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In 19 pregnancies at risk for 21-hydroxylase deficiency (21OHD) in 18 families with at least one affected child, prenatal diagnosis was performed by RFLP analysis using the enzymes Taq I and EcoRI and the DNA probes specific for the 21OH genes, the closely linked complement C4 genes and the highly polymorphic HLA class II genes DRB, DQB, and DPB. For fetal DNA analysis either chorionic villi or cultivated amniotic cells were used. In all 19 cases, a clear prenatal diagnosis was possible either with the 21OH probe alone or in most cases, by combining the results of the different closely linked loci.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a disorder with different clinical manifestations, that results from mutations in the P-450(c21) gene. Direct sequence analysis of P-4 50(c21) genes in a family demonstrates that patients with different clinical forms of congenital adrenal hyperplasia can have identical P-4 50(c21) genes, suggesting that other effects play a role in developing the different clinical forms.

Ten patients with anorchia were treated with testosterone undecanoate (40 or 80mg/d) starting at the mean age of 11.5 years (range: 10 - 14 years). The effect on Tanner stage development, growth velocity and weight gain are presented. Therapy was started with 40mg 3x/week (n= 4), 40mg 7x/week (n=6), and was increased to 80mg 7x/week (n=9). Therapy was continued for 23.7 months (range: 14 - 40 months). Stretched penile length before therapy was x = 3.35cm (range: 2.5 - 4cm), at the end of therapy 6.25cm on average (range: 5 - 8cm). Pubic hair Tanner stage 2 (PH2) was achieved after 6.7 months (range: 4 - 14 months) PH3 after 20 months (range: 12 - 29 months). Growth velocity increased to 6.7cm/year (range: 3 - 9cm/year) in the first year of treatment, concomitant weight gain was 7.2kg (range: 5.2 - 10kg). No side effects of treatment were observed. We conclude that oral testosterone undecanoate therapy starting at the age of 11 years with 40mg/d for one year and 80mg/d for a second year is simple, safe and effective and provides for a gentle induction of puberty in anorchid boys.

In 18 families at risk for the HLA-linked, 21-hydroxylase deficient form of autosomal recessive congenital adrenal hyperplasia (CAH), prenatal diagnosis (PD) was performed using two methods: (1) HLA-A,B,C typing and in the latter 11 cases also DR typing of cultured amniotic fluid cells (AFC) using the standard microcytotoxicity assay, and (2) measurement of second trimester amniotic fluid 17-hydroxyprogesterone (17-OHP) concentration using gel chromatography and radioimmunoassay. The accuracy of the prenatal predictions was confirmed by postnatal HLA typing of umbilical cord blood lymphocytes and by clinical evaluation. In 16/18 families, both HLA typing of AFC and 17-OHP measurements proved informative for PD. The predictions of both methods were concordant in 14/16 families (88 per cent). In ten of these families, a normal fetus was predicted, and in four, an affected fetus; all pregnancies were carried to term and all predictions were confirmed postnatally. In 2/16 cases (12 per cent), however, the predictions were discordant: the prenatal HLA typing indicated an affected fetus, whereas the 17-OHP values predicted a normal fetus. Both pregnancies were continued and two healthy boys were delivered. The discordance proved to be due to a 'missed' HLA antigen in one case and to serologically cross-reactive HLA antigens in the second. Finally, in 2/18 cases, prenatal assessment of fetal genotype had to rely on HLA typing alone as 17-OHP measurement was not performed in one family and in the second family the 17-OHP values obtained were not informative due to inadvertent continuation of hormone therapy to the date of amniocentesis. In both cases, the HLA typing data accurately predicted a normal fetus. In conclusion, a combination of HLA typing of cultured AFC and 17-OHP measurements of amniotic fluid permits accurate prenatal diagnosis of CAH in most cases (88 per cent). In addition, the supplementary use of HLA-DR typing of AFC as presented here for the first time proved helpful in families with HLA-A,B homozygosity due to parental sharing of antigens and can be informative for identifying HLA-B/21-OH recombinant haplotypes.

The increase of plasma cortisol (F), androstenedione (A), 17α-hydroxyprogesterone (17-OH-P) and testosterone (T) was measured after iv administration of ACTH in heterozygotes for CAH and in controls under two different conditions: Test 1: ACTH stimulation was performed without any particular preparation. Test 2: 1.5 mg of dexamethasone (dex.) was given the evening before the ACTH stimulation. Plasma F, A, 17-OH-P and T were measured by specific radioimmunoassays (RIA). Following ACTH stimulation, the increase of 17-OH-P was significantly higher in CAH-heterozygotes than in controls in both tests (P < 0.0005). Heterozygotes were characterized by a 17-OH-P increase after ACTH stimulation exceeding the +2 sd limit of the 17-OH-P increase found in controls. The detection of female heterozygotes was considerably improved by the administration of dex. before testing (test 2). In males, however, a better identification of heterozygotes was obtained without previous administration of dex. (test 1). By these tests, 100% of female (test 2) and 79% of male (test 1) CAH heterozygotes could be correctly identified. There were no significant differences in the levels of F, A and T between heterozygotes and controls except for decreased T levels in test 1 (2P < 0.01) in most male heterozygotes after ACTH stimulation.

