Increase Of 17-OHP Research Articles

Determination of estrone sulfate, testosterone, androstenedione, DHEAS, cortisol, cortisone, and 17α-hydroxyprogesterone by LC-MS/MS in children and adolescents

Quantitation of endogenous steroids and their precursors is essential for diagnosis of a wide range of endocrine disorders. Usually, these analyses have been carried out using immunoassays. However, immunoassays often overestimate concentrations due to assay interference by other endogenous steroids, especially for low concentrations. Mass spectrometry based methods offer superior specificity, accuracy, and sensitivity. We therefore present a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method with automated sample preparation for determination of 17α-hydroxyprogesterone (17OHP), cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione (A4), testosterone (T), and estrone sulfate (E1S). Samples were prepared using protein precipitation and 96-well filter plates, fully automated in a pipetting robot and analyzed by LC-MS/MS. Serum samples from 187 healthy children and adolescents aged 5-18 years were used to study hormone changes in relation to sex and pubertal stage. Lower limit of quantification for 17OHP was 0.7 nmol/L, for cortisol 11 nmol/L, for cortisone 2 nmol/L, for DHEAS 0.1 µmol/L, and for A4, T, and E1S, 0.2 nmol/L. This study showed a general increase in 17OHP, DHEAS, A4, T and E1S in both genders during puberty. In boys, A4 and T increased significantly throughout pubertal development. Girls had significantly higher A4 and E1S concentrations, while boys had higher T concentrations. No sex- or puberty-specific differences were seen in cortisol or cortisone concentrations. To the best of our knowledge, this is the first presentation of changes in serum E1S concentrations during pubertal development in healthy children.

SUN-931 Characterization of an Ovarian Steroid Cell Tumor in a VHL Patient

Most ovarian steroid cell tumors arise sporadically. However, they can also be observed as a rare manifestation of von Hippel Lindau disease. Here, we present a clinical, pathological and molecular characterization of a steroid cell tumor in a VHL patient. A 14 year old girl with molecularly confirmed diagnosis of VHL developed hirsutism and amenorrhea. Initial clinical hormonal evaluation was notable for elevated 17-OHP of 406ng/dl, androstenedione 275ng/dl, and testosterone 102ng/dl. In order to exclude congenital adrenal hyperplasia as a common cause of hirsutism in adolescents, ACTH stimulation was performed, but no increase in 17-OHP was observed. Anti Muellerian hormone, inhibin (INH) A and INH B were normal. Imaging revealed a bilobed 6cm left adnexal mass. The mass was resected en bloc via a left oophorectomy. Pathological evaluation showed multinodular steroid cell tumor with clear cytoplasm and delicate vascular meshwork. Immunoprofiling demonstrated positivity for inhibin and calretinin; while renal cell carcinoma markers were negative. All laboratory values normalized post-surgery. In addition to clinical measurements pre- and post-surgery, steroid profiles were evaluated by LC-MS/MS. Quantitative RT-PCR analysis showed robust tumor expression of enzymes facilitating the production of androgens, but not estrogens. Further preliminary analysis by exome sequencing confirmed the known germline pathogenic variant in VHL, but no additional obvious somatic driver mutations were identified. Interestingly, the NGS analysis of different specimens from the same tumor revealed multiple different single base pair variants in the VHL gene as a second hit. In summary, hirsutism in VHL patients should raise the suspicion for unusual ovarian tumors. In contrary to the usual theory of a monoclonal expansion after loss of the wt VHL allele, this tumor appeared to be oligoclonal as evidenced by different somatic VHL mutations. This could be either explained by initial parallel occurrence of several clones or that the VHL second hit is not an initial event, but the mutation instead supports tumor expansion following initial steps of tumorigenesis.

Relationships of Basal Level of Serum 17-Hydroxyprogesterone with that of Serum Androstenedione and Their Stimulated Responses to a Low Dose of ACTH in Young Adult Patients with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency

A single measurement of serum 17α-hydroxyprogesterone (17OHP) level can be unreliable because of its marked diurnal variation. We investigated the relationship of serum level of 17OHP with that of androstenedione (AD), which shows a smaller diurnal variation. And we tested whether the responses of these two hormones to low-dose ACTH stimulation are correlated in patients with 21-hydroxylase deficiency. Baseline serum 17OHP and AD levels were measured in 87 patients and a low-dose ACTH stimulation test was performed in 41 patients. The basal 17OHP level correlated positively with the basal AD level independently of sex, type of 21-hydroxylase deficiency, and the time of day of blood sampling (n = 87, R2 = 0.75, P < 0.001). The area under the curve of 17OHP and AD correlated positively with their respective basal levels. The fold-change increase in 17OHP after ACTH injection correlated negatively with the basal 17OHP level, but that of AD did not correlate with the basal AD level. The random serum 17OHP level, used in the clinic, is a reliable guide and a low-dose ACTH stimulation test provides no extra benefit for assessing the treatment adequacy in patients with 21-hydroxylase deficiency.

