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Objective: Our purpose was to determine the effects of fetal number, various ovulation induction treatments, and placental hormones on the concentration of maternal serum relaxin., Study Design: The concentrations of relaxin, human chorionic gonadotropin, estriol, and alpha-fetoprotein were determined in blood samples drawn at 16 to 18 weeks for prenatal diagnosis in 72 singleton and 115 twin pregnancies and analyzed by one-way analysis of variance, correlation analysis, and stepwise multiple linear regression of the log-transformed data., Results: The maternal serum concentrations of each of the four measured hormones were significantly higher in the twin pregnancies than in the singleton pregnancies: 1.4-fold for relaxin, 1.9-fold for human chorionic gonadotropin, 1.9-fold for estriol, and 2.2-fold for alpha-fetoprotein (all p < 0.01). The concentrations of each of the four hormones were significantly correlated with each of the others and with the number of fetuses (p < 0.01), except that estriol was not significantly correlated with human chorionic gonadotropin. The serum relaxin concentration in twin pregnancies after treatment with follicle-stimulating hormone and luteinizing hormone (menotropins) (n = 10) was 3.3-fold that in twins resulting from spontaneous ovulation (n = 89, p < 0.01). In twins resulting from in vitro fertilization or gamete intrafallopian transfer (n = 9) the serum relaxin concentration was 2.6-fold higher than in twins resulting from spontaneous ovulation (p < 0.01). The effect of clomiphene citrate (1.2-fold, n = 7) failed to reach statistical significance., Conclusions: The second fetus causes a 1.4-fold increase in the concentration of maternal serum relaxin in twin pregnancies. Induction of ovulation with menotropins causes an additional 3.3-fold increase, whereas in vitro fertilization or gamete intrafallopian transfer treatment causes an additional 2.6-fold increase over that seen in twin pregnancies that followed spontaneous ovulation.

Urinary gonadotropin peptide (UGP; beta-core fragment), a major metabolite of human chorionic gonadotropin (hCG), was shown recently to be markedly elevated in Down syndrome pregnancy between 19 and 22 weeks of gestation. To confirm and extend this finding, we obtained maternal urine and matching maternal serum samples from 14 cases of Down syndrome and six other aneuploidies between 17 and 21 weeks of gestation. UGP was measured in all these samples and in 91 singleton control urines. Results were corrected for urinary creatinine level and expressed as multiples of the control median (MOM). hCG levels were assayed in all serum samples from the cases and compared with previously established reference values. The median UGP level in Down syndrome cases was 5.34 MOM (range 2.71-12.57); 88 per cent of the values were above the 95th centile of control levels after modelling. The median maternal serum hCG level for the same cases was 2.20 MOM (range 0.84-3.40); 36 per cent of the values were above the 95th centile. The level of UGP in every case including all other aneuploidies was higher than the comparable maternal serum hCG level. Elevated UGP measurements are strongly associated with fetal Down syndrome during the second trimester and could contribute to improved Down syndrome screening protocols that are more accessible and less expensive than are currently available.

Nine centres collaborated to examine the feasibility of a screening method for trisomy 18 that was based on assigning individual risk, using a combination of maternal age and measurements of alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG). Second-trimester measurements of these analytes were obtained from 94 trisomy 18 pregnancies. In the 89 pregnancies without an associated open defect, the median levels for AFP, uE3, and hCG were 0.65, 0.43 and 0.36 multiples of the unaffected population median, respectively. The strongest individual predictor of risk for trisomy 18 was uE3, followed by hCG, AFP, and maternal age, in that order. Using a method of individual risk estimation that is based on the three markers and maternal age, 60 per cent of pregnancies associated with trisomy 18 would be detected at a risk cut-off level of 1:100, with a false-positive rate of about 0.2 per cent. One in nine pregnancies identified as being at increased risk for trisomy 18 would be expected to have an affected pregnancy. This risk-based screening method is more efficient than an existing method that is based on fixed analyte cut-off levels. Even though the birth prevalence of trisomy 18 is low, prenatal screening can be justified when performed in conjunction with Down syndrome screening and when a high proportion of women offered amniocentesis have an affected fetus.

Placental sulfatase deficiency is an X-linked metabolic defect that occurs in about 1 in 2,000 to 5,000 males. It is associated with congenital ichthyosis. In this report, the authors document a case of placental sulfatase deficiency detected during routine prenatal screening of maternal serum by the triple test: serum alpha-fetoprotein (AFP), unconjugated estriol (uE3), and human chorionic gonadotropin (hCG). At 16-weeks gestation, her AFP was 20.9 IU/mL (multiple of the median [MOM] 0.83), hCG was 14.4 mIU/L (MOM 0.42) and her uE3 was 0.01 nmol/L (MOM 0.01). The extremely low uE3 indicated a possible placental sulfatase deficiency, congenital adrenal hypoplasia, or other unknown abnormality. On receiving this information, the obstetrician obtained a family history that was consistent with ichthyosis in the maternal grandfather and his siblings. Biochemical analysis of placenta documented the lack of sulfatase activity. This case illustrates that an extremely low level of maternal uE3 should prompt investigation of the family for evidence of X-linked ichthyosis associated with placental sulfatase deficiency.

Objective: To determine if second-trimester maternal serum concentrations of unconjugated estriol (E3) and hCG are altered in pregnancies associated with fetal gastroschisis or omphalocele., Methods: Concentrations of alpha-fetoprotein (AFP), unconjugated E3, and hCG were measured in a case-control study involving 23 cases of gastroschisis, 17 cases of omphalocele, and 200 matched unaffected pregnancies., Results: As reported previously, median AFP levels were significantly higher in pregnancies with gastroschisis and omphalocele compared to unaffected pregnancies (9.42 and 4.18 multiples of the unaffected population median [MoM], respectively). The median hCG values were not significantly different for the two defects (1.10 and 1.13 MoM, respectively). Six of the cases of omphalocele were associated with other anomalies, but exclusion of these cases from the analysis did not alter the conclusions., Conclusions: Unconjugated E3 and hCG measurements are not useful in screening for, or distinguishing between, open ventral wall defects. Alpha-fetoprotein measurements alone will detect nearly all cases of gastroschisis and most cases of omphalocele.

