays of the steroids to have any confidence in the levels, while good monitoring could be guaranteed with a smaller number of assays for HPL, and SP1.

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.

To assess placental and fetoplacental function, 50 mg of dehydroepiandrosterone sulfate was administered intravenously to 11 normal obstetric patients at 35 to 40 weeks and to 36 high-risk patients at 35 to 43 weeks of pregnancy. Plasma estradiol converted from dehydroepiandrosterone sulfate by the placenta increased in all patients after infusion, with maximal concentrations of 28 to 65 ng per milliliter 30 to 60 minutes after infusion (P less than 0.01). Plasma estetrol, produced by the fetus from estradiol, increased in all patients with maximal concentrations of 0.8 to 3.2 ng per milliliter four hours after infusion (P less than 0.01). In complicated pregnancies a subnormal rise of estradiol and estetrol was highly suggestive of fetal distress whereas a normal rise was associated with no fetal distress. The simultaneous determination of estradiol and estetrol after dehydroepiandrosterone sulfate infusion may reflect placental and fetoplacental function, and may be used as an adjunct to other methods of assessing fetal well-being.

The effects of intravenous ritodrine upon plasma levels of free estradiol, estriol, estetrol, progesterone and human placental lactogen were investigated in ten third trimester pregnancies. Ritodrine

Estetrol (1,3,5(10)-estratrien-3,15α,16α,17β-tetrol; 15α-hydroxyestriol; E 4 ) is derived from both neutral and phenolic precursors which have undergone 15α-hydroxylation in the fetal liver or adrenal. Radioimmunoassays of total estrogens (TE), unconjugated estriol (E 3 ), and unconjugated estetrol (E 4 ) have been utilized to measure the concentrations of these estrogens in 46 amniotic fluid samples from 27 high-risk patients. Thirteen normal patients, 5 diabetic patients, and 9 patients with pregnancy complicated by Rh sensitization were studied. The concentration of TE in amniotic fluid in the uncomplicated pregnancies ranged from 11 to 65 μg per 100 ml. In most cases, the concentrations of E 3 and E 4 were similar, ranging from 0.5 to 2 per cent of the TE, as is expressed by a narrow range of E 3 :E 4 ratios extending from 0.8 to 4.5, with a mean of about 3.0. TE, E 3 , and E 4 concentrations in amniotic fluid obtained from pregnancies complicated by diabetes mellitus or Rh sensitization did not differ significantly from concentrations found in uncomplicated pregnancies of the same period of gestation. TE concentrations in 33 third-trimester serum samples from 6 patients ranged from 2 to 17 μg per 100 ml. In maternal serum, the concentration of unconjugated E, is usually much higher than the concentration of unconjugated E 3 , with an E 3 :E 4 ratio of approximately 9. TE concentration in amniotic fluid is similar to or higher than TE concentration in maternal serum; E 4 concentration is also higher in amniotic fluid; E 3 concentration is lower in amniotic fluid. There is little indication from this study that the concentration of unconjugated E 4 in amniotic fluid is of predictive value for the diagnosis of fetal compromise.

The conjugation and metabolism of 15α-hydroxyestrogens was studied in vivo in pregnant and nonpregnant subjects, with particular emphasis on the role of the kidney in the conjugation of these steroids. Labeled 15α-hydroxyestradiol (15α-OHE2) was administered iv to 2 pregnant women and 1 nonpregnant woman and estetrol (E4) to a pregnant woman. All pregnant patients were in the third trimester. More than 70% of the administered radioactivity was recovered in the urine in 3–5 days. The major conjugates of 15α-OHE2 were the N-acetyglucosaminides (NAG) and the sulfo-NAG (S-NAG), with glucosiduronates (GL) constituting a minor percentage of the conjugates. The major metabolite of 15α-OHE2 identified in our studies was E4, with only a small amount of 15α-OHE1 being present in one case only. The administered E4 was excreted preponderantly (ca. 82%) as the monoglucosiduronate of ring-D with small amounts of the S-GL (8–6%) and 3-sulfate (7.4%) being present in the urine. The E4 was excreted unmodified. Fu...