Licorice consumption and serum testosterone in healthy man.

We have previously found that licorice can reduce serum testosterone in healthy men. These results were not confirmed in another study, where the same amounts of licorice did not decrease salivary testosterone values. In the actual study we treated more cases with the same amount of licorice and reproduced our previous data. The mean testosterone values decreased by 26 % after one week of treatment (p < 0.01). There was also a significant increase in 17-OHP and LH concentrations and a slight, but not significant decrease in free testosterone. Licorice treatment, in addition, did not affect the response of testosterone and 17-OHP to stimulation with beta-HCG.

In vivo ovarian responses to recombinant follicle-stimulating hormone (FSH) with and without recombinant luteinizing hormone (LH) in polycystic ovary syndrome (PCOS).

Objective: To contrast the effects of recombinant FSH (rFSH) with the added effects of recombinant LH (rLH) in a 1:1 ratio on ovarian secretion of 17α-hydroxyprogesterone (17OHP), androstenedione (δ4) and testosterone (T) in women with PCOS during ovulation-induction. Design: Prospective, controlled study in a University-based fertility centre. Materials/Methods: Forty-eight clomiphene-resistant women with PCOS were allocated to receive either daily injections of rFSH (n = 24) or rFSH+LH (n = 24) in a 1:1 ratio starting: 1) on day 2–3 of progestogen-induced menses (n = 8); 2) after 6 weeks of GnRH agonist treatment (nafarelin, 400 μg b.i.d; n = 8); or 3) after nafarelin treatment as in (2) plus dexamethasone (n = 8). After the initial 5-day fixed gonadotropin regimens, the dosage was adjusted according to serum E2 levels and follicle size and number on transvaginal ultrasound assessment. hCG was given [Congruent]24 hours after the last gonadotropin dose to trigger ovulation when the E2 levels were 2000 pg/ml and at least 1 leading follicle ≥15 mm (mean diameter) was seen on ultrasound examination. Between-regimen and within-subgroup interactions in serum levels of LH, 17OHP, δ4 and T were evaluated by ANOVA with specific treatment days as a repeated measures factor. Results: Pre-study hormone levels were similar for all groupings. Nafarelin significantly suppressed LH levels, which remained at the lower limit of assay sensitivity (0.5 IU/L) during stimulation with rFSH but increased significantly to >1 IU/L when rLH was added. Independent of exogenous rLH treatment, there was a small, but uniform increase in 17OHP, δ4 and T levels in the pre-ovulatory period. As expected, a greater increase occurred in subjects on rFSH + rLH. Dexamethasone further suppressed 17OHP, δ4 and T levels and unmasked a small, but significant rise in these ovarian steroids 24 hours following the first dose of rFSH + rLH, a rise that was absent with rFSH alone. Secretion of these steroids then appeared to “catch-up” after 5 days of rFSH stimulation. Conclusions: The differing secretion patterns would suggest greater reliance on compensatory paracrine mechanisms in production of these steroids when only rFSH is given following pituitary/adrenal suppression. Ideal ranges for LH levels during gonadotropin treatment remain to be explored but suppression of LH to suboptimal levels may increase the vulnerability of some of the processes involved in follicle development in the in vivo setting. Supported by: This work was supported by a research grant from the University of Alberta and Hospitals and Serono Canada Inc.

Maternal serum androgens in human pregnancy: early increases within the cycle of conception

Previous studies have demonstrated elevations in testosterone and androstenedione initiated within the cycle of conception in pregnant non-human primates, and minimal data in the human support the same picture. In the present study we have investigated a group of patients scheduled for artificial insemination with regular menstrual cycles. For this study all patients provided blood samples at 5 days after the luteinizing hormone (LH) surges and daily through the luteal phase and into early pregnancy (n = 12). Patients who did not become pregnant served as normal controls (n = 9). We have measured 17-hydroxyprogesterone (17-OHP) as a marker of luteal activity not obscured by progesterone within the cycle of conception and testosterone and androstenedione as the major androgens. There were no significant changes in testosterone and androstenedione in the non-pregnant controls, but both testosterone and androstenedione were significantly elevated in the pregnant luteal phase, with the first increases occurring at 15 and 14 days respectively after the LH surge. Three of 12 pregnant patients did not demonstrate a dramatic increase in either testosterone or androstenedione and when examined more carefully a corresponding lack of increase in 17-OHP in those same subjects indicated less than optimal luteal activity, suggesting that these androgens were products of the corpus luteum. In three subjects in which consecutive non-pregnant and pregnant cycles were followed there was a dramatic increase from the non-pregnant luteal phase to the pregnant luteal phase indicating that the more important observation may be the concentrations of androgens in the conceptive luteal phase compared to some baseline, either previous luteal phase or even follicular phase. We have also studied changes in dehydroepiandrosterone sulphate and found that there was no significant contribution to this increase in androgens in early conception. These studies demonstrate a significant increase in both testosterone and androstenedione presumably of ovarian, specifically luteal, origin and that adrenal androgen production is not a factor in these changes.