Both a cross-sectional and a longitudinal study were performed to investigate whether or not the collection time should be taken into consideration when generating a patient's risk for fetal Down syndrome with multiple marker screening. Diurnal variations of third-trimester alpha-fetoprotein (AFP) levels and first-trimester human chorionic gonadotropin (hCG) levels have been previously reported. In addition, large episodic fluctuations of conjugated and unconjugated oestriol (uE3) as well as a diurnal variation have also been reported in the third trimester. If the levels of these analytes routinely fluctuate during the day, they could affect a patient's risk calculation for fetal Down syndrome. The longitudinal study evaluated ten non-diabetic women who underwent sequential sampling for AFP, hCG, and uE3. The cross-sectional study evaluated 1953 patients for these three markers whose time of sampling was recorded between 8.00 a.m. and 5.59 p.m. Using either study design, no significant effect was seen in the median MOM levels of the screening analytes as a function of the time of day.

The purpose of this study was to examine whether women with first trimester uterine bleeding and low serum folate have a higher incidence of spontaneous abortions and adverse perinatal outcome compared with women whose folate levels are normal. Serum folate and vitamin B12 levels were obtained on 225 women who presented with first trimester vaginal bleeding; pregnancy outcomes of those whose folate or vitamin B12 levels were low were compared with those with normal levels using the chi-square test. Of the 151 women included, 52 had low folate levels (less than 4.0 ng/ml). Their spontaneous abortion rate and perinatal outcomes were similar to those whose folate levels were normal. We concluded that in pregnancies complicated by first trimester vaginal bleeding, low folate levels do not appear to be associated with an increased risk of pregnancy loss and adverse outcome.

The application of population-based maternal serum screening to prenatal diagnosis has been shown to be practical and is now wide-spread. It has been estimated that more than 40% of genetics programs in the United States that offer second trimester maternal serum screening now use at least one analyte in addition to AFP (Dr. George J. Knight, personal communication). The studies described here suggest that additional refinements in maternal serum screening are not only possible but inevitable. The use and interpretation of the analytes AFP, uE3, and hCG continue to be examined and expanded upon (Table 1). In the future, it seems logical that other analytes may be added or may replace one or more of these currently used markers. We have demonstrated that with AFP, uE3, and hCG, a separate protocol will identify a significant portion of trisomy 18 fetuses with a minimal increase in the number of amniocenteses performed. Other significant aneuploidies, including Turner syndrome and triploidy, will be identified in screening for Down syndrome and trisomy 18. Although separate screening protocols for the detection of Turner syndrome and triploidy would not be of practical value, their detection within a screening protocol for Down syndrome is an added benefit and increases the value of the screening markers. The use of first trimester screening is being investigated. Although earlier diagnosis is attractive, routine first trimester screening for aneuploidy would necessitate the creation of an entirely new protocol for the collection of maternal serum samples and their interpretation, as well as a second round of screening for open fetal defects during the early second trimester. In contrast, an advantage of the current protocols is that screening for both types of abnormalities is possible using the same serum sample. Although research may eventually demonstrate that first trimester screening is technically feasible, an evaluation of the logistics of such screening may show it to be impractical. Obstetric implications of elevations and decreases in the new serum markers have not yet been studied carefully. It is logical, however, that just as with abnormal AFP (not explainable on a genetic basis), extreme levels of uE3 and hCG may also be associated with particular obstetric problems. Information on such deviations in analyte levels is just beginning to emerge. Finally, the study of maternal serum markers may contribute to an understanding of placental function in the second trimester, and ultimately advance our understanding of the underlying pathophysiologies of various fetal chromosomal and morphologic defects.(ABSTRACT TRUNCATED AT 400 WORDS)

Hydrops fetalis and fetal death caused by fetal parvovirus B19 infection have been reported to be associated with elevated maternal serum alpha-fetoprotein (AFP), based on a total of six cases. It has been suggested that the absence of AFP elevation may be reassuring. Maternal serum levels of the Down syndrome screening markers unconjugated oestriol and human chorionic gonadotropin in cases of fetal parvovirus infection have not been previously reported. We report four cases of hydrops fetalis and fetal death caused by fetal parvovirus infection, each with unremarkable second-trimester levels of AFP, unconjugated oestriol, and human chorionic gonadotropin.

Objective: To investigate the association between maternal cigarette smoking and serum levels of alpha-fetoprotein (AFP), unconjugated estriol (uE3), and hCG and to determine whether it is appropriate to take smoking status into account when screening for fetal Down syndrome., Methods: Smoking information was obtained from 23,668 pregnant women at the time of routine screening for fetal Down syndrome. Serum levels of AFP, uE3, and hCG were stratified by smoking status and compared. Individual risks for fetal Down syndrome were analyzed both before and after adjusting the hCG levels for the effect of smoking. The prevalence of Down syndrome in the study population was calculated for both smokers and non-smokers., Results: The average AFP, uE3, and hCG levels in women who smoked cigarettes were 3% greater (95% confidence interval [CI] 2-4%), 3% less (95% CI 2-4%), and 23% less (95% CI 22-24%), respectively, compared with levels in non-smokers. At comparable maternal ages, women who smoked cigarettes were identified as being at high risk for fetal Down syndrome 40% less often than non-smokers (4.0 versus 7.1% at a risk level of 1:250). Adjusting hCG levels for smoking status reduced the overall false-positive rate from 6.4 to 6.1% (using a term risk cutoff level of 1:250). The predicted effect on detection was small (a 0.7% increase). The prevalence of Down syndrome was lower among smokers, but the difference was not statistically significant (odds ratio 0.51, 95% CI 0.15-1.5)., Conclusions: Adjusting the serum markers used for Down syndrome screening for the effect of maternal smoking has a small effect on overall screening performance. Given the uncertainty over whether there is a lower birth prevalence among women who smoke cigarettes, such adjustment is not currently justified.

