Regulation of human placental 17 beta-hydroxysteroid oxidoreductase: mechanism of stimulation of 17 beta-estradiol formation from estrone by 5 alpha-dihydrotestosterone in homogenates and villi in vitro.

The mechanism of stimulation of 17 beta-estradiol (E2) formation from estrone (E1) by 5 alpha-dihydrotestosterone (5 alpha-DHT) in placental villi was investigated by examining; (1) if dehydroepiandrosterone (DHA) was stimulatory, (2) if NAD(P)H-generating, non-steroidal substrates stimulated E2 formation, (3) the subcellular localization of the effect, (4) if NAD(P) or NAD(P)H was required and (5) rates of 5 alpha-DHT oxidation by villi and microsomes. Although 5 alpha-DHT and DHA both inhibited the E2 to E1 reaction in villi and microsomes, only 5 alpha-DHT stimulated the conversion of E1 to E2. Glucose and lactate were slightly stimulatory when compared with 5 alpha-DHT. Stimulation of E2 formation was observed with microsomes but not with cytosol, and NAD or NADP was required. The results indicate that neither inhibition of the back reaction, E2 to E1, nor NADH or NADPH formation via the 3 beta-hydroxysteroid dehydrogenase/5-ene-3-ketosteroid isomerase reaction can account for the stimulation. It is proposed that the mechanism of stimulation involves one or more forms of membrane-bound 17 beta-hydroxysteroid oxidoreductase with NADH or NADPH formed as a product of 5 alpha-DHT oxidation being used as the cofactor for E1 reduction. This may involve a direct transfer of reduced pyridine nucleotide between enzyme molecules without equilibration with intracellular coenzyme pools.

Pyridine nucleotide levels under conditions of 5 alpha-dihydrotestosterone-stimulated 17 beta-estradiol formation from estrone and pathway of nicotinamide adenine dinucleotide biosynthesis in placental villi in vitro.

It has been reported that testosterone, 5 alpha-dihydrotestosterone and 20 alpha-dihydroprogesterone, substrates for pyridine nucleotide-dependent hydroxysteroid oxidoreductases, stimulate the conversion of estrone to 17 beta-estradiol (E2) by placental villi in vitro. Their enzyme-catalyzed oxidation generates either NADH or NADPH. If the concentration of either reduced nucleotide were rate determining in the conversion of estrone to E2, then increases in NADH or NADPH levels as the result of steroid oxidation could stimulate E2 formation. In this investigation, enzymatic assays were used to quantitate NAD, NADP, NADH, and NADPH in villous tissue from term placenta under conditions where E2 formation was stimulated by 5 alpha-dihydroxytestosterone. On the basis of concurrent observations that NAD levels varied initially and decreased in tissue samples in culture over a 24-h period, the ability of villi to incorporate [14C]nicotinic acid or [14C]nicotinamide into NAD was also examined. No changes were detected in the ratios of oxidized to reduced [14C]nicotinamide nucleotides under stimulatory conditions. NAD was formed only from nicotinic acid. The data suggest that NAD and NADP reduction, if it is the basis for stimulation, is tightly coupled to reoxidation. It would also appear that media used widely for villous tissue and cell culture may not be optimal for pyridine nucleotide synthesis.

Activation of human placental 5-pregnene-3,20-dione isomerase activity by pyridine nucleotides.

Isomerization of 5-pregnene-3,20-dione to progesterone by human placental microsomes was stimulated by NAD and NADH. Concomitant oxidation or reduction of nucleotide was not detected based on absorbance at 340 nm. Concentrations giving half-maximum activity were 0.76 microM for NADH and 24.0 microM for NAD. Vmax values with 9.28 microM 5-pregnene-3,20-dione were 22.0 nmol/min/mg protein with NADH and 65.8 nmol/min/mg protein with NAD. When isomerase was assayed as a function of 5-pregnene-3,20-dione concentration, NAD increased Vmax but had no effect on the Km value for steroid. NADP, NADPH, acetylpyridine NAD and deamino NAD did not activate nor did they compete with NAD. Exposure of microsomes to trypsin, phospholipase A2 or phospholipase C resulted in the loss of isomerase activity. Approximately 30% of the initial activity was recovered after detergent solubilization of microsomes. Hydrogen peroxide did not affect activation by NAD. The data are consistent with nucleotide enhancement of a step in the isomerization reaction other than substrate binding.