The effects of oestradiol (Oe2), oestrone (Oe1), oestriol (Oe3), oestetrol (Oe4) on the induction of the progesterone receptor (PgR) and growth of MCF-7 cells are compared. All the four oestrogens increase cell PgR concentration. Analysis of the dose-response curves shows induction by Oe2 to be 10 times and 50 times greater than Oe3 and Oe4, respectively. Oe1 and Oe2 are equally effective, even with consideration of metabolic conversion of Oe1 into Oe2. When compared with untreated cells, Oe2, Oe3 and Oe4 do not influence significantly the plating efficiency but all 3 hormones increase thymidine incorporation of cells in log phase growth. Oe2, Oe3 and Oe4 are able to rescue the growth inhibition induced by antioestrogens. The respective potency compared to Oe2 is again in the range of 10 and 50 times lower for Oe3 and Oe4, respectively. On the other hand Oe1 decreases plating efficiency, thymidine incorporation and does not rescue the growth inhibition induced by antioestrogens when the metabolic conversion of Oe1 into Oe2 is prevented. Thus, Oe3 and Oe4 behave like complete Oe2 agonists whereas Oe1 has dissociated effects, agonist on PgR induction and antagonist on cell growth.

A quantitative gas Chromatographic method for the simultaneous determination of 15α-hydroxy-estriol (1,3,5(10)-estratriene-3,15α,16α,17β-tetrol = estetrol) and estriol in pregnancy urine is described. Its specificity was investigated by various Chromatographic methods and by comparing the mass-spectra of the steroids determined with those of authentic reference standards. The accuracy and precision of the method were studied. Preliminary results obtained with 85 normal urine samples at various stages of pregnancy are presented.

The daily urinary excretion of 15α-hydroxyestriol (estetrol) and estriol was determined by a gas-liquid-chromatography method in 130 urine samples collected from 43 healthy pregnant women. The earliest sample was from the 9th week of pregnancy. The results of the estriol determination by this GLC method agree with those published before, the mean excretion of estriol being about 30 mg in 24 h towards the end of pregnancy. The mean amount of estetrol excreted in normal pregnancy urine increased from 0.28 mg in the 20th week to 2.09 mg in the 40th week of pregnancy. Some differences were observed in the rates of increase of estriol and estetrol excretion. Possible reasons for these differences are discussed. There was no significant difference in the variability of consecutive 24-h excretion of estetrol and estriol. If the collection time is shortened, the variability of estetrol excretion increases like that of estriol. The amount of estetrol excreted in maternal urine before partus is correlated with the weights of the fetus and placenta, like that of estriol.

Following the intravenous administration of labeled C19-steroids to subjects in the third trimester of pregnancy, labeled estetrol2 was isolated from the glucosiduronate fraction of the urine. In order to determine the precursor role of neutral steroids in the formation of estetrol and 15a-hydroxyestradiol, the following pairs of substrates were injected into subjects in the third trimester of pregnancy; i) (7-3H)-16α-hydroxydehydroisoandrosterone sulfate and (4-14C)-dehydroisoandrosterone sulfate; ii) (7-3H)-16α-hydroxydehydroisoandrosterone and (4-14C)-dehydroisoandrosterone sulfate; iii) (7-3H)-dehydroisoandrosterone sulfate and (4-14C)-15α-hydroxyandrostenedione; iv) (7-3H)-15α-hydroxyandrostenedione and (4-14C)-16αhydroxyandrostenedione. Urine was collected for 5–6 days and both estrogens isolated. After purification, the estetrol was identified by infrared analysis and melting point, and its radiochemical purity established by crystallization with carrier estetrol and subsequent derivative ...