Two prenatal centres in New England, routinely using a screening protocol for fetal Down syndrome that included maternal serum alpha-fetoprotein (AFP), unconjugated oestriol (uE3), and human chorionic gonadotropin (hCG) measurements in combination with maternal age, adopted a separate screening protocol for trisomy 18. That protocol identified a pregnancy as being at high risk when AFP, uE3, and hCG measurements all fell at or below specified cut-offs (0.75, 0.60, and 0.55 multiples of the median, respectively), regardless of maternal age. Among the first 19,491 women screened, 98 (0.5 per cent) were found to have values which placed them in the high-risk category. Four of these women were subsequently found not to be pregnant. In two others, samples from non-pregnant individuals were found to have been incorrectly submitted for analysis in place of the samples from the pregnant women. All of the remaining 92 women were counselled and offered amniocentesis and fetal karyotyping. Eighty-eight (96 per cent) accepted. Karyotypes or birth outcomes were available on all 92 pregnancies. Six cases of trisomy 18 and one case of Turner syndrome were identified by karyotype. One case of trisomy 18 was identified for every 14 unaffected pregnancies offered amniocentesis. In the present prospective study, an estimated 85 per cent of the cases of trisomy 18 were identified. However, given the small number of cases (six), the 95 per cent confidence interval for the detection rate is broad (40-95 per cent).

Objective: The combination of maternal serum alpha-fetoprotein, unconjugated estriol, and human chorionic gonadotropin levels and maternal age has been used to increase the sensitivity of screening for fetal Down syndrome and trisomy 18 in early-second-trimester pregnancies. We hypothesized that a unique pattern of these analytes also may be characteristic of fetal Turner syndrome, with or without hydrops., Study Design: We studied preamniocentesis, second-trimester maternal serum specimens from seven hydropic and eight nonhydropic cases of fetal Turner syndrome. Clinical and pathologic records were reviewed. Statistical analysis of the data was performed by the rank sum test., Results: In both hydropic and nonhydropic cases, alpha-fetoprotein levels were slightly reduced, and unconjugated estriol levels were markedly reduced. In hydropic pregnancies human chorionic gonadotropin levels were elevated, and nonhydropic pregnancies had low human chorionic gonadotropin levels (p = 0.001)., Conclusions: The results suggest that the morphologic defect of hydrops, rather than the aneuploidy itself, is responsible for the elevation in human chorionic gonadotropin. In conjunction with the low unconjugated estriol levels, the elevation in human chorionic gonadotropin levels will result in the prenatal identification of hydropic fetal Turner syndrome pregnancies as being at increased risk for fetal Down syndrome.

Background: Approximately 35 percent of all cases of Down's syndrome in fetuses can be detected by measuring maternal serum alpha-fetoprotein during the second trimester in the general population of pregnant women. Recent case-control studies indicate that this detection rate could be approximately doubled by measuring serum levels of unconjugated estriol and chorionic gonadotropin, which are abnormally low and abnormally high, respectively, in women carrying fetuses affected by Down's syndrome., Methods: We prospectively screened 25,207 women and adolescents in the second trimester of pregnancy and assigned each a risk of fetal Down's syndrome with an algorithm that took into account measurements of all three serum markers in combination with maternal age. On this basis, 1661 subjects (6.6 percent) were initially assigned a second-trimester risk of fetal Down's syndrome of at least 1 in 190, and 962 (3.8 percent) were offered amniocentesis for chromosomal analysis after verification of gestational age. Gestational age was determined on the basis of the first day of the last menstrual period or, when available, by ultrasonography., Results: Among the 760 women and adolescents who chose amniocentesis, 20 cases of fetal Down's syndrome were detected, along with 7 other chromosomal disorders. There was 1 additional case of fetal Down's syndrome among the 202 women who chose not to have amniocentesis. The rate of detection of Down's syndrome was thus 58 percent (21 of 36 expected cases), and the frequency of identifying a fetus with Down's syndrome in women undergoing amniocentesis was 1 per 38 amniocenteses (95 percent confidence interval, 1 in 25 to 1 in 62)., Conclusions: Measuring serum alpha-fetoprotein, chorionic gonadotropin, and estriol is more effective in screening for fetal Down's syndrome than measuring maternal serum alpha-fetoprotein alone. Such an expanded protocol can readily be incorporated into existing prenatal screening programs.

In 5 placental homogenates the Km of steroid sulfatase for DHEA sulfate increased from 15.4 in Tris buffer to 26.8 microM in phosphate (both buffers 0.1 M, pH 7.4), P less than 0.05. In 3 pooled ovarian preparations the Km increased from 14.3 microM in Tris to 33.0 microM in phosphate, P less than 0.01. There was no significant difference between the ovarian and placental values for Km in either Tris or phosphate (P greater than 0.5), and the increase in the Km produced by phosphate in ovarian tissue was not significantly different from that in the placenta (P greater than 0.5). In the placentas the Vmax in Tris was 1420 pmol/min/mg protein and this fell to 523 pmol/min/mg protein in phosphate (P less than 0.005). The Vmax was 50-fold higher in the placenta than in the ovary in either Tris or phosphate (both P less than 0.001). In the ovary, the Vmax was 27.6 pmol/min/mg protein in Tris and 11.0 pmol/min/mg protein in phosphate (P less than 0.05). The reduction of Vmax produced by phosphate in the ovary was not significantly different from that in the placenta (P greater than 0.5). The slope of the 1/v vs 1/S plot (Km/Vmax) increased 4.7-fold in the placentas and 5.8-fold in the ovaries in phosphate over that in Tris (both P less than 0.001); the increase in the placentas was not significantly different from that in the ovaries (P greater than 0.5). Phosphate ion acts as a mixed inhibitor of both placental and ovarian steroid sulfatase.