Pregnancy in Hmong refugee women.

PIP: A comparison of pregnancy course and outcome between 648 Hmong refugee women and 5278 non-Hmong controls, all of whom delivered at a Minnesota medical center in 1976-83, indicated that Hmong women were 5 times as likely to have a history of previous perinatal loss. In terms of demographic factors, Hmong women were more likely to be age 35 years or above at delivery (14% versus 2% among controls), to be grant multiparas (33% versus 3% among controls), and to be married (95% versus 61% among controls). While 59% of controls began prenatal care during the 1st trimester, only 16% of Hmong women fell into this category and 31% delayed receiving care until the 3rd trimester. A review of the obstetric histories revealed that 18.1% of Hmong women compared with 3.7% of controls had experienced 1 or more previous perinatal loss. Medical conditions found with significant frequency in the Hmong population included anemia, tuberculosis, malaria, and parasitic infestations. Preeclampsia, hypertension, diabetes, urinary and vaginal infections, and gonorrhea occurred less frequently among Hmong women than among controls. Moreover, the incidence of premature rupture of the membranes was only 4.2% among Hmong women compared to 11.8% among controls. The prematurity rate was 48.5/1000 in the study group and 117/1000 in controls; in addition, only 7.8% of Hmong infants compared to 10.9% of control infants were low birthweight (under 2500 grams). The perinatal mortality rate was similar in both groups: 14.6/1000 among Hmong infants and 15.0/1000 among controls. Contraception was accepted by 50% of the Hmong mothers, but under 10% remained users 12 months after delivery and 27% were pregnant again. The generally good pregnancy outcomes recorded among these Hmong women despite the existence of numerous high-risk factors--short stature, advanced maternal age, grand multiparity, late prenatal care, and poor nutrition--is surprising. It appears that relocation to the US has enabled this population to overcome the factors that contributed to their previous high rates of perinatal loss.^ieng

Steroid modulation of 17 beta-hydroxysteroid oxidoreductase activities in human placental villi in vitro.

Recent data are consistent with the presence of two 17 beta-hydroxysteroid oxidoreductase (17 beta-HOR) activities in placental homogenates. One is localized to intracellular membrane fractions. The conversion of 17 beta-estradiol (E2) to estrone (E1) by this enzyme can be completely inhibited in vitro by C19 and C21 steroids. The second activity is detected in microsomes, but is recovered principally in the cytosol. It also has 20 alpha-HOR activity, but has a high affinity only for C18 steroids. We used this difference to estimate the relative contributions of the two enzymes to E2, E1, and testosterone (T) metabolism by villous tissue in vitro. Fragments of tissue from vaginally delivered placentas (38-40 weeks) were incubated with [3H]E2, [3H]E1, or [3H]T as substrates and various unlabeled steroids as potential inhibitors. Approximately 40% of the E2 to E1 reaction was not inhibited by C19 steroids at 100-200 microM, whereas the conversion of T to androstenedione was inhibited by 90% or more under similar conditions. In contrast, the metabolizable C19 and C21 steroids, 5 alpha-dihydrotestosterone and 20 alpha-dihydroprogesterone, which inhibited the conversion of E1 to E2 by microsomes, stimulated E2 formation from E1 by villi. We conclude that nonspecific 17 beta-HOR accounts for approximately 60% of the E2 to E1 conversion and nearly all of the T to androstenedione conversion in villous tissue fragments. The data also suggest that net E2 formation in villi is catalyzed principally by the C18-specific 17 beta-HOR.

Inactivation of soluble 17 beta-hydroxysteroid dehydrogenase of human placenta by fatty acids.