15α-Hydroxyestriol-6,7-3H was administered to three women in the third trimester of pregnancy. About 70% of the administered dose was excreted rapidly in urine, virtually all as 15α-hydroxyestriol glucuronide. The SA of the excreted material was determined by double isotope derivative procedure, and permitted the calculation of excretion and production rates. The concentration of 15α-hydroxyestriol was also measured in four other pregnancy urines by means of double isotope derivative procedures. All excretion values of the estetrol obtained were lower than would have been expected from those reported in the literature. The excretion represented varying fractions of the production rates, raising doubts about the representative nature of urinary assays of l5α-hydroxyestriol in pregnancy.

15α-Hydroxyestriol was coupled to bovine serum albumin via the 3-O-carboxymethyl ether derivative. This antigen was used to produce an anti 15α-hydroxyestriol antiserum in rabbits which was highly specific and sensitive. Radioimmunoassay of this fetal metabolite in third trimester maternal plasma gave concentrations ranging from 1.0–4 ng/ml but none could be demonstrated in male or in non-pregnant female plasma. The free estetrol is therefore a quantitatively important component of pregnancy plasma estrogens, and its radioimmunoassay offers promise as an index of fetal viability.

A radioimmunoassay is described for the measurement of unconjugated estriol (E3) and estetrol (E4) in pregnancy serum. Specimens were obtained from about 70 women at 20–42 weeks of gestation. During the last half of pregnancy the concentration of E4 was found to increase. The mean serum concentration of unconjugated E4 during the 40th week was 2.2 ng/ml. The E3/E4 ratio was about 8 and remained nearly constant throughout pregnancy, although the increase of E4 was relatively greater than the increase of e3 during the last few weeks of gestation.

A gas-liquid chromatographic method allowing the simultaneous quantitation of urinary estetrol, estriol, estradiol, estrone, and pregnanediol in urine throughout the duration of pregnancy is presented. The method consists of an enzymatic hydrolysis, ether extraction, chromatography on Sephadex LH-20 and AGI-X2, and final quantitation of the steroids as heptafluorobutyric anhydride derivative in a gas chromatograph. Ten samples may be assayed for estetrol in one working day. The method was applied to 23 urine specimens of 13 different patients with molar pregnancies prior to evacuation and to 21 urine specimens from women with nonmolar pregnancies as controls. In 77 per cent of the molar pregnancies, no estetrol was detected, and, in the remaining 23 per cent, it was present in a much lower concentration than expected for nonmolar pregnancies. The ratio of estriol to that of estradiol plus estrone was generally lower in the molar than in the nonmolar pregnancies. No discernible pattern for pregnanediol in molar pregnancies was established. It is concluded that: (1) estetrol in some cases of molar pregnancy is synthesized to a small degree by either placental or maternal tissue and the fetal liver is not solely responsible for 15α-hydroxylation of estetrol precursors, and (2) determinations of estetrol are useful in the diagnosis of molar pregnancy.

Urinary excretion of estriol and 15α-hydroxyestriol (estetrol) was determined in 89 women whose pregnancies were complicated by toxemia, diabetes mellitus, Rh isoimmunization, intrauterine fetal death, or recurrent intrahepatic cholestasis of pregnancy. More than 300 urine samples were analyzed by a highly specific gas chromatographic method and the results were compared with values during normal pregnancies. The 2 estrogens usually gave the same correct information on the fetal state. In some cases of toxemia, the amount of estetrol was more reliable for monitoring the intrauterine fetal condition than that of estriol. In pregnancies complicated by Rh isoimmunization, the urinary excretion of estetrol bore a linear correlation to the cord blood hemoglobin concentration. The amount of estriol in the same maternal urine samples showed no correlation with the cord hemoglobin.