We investigated basal and ACTH stimulated levels of cortisol, corticosterone, 17 alpha-hydroxyprogesterone, 11-deoxycortisol and 11-deoxycorticosterone as well as plasma levels of ACTH before and during the oral administration of ketoconazole in five patients with Cushing's syndrome (3 with bilateral adrenal hyperplasia, 1 with adrenal adenoma and 1 with adrenal carcinoma) and in three controls. The influence of ketoconazole on the transformation of 3H-17 alpha-hydroxyprogesterone to 3H-11-deoxycortisol and 3H-cortisol and of 3H-11-deoxycortisol to 3H-cortisol as well as of 3H-11-deoxycorticosterone to 3H-corticosterone was also examined in slices or homogenates of normal and hyperplastic adrenal tissue from four patients. Ketoconazole induced a rise of 11-deoxycortisol and 11-deoxycorticosterone, but not of cortisol and inconsistently of corticosterone which were increased by ACTH. Thus the ratio 11-deoxycortisol/cortisol rose more after ketoconazole than after ACTH and the ratio 11-deoxycorticosterone/corticosterone rose after ketoconazole but fell after ACTH. Plasma ACTH levels were stimulated 2-50 fold by ketoconazole. Incubation studies of adrenal tissue slices with 3H-17 alpha-hydroxyprogesterone showed that ketoconazole inhibited the transformation of 3H-17 alpha-hydroxyprogesterone to 3H-cortisol but not to 3H-11-deoxycortisol so that the ratio 3H-11-deoxycortisol/3H-cortisol increased 15-80 fold. After incubation of adrenal slices with 3H-11-deoxycortisol or 3H-11-deoxycorticosterone and ketoconazole, a 2-260 fold increase of the ratios 3H-11-deoxycortisol/3H-cortisol and 3H-11-deoxycorticosterone/3H-corticosterone were also found.

Summary: Blood volume, plasma volume and red cell mass, red cell parameters, reticulocytes, serum iron, and total iron binding capacity were investigated. Fifty-nine growth-retarded children were studied: 27 children with idiopathic short stature (ISS) and 32 children with isolated growth hormone deficiency (GHD). Fifteen of the CHD patients were studied before treatment and again 3–6 months after onset of treatment with growth hormone, 3 GHD patients only before and 14 only during treatment. Blood volume, plasma volume, and red cell mass were significantly (P < 0.005) decreased by 15% in the untreated GHD patients and were normal in the children studied during treatment with growth hormone compared with the values in ISS patients. In the 15 GHD patients studied before and during treatment, blood volume, plasma volume, and red cell mass increased by 17% to normal values (P < 0.0005) on treatment with growth hormone. No significant differences were demonstrable between values related to actual body weight and to corrected weight for height. Hematocrit, mean corpuscular volume, and reticulocytes remained unchanged. Serum iron and total iron binding capacity were normal before treatment. Serum iron increased and total iron binding capacity decreased significantly during growth hormone substitution. In a pair of twins with Russell-Silver dwarfism and GHD, growth hormone increased blood volume, plasma volume, and red cell mass as in the other GHD patients. In another pair of twins with Russell-Silver dwarfism without GHD, growth the hormone was without effect on blood volume. Speculation: Our results of decreased blood volume and red cell mass in children with isolated GHD and the increase of blood volume and red cell mass during growth hormone treatment provide direct evidence that growth hormone is stimulating erythropoiesis. The less pronounced decrease of blood volume and red cell mass in our patients compared with the literature data obtained on panhypopituitary and hypophyseetomized subjects may indicate that growth hormone, thyroxin, and other hormones act synergistically on erythropoiesis. In children with panhypopituitarism or isolated GHD, blood volume, plasma volume, and red cell mass were found to be decreased by approximately 30% below normal (28, 30). Therefore, Shahidi and coworkers (30) recommended checking of blood volume in order to distinguish between dwarfism due to panhypopituitarism or GHD and idiopathic short stature. Their reports, however, include only one patient with isolved GUD (28), or do not differentiate between children with isolated GHD and panhypopituitarism (30). The effect of growth hormone on blood volume in isolated GHD has not yet been studied.