Periovulatory hormonal dynamics: relationship of immunoassayable gonadotropins and ovarian steroids to the bioassayable luteinizing hormone surge in rhesus monkeys.

The relationship between ovarian steroids and LH during the midcycle gonadotropin surge is controversial. Recent demonstration of temporal and quantitative differences in immunoassayable LH (I-LH) and bioassayable LH (B-LH) at midcycle have further clouded this issue. To evaluate the relationship of I-LH, FSH, and ovarian steroids to the onset of the midcycle B-LH surge, blood samples were obtained from five chronically catheterized rhesus monkeys at 2-h intervals for 5-6 days. The plasma was assayed for FSH, LH, 17 beta-estradiol (E2), progesterone (P4), and 17 alpha-hydroxyprogesterone (17-OHP) by RIA and for LH by a rat interstitial cell testosterone in vitro bioassay. The initiation of the B-LH surge served as time zero (to) for the temporal analysis of changes in plasma hormone levels. The I-LH and FSH surges were initiated 6.4 +/- 2.2 h (mean +/- SEM) and 5.2 +/- 1.9 h, respectively, after the onset of the B-LH surge. Although the duration of the ascending limb of the surge was similar for B-LH, I-LH, and FSH, the mean +/- SEM total duration of the B-LH surge (34.5 +/- 3.5 h) was significantly longer (P less than 0.025) than those of I-LH (24.4 +/- 5.0 h) and FSH (27.6 +/- 2.3 h). Before the onset of the B-LH surge (to - 4 h), the ratio of B-H to I-LH was unity; however, during the acme of gonadotropin secretion (to + 12-16 h), the B-LH to I-LH ratio approached 6:1. Doubling times for B-LH, I-LH, and FSH were similar during the ascending phase of the surge. Plasma E2 concentrations increased continuously from to - 40 h to to, with a mean +/- SEM doubling time of 32.6 +/- 4.7 h. Peak E2 concentrations occurred within 6 h after the onset of the B-LH surge. Plasma P4 concentrations began to increase at to - 6 h in four monkeys and at to in one monkey. Plasma P4 concentration plateaued from to to to + 24 h, then increased rapidly, with a mean +/- SEM doubling time of 20.5 +/- 2.9 h. Although there were significant individual variations in plasma 17-OHP concentrations, a definite increase in 17-OHP occurred by to - 10 h, and peak concentrations occurred at to + 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)

467 MARSHALL-SMITH SYNDROME: ANDROGEN ABNORMALITIES

In 1971 Marshall et al described a syndrome consisting of markedly accelerated skeletal maturation, relative failure to thrive and dysmorphism. Since then 11 cases have been reported. With the exception of an elevated testosterone in a neonatal female, hormonal evaluations have been normal. We present a male with Marshall-Smith Syndrome with abnormal adrenal androgen production. A.S. was noted at birth to have a bone age of 2 yrs. At age 8 mos. the bone age was 6 yrs. Noted were generalized hirsutism, prominent forehead, low set ears, shallow orbits with prominent eyes, small triangular upturned nose, hypoplasia of facial bones, long, tapered fingers, inguinal testes, small scrotum and a penile length of 5¼ cm. The following were normal for age: T4, FSH, LH, somatomedin-C, testosterone, testicular response to HCG, and androgen receptors in cultured skin fibroblasts. Seventeen hydroxyprogesterone (17-OHP) was elevated (840 ng/dl; nl 30-100) as was androstenedione (67 ng/dl; nl<50). An ACTH stimulation test was done. A 30 min. increase in 17-OHP of 23.4 ng/dl/min. suggested an adrenal enzymatic defect and hydrocortisone 20 mg/m2/day was begun. The androstenedione decreased to 13/ng/dl and on therapy has remained normal. ACTH stimulation tests were performed on both parents. The 30 min. increase in 17-OHP was elevated in both (father-13.8 ng/dl/min; mother-8.9 ng/dl/min; nl<6.5 ng/dl/min) suggesting a heterozygous state for congenital adrenal hyperplasia. It is speculated that an inherited abnormality in androgen production may be contributory to the osseous maturation seen in the Marshall-Smith Syndrome.

Linkage studies between HLA-A,B,D alleles and congenital adrenal hyperplasia (CAH)

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.

Progress in testing for heterozygosity in congenital adrenal hyperplasia (C A H )

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.