A number of serum screening protocols are either currently in use or proposed to identify pregnant women under 35 years of age who are at increased risk for a Down syndrome fetus. It has been suggested that these same screening methods be applied to gravidas over 35 years of age to identify those women who should be offered amniocentesis. To evaluate the efficacy of screening for detection of aneuploid pregnancy in this age group, the serum samples of 34 women who underwent amniocentesis and who had confirmed fetal aneuploidy were assayed for alpha-fetoprotein (AFP), hCG, and unconjugated estriol (E3). These concentrations were compared with those of 85 women with known euploid pregnancy who underwent amniocentesis for advanced maternal age. The mean multiple of the median (MoM) for each of the three markers was significantly different from control values in cases of trisomy 21 (AFP median MoM = 0.82; unconjugated E3 median MoM = 0.77; and hCG median MoM = 1.89). These differences were not found when other aneuploidies were considered. Likelihood ratios were calculated for cases and controls and examined for their ability to predict the need for amniocentesis, based on currently recommended risk levels. There was a significant difference between the mean likelihood ratio for cases of trisomy 21 compared with that of controls (mean likelihood ratio = 13.48); there was no significant difference for other aneuploidies (mean likelihood ratio = 1.08). Of the 16 cases of trisomy 21 analyzed, four would not have been diagnosed antenatally if recommendation against amniocentesis had been made based on each woman's individual age-specific risk as modified by her likelihood ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Unconjugated oestriol (uE3) and human chorionic gonadotropin (hCG) levels were determined in second-trimester maternal serum (MS) samples from 21 pregnancies associated with fetal anencephaly and 15 pregnancies associated with fetal open spina bifida. Each measurement was expressed as a multiple of the median (MoM) for unaffected pregnancies for each completed week of gestation. In pregnancies associated with anencephaly, the median value for MSuE3 was very low (0.17 MoM, range less than 0.12-0.33 MoM), suggesting a functional defect in the fetal adrenal prior to 20 weeks' gestation; the median value for MShCG was also low (0.73 MoM), although not to the same extent as for MSuE3. A biological explanation for the hCG result is not apparent. In pregnancies associated with open spina bifida, the MSuE3 and MShCG values were unremarkable, consistent with a lack of involvement of these open fetal defects in the synthesis and secretion of uE3 and hCG.

Steroid sulfatase activity was measured in 89 human samples, using dehydroepiandrosterone sulfate (DHEAS) as substrate. The lowest activity was that of follicular fluid which was significantly lower than that of other tissues tested (each P less than 0.01). The steroid sulfatase activity of ovarian tissue taken collectively (corpus luteum, stroma, and follicles) was higher than that of other tissues taken collectively (abdominal skin, uterus, and fallopian tube) (P less than 0.001), and the steroid sulfatase activity of either the follicle (P less than 0.01) or the stroma (P less than 0.05) was significantly greater than that of the corpus luteum. The geometric mean steroid sulfatase activity of the placenta was significantly higher than other tissues tested (each P less than 0.01) and was 22-fold higher than that of the follicle, the tissue with the next highest activity. These data indicate that the human ovary (particularly the stroma and follicle) is capable of utilizing DHEAS, an adrenal product, as a substrate for production of other androgens such as dehydroepiandrosterone (DHEA), androstenedione, and testosterone.

Second trimester maternal serum unconjugated oestriol levels were measured in the stored serum samples from 22 pregnancies associated with Down's syndrome and 110 unaffected control pregnancies, matched for maternal age, gestational age, duration of storage of the serum sample, smoking habits and maternal weight. The serum unconjugated oestriol level of each affected pregnancy was expressed as a multiple of the median (MoM) of its five matched controls. The unconjugated oestriol levels were significantly lower in the affected pregnancies than in the unaffected pregnancies; the median MoM was 0.79 (P less than 0.05). This association between low serum unconjugated oestriol and fetal Down's syndrome early in pregnancy raises the possibility that serum unconjugated oestriol measurement may be added to maternal age and alpha-fetoprotein measurement in the antenatal screening for Down's syndrome.

The study of seminiferous tubule--Leydig cell interactions in relation to specific germ cell stages during the cycle of the seminiferous epithelium is extremely difficult in most mammalian species due to the continual presence of different spermatogenetic stages in the testis from the onset of puberty. The problem is also compounded by the uniform distribution of both seminiferous tubules and interstitial tissue throughout the entire testis. This difficulty can be circumvented, however, by studying certain species where there is a topographical distribution of germ cell stages within the testis. The urodele amphibian Necturus maculosus exhibits a breeding cycle during which a longitudinal wave of spermatogenesis occurs along the length of the testis, resulting in a spatial and temporal segregation of differentiating germ cells. Moreover, this topographical pattern of spermatogenesis is also reflected in the degree of development of adjacent Leydig cells. This anatomical arrangement allows distinct testicular regions to be obtained using a dissecting microscope. The isolated zones, containing germ cells and Leydig cells in various stages of development, were analyzed for 17 alpha-hydroxylase, C-17,20-lyase, and aromatase activities (key enzymes for the synthesis of androgens and estrogen), estrogen binding, and cytochrome P-450 content. Functional parameters were then correlated with the morphology of Leydig cells in the various zones observed by both light and electron microscopy. It was found that there existed a distinct correlation between the state of differentiation of the leydig cells, their steroidogenic potential, and the distribution of estrogen receptors. These results in Necturus indicate indicate in this species, at least, the steroidal microenvironment of different germ cell associations may be quite specific.

Human female fatty marrow from the femur and iliac crest has the ability to convert androgen to estrogen. Homogenates of fatty marrow incubated with 19-[6,7-3H]hydroxyandrostenedione (19-hydroxyandrost-4-ene-3,17-dione) and an NADPH-generating system produced estrogen in all six samples. The major estrogen produced in five of the cases was [3H]estrone, the immediate product of the aromatizaton of 19-hydroxyandrostenedione. One case produced [3H]estradiol. The local formation of estrogens in bone marrow may be important to the closure of the epiphyses at puberty and may affect osteoporosis at the menopause.

Aromatization of [3H]androstenedione and [3H]19-hydroxyandrostenedione to [3H]estrone has been demonstrated to occur in one to two week old primary monolayer cultures of fetal rat hypothalamus. Three times more estrone is produced from 19-hydroxyandrostenedione than from androstenedione in four day incubations. Cultures treated with cytosine arabinoside have 50% less cellular protein and produce three times more estrone from either substrate than untreated cultures. Time course experiments using cultures treated with cytosine arabinoside indicate that aromatization of 19-hydroxyandrostenedione is linear for three days and can be quantitatively measured within the first two to eight hours of incubation.