The patient, diagnosed as a case of testicular feminisation in infancy, was examined at the age of 15 years because of severe symptoms of virilising puberty with poor breast development. Plasma steroid analyses revealed a 10-fold elevated androstenedione concentration (A: 1562 ng/100 ml). Testosterone (T: 266 ng/100 ml) was in the male pubertal range. Thus the A/T-ratio was far above normal. The oestrone/oestradiol ratio was also elevated (Oe1/Oe2: 10.2/2.2 ng/100 ml). A, T, Oe1 and Oe2 could not be suppressed by dexamethasone, but reacted promptly to fluoxymesterone (A: 781 ng/100 ml). hCG caused a further increase of the A/T-ratio (2220/246 ng/100 ml); ACTH did not alter the A-concentration. These findings together with simular investigations after gonadectomy suggest that the failure to convert A to T and Oe1 to Oe2 is essentially located in the testes. In vitro incubations of testicular tissue showed reduced 17-ketosteroid reductase activity in tissue slices and in the subcellular fractions microsomes and cytosole. This form of male pseudohermaphroditism can easily be detected already in infancy, if steroid analyses and stimulation tests are performed. In case of female sex assignment patients should be submitted to early orchidectomy in order to avoid virilisation in puberty.

Beschreibung eines Jungen mit Pseudohermaphroditismus maskulinus, mit normalem mannlichen Karyotyp, welcher bei der Geburt als Madchen angesehen worden war. Mit 12 Jahren Auftreten einer hochgradigen Gynakomastie. Steroiduntersuchungen wurden mit Radiogaschromatographie und mit radioimmunologischen Methoden durchgefuhrt.

In search for a biochemical marker to differentiate between adrenocortical carcinoma (AC) and adenoma (AA), plasma levels of the following steroids were studied preoperatively and postoperatively: 11-deoxycorticosterone (DOC), corticosterone (B), 11-deoxycortisol (S), and cortisol (F). Levels were measured by Sephadex LH-20 chromatography and specific radioimmunoassays. The subjects included eight children ages 2 years, 5 months to 9 years, 10 months. There were three girls and 5 boys with pseudoprecocious puberty due to adrenocortical tumors (histologically, four were AC and four, AA). The preoperative showed that DOC and S levels were elevated in all patients, F levels were elevated in four of eight children when compared with age-matched controls, whereas B was normal. Postoperatively, all levels returned to normal. The ratios of B/DOC and F/S as an index of adrenal 11 beta-hydroxylase activity were calculated. The preoperative ratios of B/DOC were markedly decreased in all patients with AC compared to controls (7.7,4.1,5.9,1.9 versus 23.5, median), but normal in three of four patients with AA (16.2, 29.6, 16.1). The F/S ratios were significantly lower in AC and AA when compared with controls. The data indicate a deficiency in 11 beta-hydroxylation in cases of adrenocortical tumors. Despite a still limited number of patients, the decreased B/DOC ratios may possibly indicate malignancy and could be helpful in distinguishing by biochemical means between benign and malignant adrenocortical tumors.

A method for the simultaneous determination of eight major corticosteroid hormones and precursors in 0.5–2.0 ml of plasma has been developed and is described in detail. After extraction of the unconjugated steroids from plasma to which tritiated steroid tracers had been added, progesterone (P), 11-deoxycorticosterone (DOC), 17-hydroxyprogesterone (17-OH-P), corticosterone (B), 11-deoxycortisol (S), aldosterone (A), cortisone (E) and cortisol (F) are simultaneously separated using eight mechanized 60 cm Sephadex LH-20 columns in parallel. Each of the isolated steroids is quantitated by radioimmunoassay after saving an aliquot for recovery counting. The combination of Chromatographic separation and appropriate antiserum resulted in high assay specificity. Water or steroid-free plasma blanks were undetectable, with sensitivities of the assays ranging between 0.020 (A) and 0.370 (E) ng/ml. There was very satisfactory precision and accuracy, with inter-assay coefficients of variation below 16.5% and linear relationships between added and found, respectively. Normal values are reported for males, females (follicular phase) and a small group of prepubertal children, revealing sex differences between the adults studied with higher A, B and P levels in females and higher 17-OH-P and E levels in males. The mineralocorticoids A and DOC were considerably higher in children than in adults. The highly practicable method is particularly useful in pediatric and experimental endocrinology.