The sensitivity of soluble, 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) of human placenta to inactivation by fatty acids was examined. Exposure to the unsaturated fatty acids oleic, arachidonic, linoleic and linolenic acid resulted in the loss of activity. Methyl and ethyl esters of oleic acid, the saturated fatty acid, stearic acid and prostaglandins E2 and F2 alpha were without effect. Inactivation by oleic acid required the fatty acid at levels above its critical micelle concentration, 50 microM, as estimated by light-scattering. Steroid substrates and inhibitors did not protect against inactivation. NAD+, NADH, NADP+ and NADPH did protect. The concentrations of NADP+, 50 microM, and NAD, 1.5 mM, necessary for complete protection were significantly greater than their respective Michaelis constants, 0.16 microM and 15.2 microM. The data suggest that soluble 17 beta-HSD can bind to fatty acid micelles and that the binding site(s) on the enzyme are at or near pyridine nucleotide binding sites.

17 beta-hydroxysteroid and 20 alpha-hydroxysteroid dehydrogenase activities of human placental microsomes: kinetic evidence for two enzymes differing in substrate specificity.

During storage at 4 degrees C, the 17 beta-hydroxysteroid dehydrogenase activity of human placental microsomes with estradiol-17 beta was more stable than that with testosterone. In order to evaluate the basis for this difference, kinetics with C18-, C19-, and C21- steroids as substrates and/or inhibitors was studied in conjunction with an analysis of the effects of detergents. Both 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activities were detected. At pH 9.0, apparent Michaelis constants were 0.8, 1.3, and 2.3 microM for estradiol-17 beta, testosterone, and 20 alpha-dihydroprogesterone, respectively, 17 beta-HSD activity with testosterone was inhibited by estradiol-17 beta, 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone, 20 alpha-dihydroprogesterone, and progesterone. In each case 90 to 100% inhibition was observed at 50 to 200 microM steroid. Activity with 20 alpha-dihydroprogesterone was similarly sensitive to inhibition by C19-steroids. By contrast, 25 to 45% of the activity with estradiol-17 beta was not inhibited by high concentrations of C19- or C21-steroids and differed from the 17 beta-HSD activity with testosterone and the major fraction of that with estradiol-17 beta by being insensitive to solubilization by detergent. These results are consistent with an association of two dehydrogenase activities with human placental microsomes. One recognizes C18-, C19-, and C21-steroids as substrates with comparable affinities. The second appears to be highly specific for estradiol-17 beta. The former activity may account for most if not all of the oxidation-reduction at C-17 of C19-steroids and at C-20 of C21-compounds at physiological concentrations by term placental tissue.

Inhibition of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activities of human placenta by steroids and non-steroidal hormone agonists and antagonists.

Various naturally occurring steroids, synthetic steroid derivatives and non-steroidal hormone agonists and antagonists were assayed as inhibitors of human placental 17 beta-HSD activities. Microsomal 17 beta-HSD was inhibited by C18-, C19- and C21-steroids. Soluble 17 beta-HSD was highly specific for C18-steroids. In contrast to the soluble activity, the microsomal enzyme also had a strong affinity for ethinylestradiol (KI = 0.3 microM) and danazol (KI = 0.6 microM); anabolic steroids and norethisterone were weaker inhibitors. Of the non-steroids tested only diethylstilbestrol and o-demethyl CI-680 were inhibitors and they showed a greater affinity for soluble 17 beta-HSD. KI-values for estradiol-17 beta, (0.8 microM), progesterone (27.0 microM) and 20 alpha-dihydroprogesterone (1.5 microM) were comparable to reported tissue levels of these compounds, consistent with a possible competition in vivo among naturally occurring C18-, C19-, and C21-steroids for the active site of microsomal 17 beta-HSD.

Characterization of a nonenzymatic component in the isomerization of 5-pregnene-3,20-dione catalyzed by human placental microsomes in vitro.

When human placental microsomes were heated in boiling water or exposed to trypsin, 30 to 40% of the 5-ene,3-ketosteroid isomerase activity was stable. Aqueous suspensions of chloroform:methanol extracts of microsomes also catalyzed isomerization of 5-pregnene-3,20-dione, activity being associated with the polar lipid fraction. The trypsin- and heat-stable activities, as well as that of resuspended microsomal lipids, showed a dependence on buffer composition and concentration. Little activity was detected in water at pH 7.0. Relative activities in various buffers were Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) greater than Pipes (1,4-piperazinediethanesulfonic acid) greater than potassium phosphate greater than Mes(4-morpholineethanesulfonic acid). The data suggest that the occurrence of membrane lipid-dependent nonenzymatic catalysis could contribute to the isotope exchange with solvent observed in previous studies of the mechanism of isomerization catalyzed by placental microsomes. The ability of the membrane lipid phase to catalyze steroid isomerization under certain conditions and the fact that this activity is subject to modifications by exogenous agents may have more general implications for an understanding of possible effects of xenobiotics on steroid hormone formation and action in vivo.