The effects of danazol on steroidogenesis in vitro in the 16-20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial 11 beta-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (KS) of 1 microM and with a lower affinity K's of 10 microM. Danazol bound to mitochondrial cytochrome P-450 with a KS of 5 microM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant KI = 0.8 microM) and the mitochondrial 11 beta-hydroxylase (KI = 3 microM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal in vitro.

Incubation parameters for a radioderivative assay for estrogen 2-hydroxylase have been examined. The assay was found to be specific and sensitive if a chromatographically purified preparation of COMT was used. Estradiol was found to be a better substrate for the 2-hydroxylase than estrone or estriol. The liver had significantly higher estrogen 2-hydroxylase activity than any other tissue examined. The estrogen 2-hydroxylase was highly localized in the microsomal fraction in both the liver and the brain. The male rat was found to have significantly more estrogen 2-hydroxylase activity in the liver than the female rat. In addition, in the male rat liver, the estrogen 2-hydroxylase activity was reversibly inducible by testosterone and was not affected by phenobarbital. In the male and female rat brain the estrogen 2-hydroxylase activities were similar.

The aromatase system associated with the mitochondrial fraction of human term placenta, present at 35--50% the specific activity of the microsomal enzyme, is substantially the same as the microsomal enzyme as determined by the following: 1) The rate of aromatization of androstenedione, 19-nortestosterone, and 16alpha-hydroxytestosterone in mitochondria was a nearly constant proportion of the microsomal rate; 2) Sensitivity to carbon monoxide was the same; 3) The magnitude of cytochrome P-450 Type I spectral interactions with androgen substrates was a constant proportion in mitochondria and microsomes; 4) Sensitivity to an antibody raised against hepatic microsomal NADPH-cytochrome c reductase was the same. When inner and outer mitochondrial membrane subfractions were prepared, the predominant aromatase activity was associated with the outer membrane preparation. This aromatase activity could not be accounted for by microsomal contamination as determined by inosine diphosphatase activity, a microsomal marker. After correction, the rate of aromatization in the outer membrane preparation was almost six times that in the inner membranes and three times that of the whole mitochondrial fraction.

The median maternal serum unconjugated oestriol level between 13 and 27 weeks gestation in 77 pregnancies associated with Down's syndrome was lower than the median level in 385 unaffected control pregnancies matched for maternal age, gestational age, and duration of serum sample storage (P less than 0.001). The median level for the affected pregnancies was 73% of that in the controls. Low unconjugated oestriol levels can be used to detect fetal Down's syndrome; at cut-off levels selected to detect at least 35% of affected pregnancies, unconjugated serum oestriol was a better screening test than either maternal age or serum alpha-fetoprotein (AFP). The use of all three variables in combination to select women with a 1:250 or greater risk of a Down's syndrome term pregnancy would yield a 45% detection rate with a false-positive rate of 5.2%. The same detection rate using maternal age alone or using age and serum AFP in combination would yield higher false-positive rates, 15% and 9.8% respectively. The addition of unconjugated oestriol to a Down's syndrome screening programme would therefore be more efficient than the use of age and AFP alone; for a given detection rate fewer women would need an amniocentesis or, for a given percentage of women having an amniocentesis, more pregnancies with Down's syndrome would be detected.

The possibility of improving the effectiveness of antenatal screening for Down's syndrome by measuring human chorionic gonadotrophin concentrations in maternal serum during the second trimester to select women for diagnostic amniocentesis was examined. The median maternal serum human chorionic gonadotrophin concentration in 77 pregnancies associated with Down's syndrome was twice the median concentration in 385 unaffected pregnancies matched for maternal age, gestational age, and duration of storage of the serum sample. Measuring human chorionic gonadotrophin in maternal serum was an effective screening test, giving a lower false positive rate (3%) at a 30% detection rate than that for maternal age (5%) and the two existing serum screening tests, unconjugated oestriol (7%) and alpha fetoprotein (11%). The most effective screening results were obtained with all four variables combined; at the same 30% detection rate the false positive rate declined to 0.5%. The new screening method would detect over 60% of affected pregnancies, more than double that achievable with the same amniocentesis rate in existing programmes (5%), and could reduce the number of children born with Down's syndrome in the United Kingdom from about 900 a year to about 350 a year.

Ferrets of both sexes were killed 8 or 5 days before expected parturition as well as 7, 15, 30, or 51 days after birth, and the activities of aromatase (using 19-[3H]hydroxyandrostenedione as substrate) and of 5 alpha-reductase (using [3H] testosterone as substrate) were assayed in whole homogenates of preoptic area plus anterior hypothalamus (POA), mediobasal hypothalamus (MBH), temporal lobe (TL), and cerebral cortex. Aromatase and testosterone 5 alpha-reductase activities were also measured in these regions in adult gonadectomized male and female ferrets. Compared with adults of both sexes in which aromatase activity was low in all brain regions studied, fetal ferrets had high levels of aromatase activity in POA plus MBH and in TL. At these prenatal ages, aromatase activity in POA plus MBH was significantly higher in males than in females. Aromatase activity in POA, MBH, and TL remained high in both sexes on postnatal days 7, 15, and 30, before declining by postnatal day 51. Cortical aromatase activity was uniformly low across all perinatal ages. The existence of a sex difference in aromatase activity in fetal POA plus MBH cannot be explained by a concurrent sex difference in circulating testosterone. Administration of testosterone to pregnant female ferrets over days 30-41 of gestation caused 150- to 350-fold increases in maternal plasma concentrations of testosterone and 2- to 5-fold increases in fetal plasma testosterone. However, aromatase activity was not affected in the POA and MBH of fetuses or mothers, although activity was significantly increased in the TL of mothers given testosterone. Furthermore, castration of neonatal or adult breeding males decreased plasma androgen levels by factors of 8 and 480, respectively, but resulted in only modest reductions in POA, MBH, and TL aromatase activity (a significant reduction occurred only in the adult male TL). Relatively high levels of testosterone 5 alpha-reductase activity were found in all brain regions across all perinatal ages, as well as in gonadectomized adult ferrets; there was no sex differences at any postnatal age studied. Prenatally, males had higher levels of 5 alpha-reductase activity than females only on day -8 in the POA plus MBH. The results show that estrogen and 5 alpha-reduced androgens can be synthesized in the brains of ferrets of both sexes during the perinatal period of sexual differentiation. A functional role for this neural metabolism of androgen remains to be demonstrated in this carnivorous species.