Extract: Plasma concentrations of estrone and estradiol were determined by radioimmuno-assay of cord blood from 17 neonates and of venous blood of 15 neonates, 61 boys and 69 girls of all developmental stages, 27 healthy women, 20 healthy men, and 89 male and female adolescents with different disorders of sexual development. In cord blood both estrone and estradiol averaged 15,000 pg/ml. Blood drawn from neonates shortly after birth showed levels between 300 and 500 pg/ml. These levels fell to 5–15 pg/ml within a few days after birth, which is the normal range for both sexes up to 7 years. Before puberty, estrone rises earlier in girls than in boys. The increase of estradiol follows the onset of puberty. Boys reach adult levels in stage IV, whereas girls in stage V still have lower estrogen levels than mature women. We did not find higher levels in 29 adolescents with gynecomastia than in normal adolescents at the same stage of puberty. We observed abnormally high estrogens in children with congenital adrenal hyperplasia, but, in spite of some very high levels, we did not observe premature thelarche or gynecomastia in children with this disease. Six females and 10 males with panhypopituitarism, aged from 10 to 19 years, revealed low values within the infantile range. Six boys between 5 and 14 years who suffered from anorchia had very low levels of estrogen, especially estradiol (which was nearly undetectable). In contrast to patients with functioning testicle tissue, the patients with anorchia did not show any increase of estrogens after stimulation with human chorionic gonadotropin. Abnormally low levels were found in females with Turner's syndrome, but only in those with 45, XO karyotype. Patients with 45, XO/46, XX, or 46, XXp– karyotype had high levels of estrogen which corresponded to clinical signs of advanced puberty. Speculation: As demonstrated by our results, the radioimmunologic method enables us to measure the low estrogen levels which are found in children. This will permit a more intensive examination of the pituitary-gonadal and pituitary-adrenal interrelation in the future.

Plasma testosterone was investigated by gas-liquid chromatography with an electron capture detector in a cross-sectional study in childhood and during puberty in 214 boys. The testosterone values were correlated with sexual characteristics such as axillary hair, pubic hair, testicular size and bone age. In 22 individuals during puberty a longitudinal study was started, indicating a very steep increase of plasma testosterone between 40 and 240 ng/100 ml. This stage is mostly passed through very rapidly within 10 months. Plasma testosterone, growth velocity and weight gain were correlated in two boys during puberty. The peak of growth spurt occurs between 50 and 170 ng of plasma testosterone.

To investigate the adrenocortical suppression caused by the anesthetic etomidate, plasma levels of progesterone (P), 17-hydroxyprogesterone (17-OHP), 11-deoxycorticosterone (DOC), corticosterone (B), aldosterone (Aldo), 11-deoxycortisol (S), cortisol (F), and cortisone (E) were measured simultaneously before and after a short-term ACTH stimulation test in a 6.5-year-old boy whose convulsions could be kept under control only with constant etomidate infusions. During etomidate therapy, plasma levels of DOC and S were extremely elevated, the progestins P and 17-OHP were slightly elevated, whereas B and Aldo were in the lower normal range, and F and E were markedly decreased. A short-term ACTH stimulation test during etomidate infusion gave a blunted response of B, Aldo, F and E, whereas the level of DOC remained high and S even further increased. P and 17-OHP showed a positive response to ACTH. The ratios of B/DOC and F/S, which reflect adrenocortical 11 beta-hydroxylase activity, were extremely decreased during etomidate and did not change after ACTH stimulation. In contrast, the ratios of DOC/P and S/17-OHP, which reflect 21-hydroxylase activity, were elevated and remained elevated after ACTH stimulation. After discontinuation of etomidate therapy, all the baseline steroid levels were somewhat elevated, but responded normally to ACTH. These results demonstrate that etomidate causes a specific and reversible blockade of the 11 beta-hydroxylation of adrenal steroid synthesis.

Butenandt, O., Bidlingmaier, F. and Knorr, D. (Department of Paediatrics, University of Munich, Germany). Effect of different human growth hormone preparations on nitrogen retention in hypopituitary children. Acta Paediatr Scand, 63: 549, 1974.—The biologic activity of three different preparations of human growth hormone (HGH) obtained with the methods of Raben, Reisfeld and Roos respectively were compared in nitrogen retention tests in 18 patients with hypopituitarism and in 17 children with familial shortness of stature. HGH Raben produced a mean nitrogen retention of 30.9% in 11 hypopituitary patients and no retention in the control group, HGH Reisfeld was followed by a retention of 19.7% in the 9 hypopituitary patients and an elevated excretion of 12% in the control group, and HGH Roos evoked a retention of 33.2% only in the 9 hypopituitary children. HGH Reisfeld had a lesser response in consecutive tests with different HGH preparations in the same patients than the other two HGH preparations. The dilution curve of HGH Reisfeld obtained with a double antibody radio immunoassay did not parallel a dilution curve of a reference hormone from the National Institute of Health whereas the other two preparations had identical dilution curves. It is concluded that the method of extraction alters the biologic activity of the hormone as well as the physico-chemical properties.