15α-Hydroxyestradiol (15α-OHE2) was given iv to two pregnant women at 31 and 33 weeks gestation and urine was collected for 6 days. Excretion was almost complete during this period (93% and 96%). Separation of metabolites according to conjugate types was effected by chromatography on alumina and Sephadex G-10 and in one instance, DEAE-Sephadex A-25. Individual conjugates were purified by Celite partition chromatography. Each metabolite was assayed by treatment with an appropriate enzyme and subsequent purification of the aglycone, usually by Celite chromatography and isotope dilution techniques. About 70% of the radioactivity was identified. The predominant conjugates were the 15-N-acetylglucosaminides (35%) and 3-sulfate-15-N-acetylglucosaminides (21%) of 15α-hydroxyestrone (15α-OHE1 and 15α-OHE2. No other N-acetylaminoglucosides were detected. The major glucosiduronates (14%) were those of 15α-OHE1, 15α-OHE2, estetrol (E4) and of a metabolite hitherto not reported in the human, 15α-OHE2-17α. Th...

The preparation of the tritium labelled fetal metabolite estra-1, 3, 5 (10)-3, 15α, 16α, 17β-tetrol 6,7-3H is described. The material was prepared by oxidation of the tetrol tetraacetate lb to the 6-keto derivative II. Reduction of the 6-ketone to the 6-hydroxy compound III and dehydration with dimethylsulfoxide gave the Δ6 derivative III. Reduction of III with tritium followed by hydrolysis gave the tetrol-3H with a specific activity of 43 curies/mmole.

The urinary metabolites of intramuscularly administered 17β-oestradiol-4-14C were studied in three infants born with multiple malformations. Of the radioactive material recovered from the urine less than 5 per cent was in an ether-soluble (»free«) form. Following enzyme hydrolysis, an average of 46 per cent became ether-extractable. Of the remaining material all but some 2 per cent was butanol-soluble. The pattern of urinary metabolites was similar in all subjects. Oestrone, 17β-oestradiol and possibly 2-methoxyoestrone accounted for less than 5 per cent of the total urinary radioactive material. Oestriol, 16α-hydroxyoestrone and 16-oxo-17β-oestradiol comprised an important group of metabolites, representing together some 20 per cent of the total. However, the quantitatively most important metabolite was a hydroxylation product somewhat more polar than 2-hydroxyoestriol. This compound accounted for 16 per cent of the total. It is concluded that the metabolism of 17β-oestradiol in early infancy is characterized by extensive hydroxylation reactions. This pattern of metabolism may indicate one of the mechanisms by which the foetus is protected against steroids with high biological activity.

In order to determine the precursor role of neutral steroids in the formation of estetrol in man the titled compound was prepared as a substrate by two different routes. First, 17, 17-ethylenedioxy-5α-androst-15-ene-3β, 5-diol (I), readily available by the known method, was employed as a starting material. Transformation into the 15α, 16α-glycol structure was attained by osmium tetroxide oxidation toward the Δ15 double bond. Dehydration with thionyl chloride provided a mixture of the Δ4 and Δ5-3β, 15α, 16α, 17β-tetraols (V), which was in turn led to the 15, 16-acetonide (VI). Oppenaner oxidation followed by removal of the dioxolane group afforded the Δ4-3-ketosteroid (VIIIa). Alternatively, 3β, 5β-dihydroxyandrost-15-en-17-one ethylene ketal (XIV), derivable from dehydroepiandrosterone in several steps, was similarly converted into androst-4-ene-3β, 15α, 16α, 17β-tetraol (XXa). Selective oxidation of the hydroxylic group at C-3 with N-bromoacetamide provided the desired compound (VIIIa). The latter synthetic route has proved to be more favorable in respect to overall yield.