Inactivation of microsomal 17 beta-hydroxysteroid dehydrogenase by phospholipase C: rates of phospholipid hydrolysis and enzyme inactivation, and effects of phospholipids.

When guinea-pig liver microsomes were exposed to phospholipase C the rate of phospholipid hydrolysis exceeded the rate of decrease in 17 beta-hydroxysteriod dehydrogenase (17 beta-HSD) activity. The time-course of the decrease in 17 beta-HSD activity was biphasic. An initial more rapid decrease (30-50% of total) was associated with the major extent (85%) of phospholipid hydrolysis. Subsequently, a second, slower phase of 17 beta-HSD inactivation was observed. The addition of purified phospholipids did not reactivate 17 beta-HSD but did protect against the inactivation seen in the second phase. The diacyglycerides produced by phospholipase C action remained associated with the microsomes. It is proposed that the differences in the rates of 17 beta-HSD inactivation reflect variations in the distribution of a single form of 17 beta-HSD among differing membrane fractions rather than the existence of multiple enzyme forms. The stabilizing effects of phospholipids may be due to their ability to prevent changes in lipid-lipid, lipid-protein and protein-protein interactions resulting from diacylglyceride formation. Resuspended microsomal lipids (chloroform-methanol extracts) inactivated 17 beta-HSD suggestive of the presence of endogenous lipid modulators of enzymatic activity.

Adolescent contraceptive use: experience in 1,762 teenagers.

Retrospective analysis was done on 1,762 adolescent family planning patients who used the St. Paul Maternal and Infant Care Project Teen Clinic from 1969 through June 1979. Factors evaluated included method of contraception, demographic data, and continuation rates calculated by Life Table Method. A further comparison was done on 403 of these patients who initiated their family planning care at a project satellite clinic located within a local high school.

PIP: Retrospective analysis was done on 1762 adolescent family planning patients who used the St. Paul Maternal and Infant Project Teen Clinic from 1969 through June 1979 in the attempt to analyze their patterns of contraceptive usage. The contraceptive usage of a subgroup of 403 adolescents who received their initial services and follow-up in a high school clinic was further analyzed and compared to that of the overall group. Factors evaluated included method of contraception, demographic data, and continuation rates calculated by the Life Table Method. The ages of the study population at the time of their 1st visit in the Teen Birth Control Clinic ranged from 12-19 years. 85% were white, 10% black, and the remaining 5% were members of other races. 89.2% were nulliparas, and the remainder had had 1 or more abortions or 1 or more term pregnancies. 58% were already pregnant at the time of the initial visit and were referred for pregnancy counseling. Of these, 9 had an abortion and subsequently returned to the Clinic for contraception. Oral contraceptives (OCs) were the initial method chosen by 86.2% of those adolescents starting contraceptions. 10.3% chose other methods. Of the total study population, 889 patients (50.5%) were released from follow-up as active contraceptors. 381 patients were lost to follow-up. Among the high school group, the distribution of number of users of contraceptives who chose OCs, IUDs, and other methods was similar to that of the overall teen clinic population. Similarly high continuation rates were found in both the overall teen clinic population and the high school subgroup.

Phospholipase A2 inactivation of microsomal 17 beta-hydroxysteroid oxidoreductase: rates of phospholipid hydrolysis and enzyme inactivation, effects of hydrolysis products and properties of the phospholipase A2-treated enzyme.

Exposure of guinea pig liver microsomes to phospholipase A2 resulted in the nearly complete loss of 17 beta-hydroxysteroid oxidoreductase (17 beta-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17 beta-HSD indicating that they may contribute to the inactivation by phospholipase A2. If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17 beta-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis per se results in a destabilization of 17 beta-HSD resulting in the subsequent activity loss. The inactivation of 17 beta-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism in vivo.