The maternal adrenal cortex seems to be involved in the adaptation to pregnancy. To study in detail adrenocortical secretion during pregnancy, we measured plasma aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, and cortisone simultaneously by RIA after extraction and automated Sephadex LH-20 chromatography of 10 normal pregnant women longitudinally throughout pregnancy at weeks 8-10, 14-17, 21-24, 28-32, and 38 as well as at the time of admission to the delivery room. The mean plasma progesterone and 17-hydroxy-progesterone concentrations increased from 37.2 +/- 6.5 (+/- SE) and 8.2 +/- 1.0 nmol/L, respectively, in early gestation to maximum levels of 138.0 +/- 25.7 and 22.8 +/- 2.2 nmol/L at week 38 (P less than 0.01). Plasma glucocorticoid levels rose 2- to 3-fold (P less than 0.01) from weeks 8-10 (corticosterone, 18.5 +/- 5.4; 11-deoxycortisol, 1.9 +/- 0.2; cortisone, 24.2 +/- 4.2; cortisol, 195.5 +/- 37.6 nmol/L) to week 38 (corticosterone, 42.9 +/- 11.2; 11-deoxycortisol, 4.6 +/- 0.5; cortisone, 71.5 +/- 13.6; cortisol, 420 +/- 63 nmol/L). Similarly, plasma mineralocorticoid levels increased 5- to 7-fold (P less than 0.01) from weeks 8-10 (11-deoxycorticosterone, 0.69 +/- 0.12; aldosterone, 0.41 +/- 0.08 nmol/L) to maximum levels at week 38 (5.3 +/- 0.9 and 2.1 +/- 0.3 nmol/L, respectively). At the time of admission to the delivery room, plasma 11-deoxycortisol, corticosterone, and cortisol concentrations were higher (P less than 0.02) than at 38 weeks, but plasma progestin and mineralocorticoid concentrations were not. We conclude that the source of the elevated maternal corticosteroid levels in pregnancy in addition to the estrogen-mediated rise in corticosteroid-binding globulin is the maternal adrenal cortex itself. The peak glucocorticoid levels at admission to the delivery room reflect increased maternal and fetal stress with the onset of labor.

Plasma levels of aldosterone, corticosterone, 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, and cortisone were measured simultaneously by a micromethod of multisteroid analysis in eight vaginally delivered premature infants (PI) of 33-36 wk gestation with uneventful peri- and postnatal course. Mean concentrations (ng/ml) in umbilical arterial and in peripheral venous or capillary plasma sampled longitudinally at age 2 h to 7 days were compared with the same kind of data obtained from a group of 12 term infants (TI) who served as controls. Mean aldosterone was two to five times higher in PI than in TI (umbilical artery, 2 h to 7 days; p less than 0.05), whereas 11-deoxycorticosterone was lower in PI from 2 h (p less than 0.01) until 7 days (NS). Corticosterone was significantly higher in PI than TI at 6 and 24 h after birth, whereas cortisol was slightly lower (NS) in PI in umbilical artery and 2 h after birth, but higher (p less than 0.02) at 6 h, showing less variation in PI than in TI. 17-Hydroxyprogesterone levels in PI were two to three times higher (p less than 0.02) during 6 h until 7 days after birth. The data suggest that PI are able to maintain high aldosterone levels in the early neonatal period. Higher levels of the active glucocorticoids (cortisol and corticosterone) seen after delivery point to a more stressful extrauterine adaptation of PI. Furthermore, the data demonstrate that the adrenal cortex is fully functioning in premature infants (33-36 wk gestation) as well as in term infants.

We applied the ACTH-stimulation test developed in our laboratory for the detection of heterozygous carriers of the 21-hydroxylase deficiency gene to patients suffering from hirsutism (n=89), premature pubarche (n=75), early puberty (n=37), and precocious puberty (n=22). While, in the general population, this test is positive in less than 2%, we found in 33% of hirsute patients, in 41% of patients with premature pubarche, and in 33% of patients with early puberty a hormonal response similar to the one seen in heterozygous carriers for the 21-hydroxylase defect. In contrast, only 18% of patients with precocious puberty responded abnormally. Thus we speculate that, at least in some patients with hirsutism, premature pubarche, and early puberty, heterozygosity for the 21-hydroxylase defect plays a major role in the pathogenesis of these disorders.

The pharmacodynamics of plasma testosterone (T) and androstenedione (A) levels were studied in ten hypogonadal boys after oral administration of testosterone undecanoate (TU). Plasma T and A levels were measured by specific radio-immunoassays. Six hours after a single dose of 120 mg TU, there was a significant increase (P < 0.005) in plasma T and A with a median T peak level of 940 ng/100 ml. Furthermore, twelve agonadal boys treated with a mean dose of 60 mg TU/day were examined over a period of 18–24 months. During this therapy, plasma T and A levels were significantly higher than before (P < 0.005), whereas plasma levels of LH and FSH did not decrease significantly. With the exception of one anorchic boy, all patients showed signs of sexual maturation, such as growth of pubic and axillary hair, and steady development of bone age during oral TU treatment.