Aromatization was measured in testicular microsomal preparations obtained from rats treated 3--4 days with FSH, hCG, or vehicle, hCG, but not FSH, was found consistently to stimulate testicular aromatase activity at least 10-fold. As a marker for FSH action, epididymal androgen-binding protein was assayed and found to be 3 times higher in FSH-treated rats than in either hCG or control rats. hCG, but not FSH or vehicle, stimulated serum testosterone levels more than 100-fold. In all groups, aromatase activity in the microsomal fraction was at least 6 times higher than that found in the mitochondrial fraction. In experiments in which testicular compartments were separated, microsomal preparations from interstitial tissue of hCG-treated rats had 5--7 times more aromatase activity than microsomes from seminiferous tubules and 2--3 times more activity than microsomes from whole testes. It is concluded the hCG administered in vivo can stimulate testicular aromatase activity in immature rats, and the increase in activity is localized in the interstitial tissue.

The rate of adrenal protein degradation appears to be slower in rats to which ACTH (adrenocorticotrophin) has been chronically administered. As measured by the exponential decay of radioactively labelled adrenal protein in vivo, the mean half-lives of total protein and of mitochondrial, microsomal and 18000g-supernatant protein were significantly longer in ACTH-treated animals. Experiments in which either [(3)H]leucine or NaH(14)CO(3) was used to label proteins showed that of the fractions studied, the effect on mitochondrial protein degradation was most pronounced. The half-lives of the same subcellular fractions in rat liver were not affected by ACTH. The possibility that the results might have been caused by changes in radioisotope reutilization and pool size is discussed.

When the plasma concentrations of estrone sulfate (E1S) were measured in five menstrual cycles, the highest concentrations were found on the day of LH peak (14.25 nmol/l +/- 2.94 [SE]). Peak levels of E1S were 20 times higher than the highest E2 levels measured (0.769 +/- 0.276 nmol/l). To determine whether E1S can be metabolized by adult and fetal tissues we examined estrone (E1) sulfatase activity in brain and other tissues. E1 Sulfatase activity was present in all tissues studied including adult endometrium, fat and skin. When the rate of sulfatase activity was measured in homogenates of fetal hypothalamus, frontal cortex and pituitary (n = 4), the hypothalamic activity (306.0 +/- 39.1 [SE] pmol/min/mg protein) was significantly higher than that of the frontal cortex (127.4 +/- 19.4, P less than 0.002) or pituitary (193.7 +/- 43.3, P less than 0.03). This was not apparent in the adult (n = 2) where the enzyme activity was similar in the hypothalamus (413.9 +/- 27.3) and frontal cortex (446.3 +/- 82.2) and lower in the pituitary (98.2 +/- 19.2). The Km for E1 sulfatase in the fetal frontal cortex was 28.9 microM. The high E1 sulfatase activity in estrogen responsive target tissues, particularly fetal hypothalamus, accompanied by a large circulating reservoir of E1S, suggest that this enzyme could possibly have a regulatory role in controlling the level of intracellular estrogens and in modulating their intracellular function.

Experiments were designed to identify the neural cell type(s) responsible for the aromatization and 5 alpha-reduction of androgens in the rat hypothalamus. Primary cultures of fetal rat hypothalamic cells, which had enhanced neuronal morphology, were treated at various times after plating with kainic acid (KA), a neurotoxic agent which selectively destroys neuronal cells. Neuronal morphology was disrupted in a time (0-6 days)- and dose (10(-4)-10(-2) M)-dependent fashion after KA treatment, with no apparent change in the appearance of the flattened, underlying non-neuronal cells. KA treatment for 4 days decreased aromatization by 94% in a dose-dependent fashion (10(-4)-10(-2) M KA), while 5 alpha-reduction declined by no more than 25%. A 6-day time course with 10(-3) M KA showed a dramatic decline in aromatization and no alteration in 5 alpha-reduction. In control experiments, substance P, a neuronal peptide, declined after KA treatment while the activity of glutamine synthetase, a glial enzyme, did not change. We conclude from these results that aromatase is localized primarily to neuronal cells in the hypothalamus while 5 alpha-reductase is confined primarily to non-neuronal cells.

When measured in vitro using human placental microsomal preparations, the aromatization of 16alpha-hydroxytestosterone to estriol and androstenedione to estrone and estradiol proceeds at almost identical initial rates. Important differences between 16alpha-hydroxytestosterone and adrostenedione aromatization are evident, however. While substantial findings have implicated cytochrome P-450 in placental aromatization, the aromatizaiton of androstenedione is insensitive to CO although it is competitively inhibited by metyrapone. 16alpha-Hydroxytestosterone aromatization, in contrast, is inhibited 50-60% by CO and is strongly inhibited by metyrapone. 16alpha-hydroxytestosterone aromatization is strongly inhibited in a competitive manner by androstenedione, while 16alpha-hydroxytestosterone has essentially no effect on androstenedione aromatization, althogh at very high 16alpha-hydroxytestosterone concentrations (65 muM) and subsaturating androstenedione concentrations, 16alpha-hydroxytestosterone appears to noncomptitively inhibit androstenedione aromatization. The apparent Km for the aromatization of androstenedione is 95 nM and for 16alpha-hydroxytestosterone, 7 muM. Both androstenedione and 16alpha-hydroxytestosterone cause type I spectral perturbations associated with binding to cytochrome P-450 when added to placental microsomes; however, the deltaA390-420 is twice as great in response to saturating amounts of androstenedione than in response to 16alpha-hydroxytestosterone. If androstenedione is added to 16alpha-hydroxytestosterone, the same spectral change as that caused by androstenedione alone is expressed. The apparent spectral dissociation constant for androstenedione is 93 nM while for 16alpha-hydroxytestosterone it is 11muM; both essentially the same as the comparable apparent Kms for aromatization. The evidence suggests the presence of two aromatase P-450's in human placenta.