5hydroxyoestriol) offers a possible solution to this problem, as its production is almost entirely dependent upon a functioning foetal liver [2] and initial investigations have indeed indicated that the measurement of this steroid may provide a useful index of foetal viability [3,4]. The antisera used in these earlier investigations were raised against an antigen in which the steroid was linked through the three-position and showed reasonable specificity for the D ring; the cross-reaction for oestriol being 2.5%

Simultaneous determinations of unconjugated estradiol (E2), estriol (E3) and 15 alpha-hydroxyestriol (E4) levels in maternal serum were studied serially to ascertain the significance of these estrogens in the feto-placental unit. The samples of serum were collected serially from 25 normal and 44 abnormal pregnancies. In normal pregnancy, these estrogen levels increased throughout pregnancy, especially E3 and E4 nearing the term. In 15 cases of IUGR pregnancy (including 4 cases of perinatal death), E2 levels were mostly low (less than M -- S.D.), E3 was within normal limits (M +/- S.D.) or low, and E4 was either high (greater than M + S.D.) or relatively low, and normal. In 9 cases of twin pregnancy, most E2 levels were within normal limits, while E3 and E4 were remarkedly high. The results signified that E2 indicated placental function, that E3 indicated placental and fetal function, and that E4 indicated fetal function.

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.

Based on a review of the literature, this paper clarifies the pharmacologic properties of shortacting estrogens and their role in physiology and medicine. Shortacting estrogens display mixed agonist-antagonistic properties when injected in saline. The mixed estrogenic function results from the rapid clearance of these compounds from target tissue. When administered by pellet implant, however, shortacting estrogens act as full agonists. Both the uterotropic and vaginotropic response patterns of these compounds are detailed. Shortacting estrogens stimulate early uterotropic responses while having little effect on true uterine growth when administered by injection in saline. Thus, they have no antagonistic action when examined by shortterm uterotropic assays, but display partial antagonism when longterm uterine growth assays are used. Previous research has suggested that shortacting estrogens would not be effectual or antagonistic if present in a continuous fashion which would result in constant or longterm occupancy of the estrogen receptor. Estradiol, however, does manifest these properties when injected. Shortacting estrogens do not act as antagonists on vaginotropic responses as they do uterotropic responses. The paper also reviews the functions of these compounds in various physiological states, including blood binding, metabolism, menstruation, and pregnancy. Finally, clinical considerations are discussed. Estriol has an apparent selective effect on vaginotropic events. It has been effective in correcting symptoms of menopause, for example. However, estriol is not believed to have a protective effect against breast cancer. When it is present in a continuous fashion, estriol acts as an estrogen, thereby ruling out such an effect.

A pregnancy with placental sulfatase deficiency was suspected when a 36-year-old patient at 41 weeks of gestation was found to have extremely low urinary estriol excretion and an otherwise normal prenatal course. The maternal plasma levels of estriol and estradiol 17-beta (E2) were extremely low and estetrol (E4) was undetectable (less than 40 pg/ml), whereas dehydroepiandrosterone sulfate (DS) was normal. THE AMNIOTIC Fluid DS concentration was 22-fold higher than the mean of normal pregnancy, while that of dehydroepiandrosterone (D) and androstenedione (A) was normal. Following intravenous infusion of 50 mg DS, no rise of plasma E2 was noted and plasma E4 levels remained undetectable. At 42 weeks of pregnancy, after induction of labor failed, a healthy male infant was delivered by cesarean section. The umbilical vein (UV) and umbilical artery (UA) levels of DS were extremely high, and those of E2 and E4 were subnormal. The UA level of A was normal and the levels of D and testosterone were slightly elevated. In vitro studies of placental microsomes and the 10,000 x g supernantant confirmed the diagnosis of placental sulfatase deficiency. The infant at 6 months of age had normal growth and development and normal peripheral plasma DS concentration.