Congenital adrenal hyperplasia (CAH) is a disorder of Cortisol and aldosterone biosynthesis that mostly results from a 21-hydroxylase enzyme deficiency [1]. There is an incidence of about 1: 7000 to 1: 10 000 in central Europe [2–4]. It is characterized by increased plasma levels of 17-hydroxyprogesterone and elevated adrenal androgens, causing virilization.

We reported previously reference values for seven corticosteroids in the plasma of term neonates and their mothers, including values for aldosterone (Aldo) that appeared high compared to the values in the literature. Plasma 11-deoxycortisol (S) was not measured in that longitudinal study. Using an improved technique of chromatographic separation of Aldo and S, the Aldo levels were measured again, and S levels were newly determined in stored plasma samples of the 12 newborn infants of our original report. The mean concentrations were: (Formula: see text). These plasma Aldo values should replace those of our original report. Thus, both plasma Aldo and S levels decline in the first week of life in normal infants.

Corticosteroids (CS) are known to be essential for fetal organ maturation and seem to play an important role in both the initiation of parturition and the postnatal adaptation of the human neonate. Pharmacologically, CS are widely used for enhancing fetal lung maturation prior to premature delivery. However, knowledge of endogenous CS and precursor levels throughout fetal and perinatal life and their response to exogenous CS is limited. Therefore, using automated liquid column chromatography plus specific radioimmunoassays, unconjugated aldosterone (Aldo), corticosterone (B), 11-deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), 11-deoxycortisol (S), cortisol (F) and cortisone (E) were simultaneously followed in 70 amniotic fluid (AF) control samples throughout pregnancy, and in cord and neonatal plasma longitudinally during the first week of life. From 14 to 38 weeks, AF levels of all measured steroids except E rose by 2 to 12-fold on the average (allP

Mature female rabbits immunized with T-3-BSA developed highly specific antisera against T. Animals with the highest antibody titres revealed elevated plasma T-levels up to 2 000 ng/dl (normal: 15 ng/dl). Nevertheless, mating was successful and pregnancy uncomplicated. Immediately after normal delivery, the newborns were bled and fixed in Bouin's solution. In all neonatal plasma samples high concentrations of antibodies against T were found with identical characteristics as in the maternal plasma. T-levels were excessively elevated: up to 2 500 ng/dl in females and up to 25 000 ng/dl in males (normal: 20 ng/dl). Histological examination, however, revealed normal sex differentiation in the females, but inhibited male sex differentiation in the males: partial retrogression of Wolffian duct derivatives, absence of prostatic glands, feminized phallus with reduced corpus fibrosum, and marked hypospadias; testicular differentiation was normal, Mullerian ducts were absent. Our experiments indicate that hormones essential for normal fetal development can be neutralized in the fetus by their respective antibodies raised in the mother.

The small adrenal cortex of SGA infants may play a role in the pathogenesis of their frequent postnatal problems. We therefore evaluated adrenocortical function of SGA infants at birth and during postnatal adaptation. In 22 vaginally delivered term infants (SGA

Influence of the Mode of Delivery on Umbilical and Postnatal Plasma Levels of 7 Corticosteroids in the Newborn

To evaluate the development of the HPG axis the method of immunologic hormone inactivation was used to study male rat fetuses at different ages of gestation. From the 12th day of gestation pregnant rats were passively immunized against testosterone (T) by daily injections of rabbit antiserum with high binding capacity for T. Fetuses were studied on each day of gestation starting at day 18. Antibodies against T were detected in all fetal plasma samples. While the reduction of circulating free T by antibody binding did not affect the differentiation of the male genital tract it markedly stimulated the endocrine activity of the testes. At day 19 and all subsequent days of gestation the testicular T content -an index of circulating go-nadotropins- was significantly higher in immunized fetuses than in controls. However, this difference was not yet apparent at day 18 when the normal fetal rat testis shows its peak activity. These results indicate that in the male rat fetus the negative feedback between gonads and hypothalamo-pituitary system develops between day 18 and 19 of gestation when the differentiation of the genital tract is already well advanced.

Urinary estrone (E1) and estradiol (E2) glucuronide and sulfate were determined separately by RIA in 44 newborn and young infants (26 boys, 18 girls). In 7 cases the estrogen excretion was followed up over one month. The conjugates were hydrolyzed enzymatically; the estrogens were purified and separated by Sephadex LH 20 chromatography. During the first month, children excreted more sulphated than glucuronized estrogens with a ratio of 8:2, but within the first year, this ratio was reversed to 3:7. In both sexes E1 + E2 excretion was 10 times higher during the first 2 months than at the end of the first year. Boys showed a sharp decline during the third month of life; girls, however, did not reach low values until the sixth month. While the E2/E1 ratio in boys was one, girls showed predominance of E2-excretion with a mean ratio of 2.5:1 during the first 6 months of life.