The effects of danazol on steroidogenesis in vitro in the rat testis were examined by studying: 1) androgen synthesis in rat Leydig cells cultured with danazol, 2) danazol binding to rat testis microsomal cytochrome P-450, and 3) enzyme kinetics of danazol inhibition of the microsomal enzymes of testicular steroidogenesis. Concentrations of danazol as low as 1 micrometer suppressed LH-stimulated testosterone and androstenedione production in cultured Leydig cells. The addition of danazol to a preparation of testicular microsomes elicited a type I cytochrome P-450 binding spectrum, with an apparent spectral dissociation constant (Ks) of 4.8 micrometer. Danazol inhibited progesterone and 17alpha-hydroxy-progesterone binding to microsomal P-450 with apparent spectral inhibition constants of 2.4 micrometer and 2.8 micrometer, respectively. Danazol competitively inhibited 3beta-hydroxy-delta5-steroid dehydrogenase-isomerase (apparent enzymatic inhibition constant, KI = 5.8 micrometer), 17alpha-hydroxylase (KI = 2.4 micrometer), 17,20 lyase (KI = 1.9 micrometer), and 17beta-hydroxysteroid dehydrogenase (KI = 4.4 micrometer). These findings indicate that low concentrations of danazol directly inhibit steroidogenesis in the rat testis in vitro.

The intratesticular localization of enzymes of androgen and estrogen biosynthesis was studied in the ground squirrel (Citellus lateralis). In mature animals, interstitium and tubules were isolated by manual dissection. Microsomes were prepared and enzymes assayed by analysis of product formation after incubation with appropriate 3H-labeled substrates. In the immature testis, tubules and interstitium are not readily separable; thus, distribution was inferred after analysis of whole testicular microsomes from control, follicle-stimulating hormone (FSH)-treated, and luteinizing hormone (LH)-treated animals. To verify the cellular composition of tissues and the status of steroidogenic organelles in Leydig and Sertoli cells, samples were also analyzed by light and electron microscopy. In mature squirrels, enzymes of androgen biosynthesis were concentrated in the interstitium; however, levels present in the tubules were sufficient to account for a substantial fraction of whole testicular activity (1/3 to 1/5). By contrast, virtually all of the testicular aromatase was accounted for by that in the seminiferous tubules. The purity of these fractions was checked by light microscopy; they showed little cross-contamination. In whole testicular microsomes of immature squirrels, androgen biosynthetic enzymes had a much lower specific activity than in mature animals; however, the opposite was true for aromatase, its activity being approximately 5-fold higher in prepubertal animals. Luteinizing hormone treatment markedly stimulated hydroxylase and lyase but not aromatase. Luteinizing hormone also induced an increase in Leydig cell size and a dramatic proliferation of smooth endoplasmic reticulum. These changes were correlated with increased serum testosterone. As shown previously in rats, 3 beta-hydroxysteroid dehydrogenase was independent of LH control. Follicle-stimulating hormone had no effect on any of the enzymes studied, but induced some increase of agranular reticulum in Sertoli cells. Results from immature squirrels thus corroborate data from mature animals, showing a predominant interstitial location of androgen biosynthetic enzymes. While we cannot explain the absence of FSH stimulation of aromatase activity, the data do not refute the findings in mature animals showing a predominant tubular location of this enzyme. We conclude that the distribution of steroidogenic enzymes in the testis of squirrels differs in several important respects from rats, although both are members of the order Rodentia.

Sex and regional differences in aromatase activity were characterized in brains of rats sacrificed on embryonic days 18 (E18) and 20 (E20) and on postnatal days 2, 4 and 13 (D2, D4, D13). Aromatase activity was measured in vitro in homogenates of the hypothalamus/preoptic area (HPOA) and temporal lobe (TL) from individual rats, using [3H]19-hydroxyandrostenedione as substrate in the presence of NADPH. The apparent Km of aromatase for 19-hydroxyandrostenedione in TL at D4 was similar in males (34 nM) and females (22 nM,). Aromatase activity in the HPOA was highest prenatally (E18) and gradually declined to low levels by D13. Aromatase activity in the TL remained constant from E18 to D2, increased significantly on D4, and subsequently declined to low levels by D13. The level of aromatase activity was significantly greater in males than in females on E18 and D4 in the HPOA and on D4 in the TL. Differences in aromatase activity within regions of the HPOA were studied at E20 and D4. At both ages, the activity was highest in the preoptic area, lower in the anterior hypothalamus (AH), and lowest in the posterior hypothalamus. Aromatase activity was significantly higher in males than in females in the AH, but only on D4. The contiguity of males in utero was not correlated with aromatase activity in brain regions of adjacent female fetuses. The present results indicate that significant sex differences in aromatase activity exist in specific brain regions only at discrete times during perinatal development.(ABSTRACT TRUNCATED AT 250 WORDS)

In the spiny dogfish (Squalus acanthias), germ cells and adjacent steroidogenic elements are topographically segregated within the testis according to stage of development. In the experiments reported here, we have taken advantage of this favorable anatomical arrangement to demonstrate stage-specific variations in steroidogenic enzymes and estrogen receptor number. The testes of Squalus collected in July-November were sectioned transversely and further subdivided into three zones as follows: I) germinal bed plus immature lobules with spermatogonia; II) lobules with primary or secondary spermatocytes; III) lobules with spermatids or mature spermatozoa. The morphology of these zones was verified by light microscopy and, in a separate study, by electron microscopy. Through the course of spermatogenesis, Sertoli cells increased dramatically in size and in the abundance of steroidogenic organelles. By contrast, interstitial tissue was sparse in all stages of development, and only relatively undifferentiated Leydig-like cells were present. Microsomes prepared from each zone were incubated with [3H] progesterone, [3H]17 alpha-hydroxyprogesterone, or [3H]androstenedione to evaluate androgen and estrogen biosynthetic potentials. Based on product formation, 17 alpha-hydroxylase and C-17,20-lyase activities increased progressively from less mature to more mature regions (zone III greater than II greater than I), whereas aromatase was greatest in regions undergoing meiosis or early spermiogenesis (zone II). These enzymes were not detected in semen, although C21 substrates were converted to unidentified polar metabolites in high yield. Estrogen receptors were concentrated in immature zones (zone I greater than II greater than III), and the percentage of occupied receptors revealed the same distribution. Semen and epigonal tissues were receptor negative. We conclude that Sertoli cells are responsible for steroidogenesis in Squalus testis and that hormone production is keyed to the spermatogenic cycle. The data are consistent with an important role for androgens during spermatid maturation and/or after sperm release, but reveal that estrogen actions are expressed primarily during early spermatogenic stages. This report demonstrates the usefulness of unconventional animal models for obtaining new information of general relevance.