Although several studies have been reported on the synthesis of steroids in the fetus, the metabolism and conjugation of estrogens in the fetus is not completely known. In this study serum levels of estetrol (E4), estetrol glucosiduronate (E4-G), estriol (E3) and estriol-16-glucosiduronate (E3-16-G) were measured by R.I.A. in maternal peripheral vein blood (MPV), umbilical artery blood (UA) and umbilical vein blood (UV) at delivery. The same steroids were also measured in various fetal organs (liver, kidney, intestine, adrenal, lung and placenta) in the second-trimester. After 4-14C-E2, 4-14C-E3 and 6, 9 (n)-3H-E4 were incubated with homogenates of various human fetal organs, the metabolites were analyzed with an authentic sample. 15, 15-D2-estradiol-17 beta (d2-E2) was incubated with human second-trimester fetal liver and the metabolites were analyzed with GC-MS. It was concluded that: 1) E3-16-G was higher in UA and UV than in MPV, suggesting active glucosiduronation in the fetus. 2) Higher levels of E4 and E4-G in UA and UV than in MPV suggest the production of E3 and E3-16-G in the fetus. 3) Levels of E4 and E4-G were higher than those of E3 and E3-16-G in various fetal organs in the second-trimester. 4) E4 and E4-G were especially high in fetal liver and intestine, and E3 and E3-16-G were high in fetal kidney, liver and intestine. 5) The level of E3-16-G was higher than that of E3 in these organs. 6) The second-trimester fetal liver conjugated E2, E3 and E4 to each glucosiduronate. 7) The second-trimester fetal kidney conjugated E2 and E4 to each sulfate, but E3 to E3-16-G. 8) 15-Hydroxylation of d2-E2 was demonstrated in the incubation with homogenate of second human fetal liver. 9) The active glucosiduronation of E3 in the fetus was thought to inactivate a large amount of E3 transported from the placenta.

A radioimmunossay (RIA) for the measurement of both unconjugated and total serum estetrol has been developed, using an antiserum to an E4-3-conjugate and a 125I-radioiodinated E4 tracer. Assay of dried ethyl ether extracts was used for the determination of unconjugated E4, while a direct measurement of unextracted hydrolyzed serum in the presence of 0.3% 8-anilino-1-naphthalene sulphonic acid (ANS) proved adequate for total E4. Assay reliability was evaluated and the procedure standardized through a series of tests aimed at assessing accuracy, sensitivity and precision. No steroidal interference was found to practically affect the assay (0.3% estriol cross-reactivity), nor were solvent and sample blanks observed in the case of unconjugated E4. For total E4 assay, the sample blank effects were acceptably overcome by using hydrolyzed male serum and 0.3% ANS, as a standard diluent. An interassay variability amounting to approximately 10 and 6% resulted for unconjugated E4 and total E4 RIA, respectively. A number of serum samples (285 for unconjugated E4, 147 for total E4) randomly collected throughout normal pregnancy were assayed. The unconjugated E4 levels at 15th week and at term were 62.7 +/- 22.6 and 766.5 +/- 208.2 (SD) pg/ml, respectively. Total E4 was about 6--7 times higher than the levels of free E4 and increased 7 times from the 15th week to term.

To assess the significance of measurement of plasma estetrol after dehydro-epiandrosterone sulfate (DHAS), 100 mg of DHAS was injected 30–60 minutes (7 cases), 120 minutes (6 cases), 180 - 240 minutes (5 cases) before delivery into 18 normal obstetric patients (37–39w) undergoing elective repeat cesarean section. At delivery, maternal peripheral vein (MV), umbilical vein (UV), and umbilical artery (UA) blood and amniotic fluid (AF) were obtained, and unconjugated estetrol in each compartment was measured. A significant increase of plasma estetrol concentration was observed in MV, UV, and UA, with the maximal levels at 180–240 minutes, but not in AF. The increase of plasma estetrol concentrations were higher in UV and UA than in MV. However, no significant difference in the plasma estetrol concentration was seen between UV and UA after DHAS injection. A significant correlation was found between individual MV and UV plasma estetrol concentrations after DHAS injection. The data indicate that determination of a maternal plasma estetrol rise after DHAS injection reflects fetoplacental function and may provide further useful information to evaluate fetal well being.