Recently we have demonstrated inhibition of masculine differentiation in male offspring of female rabbits actively immunised against T. After passage through the placenta the T-antibodies not only inhibit the entry of T into the fetal target cells but also protect it from metabolism. Thus, besides marked feminisation, 1000-fold elevated plasma T levels in the male fetuses are observed. To assess the influence of T-antibodies on fetal testes we studied histologically the testes of 7 neonates born by 3 T-immunised rabbits and of 3 controls. No influence on testicular differentiation could be observed. However, the neonates immunologically deprived of T showed significantly enlarged interstitium and the number of Leydig cells was twice as high as in the controls. These findings indicate increased stimulation of the testes in these animals and suggest that normally even in the fetus a negative feedback is acting between testes and hypothalamo-pituitary system, which is interrupted by T-antibodies. Obviously T plays an important role in the control of the HPG system in the male fetus.

In our first study we found binding sites for ANP on human platelets. Binding studies on these easily accessible cells could be a useful model to investigate receptor-status in patients with elevated ANP plasma levels. We reported high ANP levels in neonates and in patients with heartfailure. Therefore we studied the number of binding sites on platelets in venous umbilical blood of term infants (n = 10), in adults (n = 15) with heartfailure and in controlsubjects (n = 18). For the binding studies 40 × 106 platelets were incubated with 10.000 cpm 125J-ANP and increasing concentrations of unlabeled ANP. Time of incubation was 120' at room temperature. Separation of bound and free tracer was performed by filtration through Whatman CF/G filters. In neonates we found 18-32 and in patients with heart failure 8-30 binding sites/platelet. In comparsion with control subjects (15-30 binding sites/platelet) there was only in patients with heartfailure a trend for reduction of binding sites (p < 0.1). The Kd (14 ± 8 pM) was similiar in all subjects studied. In our results there was no convincing evidence of “receptor down regulation” in the presence of high ANP levels as described in in-vitro experiments.

17-Ketosteroid Reductase Deficiency - Plasma Steroids and Incubation Studies with Testicular Tissue

The observation by Dupont et al.of close linkage between CAH and the HLA-complex prompted us to study the inheritance of HLA-haplotypes and the deficiency of C21-Hydroxylase(C21-H) in 12 families with CAH children.All family members were tested for CAH heterozygosity by the increase of plasma 17-OHP after ACTH stimulation.An increase of 17-OHP > 200 ng/dl was taken as evidence for CAH-heterozygosity.HLA-A,B,D typing was done by established serological and cellular methods.In 3 families with 2 CAH cases, we found identical HLA-A,B,D in the affected siblings. There was a good correlation between the results of the heterozygosity test and the segregation of HLA-haplotypes, in some cases over 3 generations.The distribution of HLA-A,B,D alleles in the 12 unrelated CAH-patients was not significantly different from controls,indicating that there is no positive association between C21-H deficiency and antigens of the HLA-series.Our data support the concept of close linkage of CAH and HLA allowing more precise prenatal diagnosis of CAH and identification of heterozygotes.

Increased mortality with long-term E infusions in intensive care patients has been reported and evidence is accumulating that E causes reversible adrenocortical suppression with decreased serum cortisol levels. A direct inhibition of adrenal steroidogenesis has been suggested, but so far data on the mechanism of this action are lacking. A 6 1/2 year old boy with convulsions due to encephalitis was treated with constant E infusions (1 - 2 mg/kg/h). Prior to E administration, adrenocortical function was normal as tested by an ACTH-test. 5 days after institution of therapy and 14 days after discontinuation, plasma levels of 11-deoxycorticosterone (DOC), corticosterone (B), 11-deoxycortisol (S) and cortisol (F) were measured by RIAs after Sephadex LH-20 chromatography. During E therapy DOC and S levels were highly elevated, while B was in the lower normal range and F was markedly decreased. Likewise the ratios of B/DOC and F/S, which reflect adrenocortical 11 β-hydroxylase activity, were extremely decreased (B/DOC:0.06 vs 27 in controls; F/S:0.009 vs 230 in controls). After discontinuation of therapy, the absolute values as well as the ratios returned to normal. These results show, that E is a potent inhibitor of adrenal 11 β-hydroxylation.Increased precaution seems to be necessary when this drug is used in patients who are under considerable amount of stress.