Experiments were conducted to study the regulation of the developmental pattern of aromatase in the forebrain of the perinatal rat. Two experimental designs were used: aromatase measured in primary cultures of fetal hypothalamic cells and in cell-free preparations of forebrain tissue excised at varying ages. In cultured cells, aromatase decreased logarithmically at a slow rate (t1/2 = 7.8 days). Norepinephrine caused a pronounced dose (4 x 10(-6) M) and time-dependent (2-6 days) drop in aromatase without affecting the levels of 5 alpha-reductase or substance P. In isolated tissue, aromatase activity was compared with the concentrations of norepinephrine and dopamine in the forebrain of males vs females at different perinatal ages and in discrete forebrain areas at postnatal day 4. In no case was a sex difference in catecholamines seen. An overall developmental decline in aromatase was associated with developmental increases in catecholamine levels. Acute treatment with the beta-agonist, isoproterenol, had no effect on brain aromatase activity.

The interaction of 19-norethindrone [4-estren-17 alpha-ethinyl-17 beta-ol,3-one (NET)] with human placental microsomes was investigated using enzymatic and spectral techniques. The incubation of [6,7-3H]norethindrone with human placental microsomes, NADPH, and molecular oxygen resulted in the production of ethinyl estradiol [1,3,5-(10)estratrien-17 alpha-ethinyl-3,17 beta-diol (EE)]. The reaction was linear with respect to time and protein concentration. Androstenedione inhibited the enzymatic aromatization of NET to EE. The product was identified by thin layer chromatography, recrystallization to constant specific activity, and derivative formation. No acid or base was used in any step of product identification. To ensure that spontaneous aromatization of metabolites of NET did not contribute to our results, representative samples were treated with sodium borohydride before processing. Sodium borohydride reduces the 4-en-3-one grouping of the A-ring, thereby preventing chemical aromatization. Sodium borohydride treatment did not reduce our observed yields of EE from NET. The addition of NET to a preparation of solubilized, partially purified placental microsomal cytochrome P-450 yielded a type I cytochrome P-450 binding spectrum. The apparent spectral dissociation constant for NET binding to cytochrome P-450 was 28 microM. These results suggest that NET is enzymatically aromatized to EE by human placental microsomes.

Male ferrets born in the laboratory received subcutaneous Silastic capsules containing either the aromatase inhibitor, androst-1,4,6-triene-3, 17-dione (ATD), the 5 alpha-reductase inhibitor, testosterone-17 beta-carboxylic acid (17 beta C), or no hormone, for 15 days beginning on the day of birth; an additional group of females received empty Silastic capsules. All ferrets were gonadectomized when 11 weeks of age and were subsequently tested for masculine sexual behavior after a latin-square sequence of treatments with subcutaneous Silastic capsules containing testosterone (T), estradiol (E), or dihydrotestosterone (DHT). After T, control males displayed significantly more neck gripping, mounting and pelvic thrusting than control females, and males treated neonatally with ATD or 17 beta C were no less responsive than control males. After DHT, little masculine sexual behavior was shown by any group. After E, the duration of mounting was significantly longer in control and ATD males than in control females or 17 beta C males. Subsequently, however, there were no differences between control and 17 beta C males on any parameter of masculine sexual performance, when they were retested sequentially after subcutaneous implantation of E followed by E + DHT. Additional groups of newborn male and female ferrets received subcutaneous capsules containing either ATD, 17 beta C, or no hormone and were killed on postnatal day 7. Administration of ATD, but not 17 beta C, strongly inhibited aromatase activity in the hypothalamus + preoptic area. In all groups, the formation of significantly inhibited cortical 5 alpha-reductase activity. Plasma concentrations of T were equivalent on postnatal day 7 in males given each of the neonatal treatments. These results suggest that behavioral masculinization in the male ferret results primarily from the neonatal action in brain of T itself, and not from its estrogenic or 5 alpha-reduced androgenic metabolites.

The microsomal fraction isolated from the testis of the urodele amphibian, Necturus maculosus, is very rich in cytochrome P-450 and three cytochrome P-450-dependent steroidogenic enzyme activities, 17 alpha-hydroxylase, C-17, 20-lyase, and aromatase. In this study, we investigated aspects of these reactions using both spectral and enzyme techniques. In animals obtained at different points in the annual cycle, Necturus testis microsomal P-450 concentrations ranged from 0.6-1.8 nmol/mg protein. Substrates for the three enzymes generated type I difference spectra; progesterone and 17 alpha-hydroxyprogesterone appeared to bind to one P-450 species while the aromatase substrates, androstenedione, 19-hydroxyandrostenedione, and testosterone, all bound to another P-450 species. Spectral binding constants (Ks) for these interactions were determined. Michaelis constants (Km) and maximum velocities were determined for progesterone 17 alpha-hydroxylation, 17 alpha-hydroxyprogesterone side-chain cleavage, and for the aromatization of androstenedione, 19-hydroxyandrostenedione, and testosterone. Measured either by spectral or kinetic methods, progesterone, androstenedione, and 19-hydroxyandrostenedione were high affinity substrates (Ks or Km less than 0.3 microM), while 17 alpha-hydroxyprogesterone and testosterone were low affinity substrates (Ks or Km = 0.6-4.8 microM). As evidence for the participation of cytochrome P-450 in these reactions, carbon monoxide was found to inhibit each of the enzyme activities studied. The activity of NADPH-cytochrome c reductase, a component of cytochrome P-450-dependent reactions, was also high in Necturus testis microsomes.