Pregnancy in the underweight woman. Course, outcome, and growth patterns of the infant.

The obstetric performance and pregnancy outcome of 354 underweight patients were compared with matched control subjects of normal weight. The growth patterns of their infants were also compared. The underweight women had significantly higher rates of cardiac/respiratory problems, anemia, PROM, and endometritis but were less prone to develop pre-eclampsia. Prematurity and low Apgar scores were significantly more frequent in the infants of underweight women. There was no difference in the frequency of IUGR and in perinatal mortality rates. The mean birth weight of the infants of underweight women was 231 grams less than that of infants of control subjects. Underweight women, particularly if they were anemic, had a higher incidence of low-birth-weight infants despite adequate weight gain. AGA infants of underweight women were more likely to be below the twenty-fifth percentile for weight correlated with length by 12 months of age.

A Simplified antepartum risk-scoring system.

The effectiveness of a simple antepartum risk-scoring system was evaluated in 2085 consecutive deliveries. Neonatal morbidity was observed in 42.1% of infants of mothers classified as high risk (score greater than or equal to 7) compared to 12.5% of infants of mothers classified as low risk (score less than 7). No neonatal deaths were observed in the low-risk group, compared with 34 in the high-risk group (P less than 0.001). Of all perinatal deaths, 88.6% occurred in the high-risk group. The perinatal mortality rates for low- and high-risk pregnancies were 7.2 and 63.3, respectively, per 1000 live births.

Antepartum fetal testing. I. The oxytocin challenge test.

Two hundred seventy-eight high-risk patients were managed by a comprehensive assessment of the anatomic, biochemical, and functional environment of the fetoplacental unit utilizing predetermined guidelines. Decisions to terminate pregnancy were reserved for patients who demonstrated a positive OCT. Perinatal outcome in patients with positive OCT's was significantly worse than in patients who did not have a positive OCT. Patients with suspicious OCT's frequently had positive OCT's and were more likely to bear growth-retarded infants, whereas negative OCT's in general were associated with a favorable outcome. Correlation of estriol excretion with the OCT and perinatal outcome was inconsistent. There were a total of four prenatal deaths, all of which were considered unpreventable.

Antepartum fetal testing. II. The acceleration/constant ratio: a nonstress test.

For a period of one-half hour of undisturbed fetal monitoring, periodic changes of the FHR in response to spontaneous fetal movements (FM's) were recorded. FM's in patients who subsequently had positive OCT's were less likely to show accelerations (p = 0.001) and more likely to show variable decelerations (p = less than 0.001) and no change (p = less than 0.001) in the FHR when compared with patients who did not have a positive OCT. A ratio between the number of FM's associated with accelerations and the sum of FM's associated with no change and decelerations was defined as the acceleration/constant (A/C) ratio. The outcome in patients who exhibited reactive tests (i.e., A/C ratio greater than 1) was more favorable than the outcome in patients with nonreactive tests (i.e., A/C ratio less than or equal to 1). Patients with positive OCT's universally showed nonreactive tests, whereas patients with false positive OCT's were more likely to have reactive tests. Evidence is presented to suggest that the A/C ratio is more predictive of the intrauterine environment than the OCT.

Prenatal care for pregnant adolescents in a public high school.

Two groups of pregnant adolescents enrolled in the St. Paul, Minnesota Maternal and Infant Care Project were involved in this study. A retrospective analysis of obstetrical summary sheets of delivered pregnant adolescents was conducted to demonstrate the relationship of the availiability of a comprehensive, interdisciplinary program of prenatal care in a regular public school setting to the achievement of early and continuous prenatal care and to the minimizing of obstetrical complications of the pregnant adolescents who were students in the school. A total sample of 36 students who received prenatal care in the school clinic (study group) from 1973 to 1976 was compared with a random sample of 36 adolescent patients (matched for race) who received care at a non-school clinic (comparison group). The data demonstrated that the study group initiated care earlier and had more total prenatal visits than did the comparison group. Also demonstrated were fewer obstetrical complications in the study group than in the comparison group. The comparison group had more low birth weight infants and more complicated deliveries than did the study group. Therefore, the results of this study support the initial objective and may have significance for educators and health personnel.