In order to obtain an improved differentiation between heterozygotes and normals, the increase of 17-OHP in plasma of CAH parents (n=30) and of controls (n=31) was measured after ACTH (Synacthen) stimulation i.v. at 60' under standardised basal conditions. The ACTH stimulation was performed after suppression of the adrenal cortex with 1.5 mg dexamethasone (Dex.) given the previous evening. Females were tested between days 3 and 8 of the menstrual cycle. Plasma 17-OHP was measured by a specific RIA. The mean increase of 17-OHP was significantly higher in heterozygotes than in normals (p=0.0005). In the control group, 100% of the males and 89% of the females showed an increase below 200 ng/d1. Assuming an increase of 200 ng/d1 as the dividing line for the differentiation, 54% of the CAH fathers and 100% of the CAH mothers were found to be heterozygotes. Because of the unsatisfactory differentiation of CAH fathers, we now carry out our former test without Dex. administration in males. However, for the detection of female heterozygotes, the test described here is a marked improvement.

To clarify the origin of estrogens in infant blood, we measured E1 and E2 in the gonads of 50 girls and 64 boys deceased by sudden death between birth and 2 years of age. In both sexes, E2 was the major gonadal estrogen which exceeded E1 almost tenfold in the ovaries and twice in the testes. Thus, there was a clear sex difference in gonadal estrogen concentrations. On an average, girls had 8 times higher E2 and twice as high E1 concentrations in their gonads than boys. As in plasma, E2 concentrations were highest in ovaries of 2 to 4 months old girls (2 - 55 ng/g) and in testes of 1 to 3 months old boys (0.6 - 6.4 ng/g). Ovarian E2 concentrations decreased to values below 3 ng/g until the end of the 1st year of life, testicular E2 reached values below I ng/g already by 6 months of age. The gonadal steroidogenic activity paralleled changes in gonadal morphology. Ovarian weights showed a similar pattern of rise and fall as the E2 concentrations, the biggest ovaries consisting of multiple macroscopic cysts. In boys, testicular E2 closely correlated with Leydig cell development and testicular testosterone concentrations. We conclude that the surge of E2 in the blood of infant girls and boys originates from ovarian follicles and testicular Leydig cells, respectively, which develop under the influence of the characteristic post-natal gonadotropin surge.

In our first studies we found elevated plasma levels of ANP and its second messenger cGMP in newborns and in children with cardiac diseases. Despite these high levels there is no effect on the water and electrolyte excretion. The lacking response to ANP in these patients may be explained by an enhanced release of other volume regulating hormones. To prove this hypothesis we measured plasma and urinary levels of ANP and cGMP as well as urinary excretion of aldosterone (Aldo) and vasopressin (AVP) in 22 healthy infants and 26 infants with cardiac diseases. Urine was collected for 8 hours from 10 p.m. to 6 a.m. In children with cardiac diseases we found significantly higher plasma levels of ANP ( = 225 ± 65 pg/ml) and cGMP ( = 8,5 ± 4,5 pmol/ml) and urinary levels of cGMP ( = 30 ± 5 nmol/8h/kg), Aldo ( = 800 ± 20 pg/8h/kg)and AVP (1100 ± 250 pg/8h/kg) than in control infants (p < 0.001). There was no correlation between ANP or cGMP levels and water- and sodiumexcretion.

E1, E2, LH and FSH were measured radioimmunologically in 81 plasma specimens of 28 sexually precocious girls 1 to 10 years of age. Prior to treatment in these girls the mean E1 value (15 pg/ml) was within the infantile normal range, but the mean values of E2. (26 pg/ml), LH (1, 2 ng/ml) and FSH (1, 6 ng/ml) were significantly elevated. However, out of 63 plasmas only 31 showed elevated E2, 24 LH and 34 FSH values above the normal range for age. Grouping the values according to breast development revealed considerably lower estrogens and gonadotropins in patients than in normally maturating girls of the same developmental stage. During treatment with CY (mean daily dose: 50 mg/m2) the mean estrogen and gonadotropin levels of 8 patients decreased significantly (E1 : p

Effect of opioid(pentazocine) and opioid-antagonist (naloxone) administration on plasma β-LH,FSH and Prolactin(PRL) levels in the perinatal period

Since corticosteroids have been shown to be essential for fetal organ maturation, knowledge of their endogeneous levels during fetal life is fundamental. Therefore, unconjugated progesterone(P) and 17α-hydroxyprogesterone(OHP), 11-deoxycorticosterone (DOC) and 11-deoxycortisol(S), corticosterone(B) and cortisol(F), aldosterone(A) and cortisone(E) were simultaneously determined by specific RIAs after automated LH-20 chromatography in 54 control samples of amniotic fluid obtained at 16 to 40 weeks of pregnancy. Whereas the progestins P and OHP increased significantly (p