Intravenous injections of 50 mg dehydroepiandrosterone sulfate (DHEAS) were given to 7 women with normal pregnancies and 5 with pathologic pregnancies and the serum levels of 17 beta-estradiol and estetrol were assayed before and at 15 or 30-minute intervals for three hours after the injection. All tests were carried out during the 25th to 36th week of amenorrhea. Serum estradiol rose rapidly in normal subjects and remained high to the end of the test. In patients with gestational pathology the estradiol pattern was not significantly different from that of the controls. Esterol plasma levels showed a biphasic pattern with an initial rise at 30 min. and a second rise at 90 min. in normal pregnancies, whereas in pathologic pregnancies this response was either lacking completely or was markedly reduced compared to the controls.

A simultaneous microdetermination of unconjugated estrone, estradiol, estriol and estetrol in serum or amniotic fluid by High Performance Liquid Chromatography with an Amperometric Detector is described. Steroids in serum or amniotic fluid were extracted with 10 volumes of ethyl ether, and then ether extract was evaporated to dryness under N2 gas. After defatting with a mixture of 50% methanol/n-hexane, the methanol phase was evaporated to dryness under N2 gas. The residue was applied to microcolumn packed with 2 ml volume of Sephadex LH-20 in the eluting solvent benzene/methanol (85:15). Fractions contained estrone and estradiol; estriol and estetrol were collected and then evaporated to dryness under N2 gas. The sample solution was applied to HPLC using a reverse phase ODS column and acetonitrile: 0.1M KH2 PO4 47:53 for estrone and estradiol fraction, and 30:70 for estriol and estetrol fraction as a mobile phase, respectively. The fraction of each estrogen was separated completely within a 20 minute period. The limit of detection of estrone, estradiol, estriol and estetrol was 50 pg, respectively.A simultaneous microdetermination of unconjugated estrone, estradiol, estriol, and estetrol in serum or amniotic fluid by high performance liquid chromatography (HPLC) with an Amperometric Detector is described. Steroids in serum or amniotic fluid were extracted with 10 volumes of ethylether, and then ether extract was evaporated to dryness under N2 gas. After defatting with a mixture of 50% methanol/n-hexane, the methanol phase was evaporated to dryness under N2 gas. The residue was applied to microcolumn packed with 2 ml volume of Sephadex LH-20 in the eluting solvent benzene/methanol (85:15). Fractions contained estrone and estradiol; estriol and estetrol were collected and then evaporated to dryness under N2 gas. The sample solution was applied to HPLC using a reverse phase ODS column and acetonitrile: 0.1M KH2PO4 47:53 for estrone and estradiol fraction, and 30:70 for estriol and estetrol fraction as a mobile phase, respectively. The fraction of each estrogen was separated completely within a 20 minute period. The limit of detection of estrone, estradiol, estriol, and estetrol was 50 pg, respectively. (author's)

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 bolus iv injection of 50 mg dehydroepiandrosterone sulfate (DS) followed by a constant 2-h infusion of 200 eg DS/min was administered to 5 pregnant patients at term and to 5 postpartum patients. Its effect on plasma androgen and estrogen concentrations wasdetermined by frequent plasma hormone determinations. At the end of the constant infusion plasma DS concentrations were 7-fold higher than pre-treatment levels in both postpartum and pregnant patients. The metabolic clearance rates (MCR) of the infused DS for pregnant and postpartum patients were 1.2 and 1.0 liter/kg/day, and the half-lives 146 and 179 min, respectively (P > 0.05). A 3-fold rise of plasma dehydroepiandrosterone concentrations, which was observed in pregnant as well as postpartum patients, suggested that the rise of plasma dehydroepiandrosterone was due to peripheral rather than placental metabolism of DS. The rise of plasma androstenedione and testosterone following DS infusion, which was greater in postpartum than in pregnant patients...