Peptide hormone analysis in diagnosis and treatment of Differences of Sex Development: joint position paper of EU COST Action 'DSDnet' and European Reference Network on Rare Endocrine Conditions.

Differences of Sex Development (DSD) comprise a variety of congenital conditions characterized by atypical chromosomal, gonadal, or anatomical sex. Diagnosis and monitoring of treatment of patients suspected of DSD conditions include clinical examination, measurement of peptide and steroid hormones, and genetic analysis. This position paper on peptide hormone analyses in the diagnosis and control of patients with DSD was jointly prepared by specialists in the field of DSD and/or peptide hormone analysis from the European Cooperation in Science and Technology (COST) Action DSDnet (BM1303) and the European Reference Network on rare Endocrine Conditions (Endo-ERN). The goal of this position paper on peptide hormone analysis was to establish laboratory guidelines that may contribute to improve optimal diagnosis and treatment control of DSD. The essential peptide hormones used in the management of patients with DSD conditions are follicle-stimulating hormone, luteinising hormone, anti-Müllerian hormone, and Inhibin B. In this context, the following position statements have been proposed: serum and plasma are the preferred matrices; the peptide hormones can all be measured by immunoassay, while use of LC-MS/MS technology has yet to be implemented in a diagnostic setting; sex- and age-related reference values are mandatory in the evaluation of these hormones; and except for Inhibin B, external quality assurance programs are widely available.

The Long-Term Outcome of Boys With Partial Androgen Insensitivity Syndrome and a Mutation in the Androgen Receptor Gene.

BACKGROUND: In boys with suspected partial androgen insensitivity syndrome (PAIS), systematic evidence that supports the long-term prognostic value of identifying a mutation in the androgen receptor gene (AR) is lacking. OBJECTIVE: To assess the clinical characteristics and long-term outcomes in young men with suspected PAIS in relation to the results of AR analysis. METHODS: Through the International Disorders of Sex Development Registry, clinical information was gathered on young men suspected of having PAIS (n = 52) who presented before the age of 16 years and had genetic analysis of AR. RESULTS: The median ages at presentation and at the time of the study were 1 month (range, 1 day to 16 years) and 22 years (range, 16 to 52 years), respectively. Of the cohort, 29 men (56%) had 20 different AR mutations reported. At diagnosis, the median external masculinization scores were 7 and 6 in cases with and without AR mutation, respectively (P = .9), and median current external masculinization scores were 9 and 10, respectively (P = .28). Thirty-five men (67%) required at least one surgical procedure, and those with a mutation were more likely to require multiple surgeries for hypospadias (P = .004). All cases with an AR mutation had gynecomastia, compared to 9% of those without an AR mutation. Of the six men who had a mastectomy, five (83%) had an AR mutation. CONCLUSIONS: Boys with genetically confirmed PAIS are likely to have a poorer clinical outcome than those with XY DSD, with normal T synthesis, and without an identifiable AR mutation. Routine genetic analysis of AR to confirm PAIS informs long-term prognosis and management.

LC-MS/MS based determination of basal- and ACTH-stimulated plasma concentrations of 11 steroid hormones: implications for detecting heterozygote CYP21A2 mutation carriers.

OBJECTIVE: Heterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry. DESIGN: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared. METHOD: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years). RESULTS: Heterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30  min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%. CONCLUSION: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism.

Hypophosphatemic rickets with hypercalciuria due to mutation in SLC34A3/NaPi-IIc can be masked by vitamin D deficiency and can be associated with renal calcifications.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by mutations in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. Despite increased urinary calcium excretion, HHRH is typically not associated with kidney stones prior to treatment. However, here we describe two sisters, who displayed nephrolithiasis or nephrocalcinosis upon presentation. The index patient, II-4, presented with short stature, bone pain, and knee X-rays suggestive of mild rickets at age 8.5 years. Laboratory evaluation showed hypophosphatemia, elevated 1,25(OH) (2) vitamin D levels, and hypercalciuria, later also developing vitamin D deficiency. Her sister, II-6, had a low normal serum phosphorous level, biochemically vitamin D deficiency and no evidence for osteomalacia, but had undergone left nephro-ureterectomy at age 17 because of ureteral stricture secondary to renal calculi. Nucleotide sequence analysis of DNA from II-4 and II-6 revealed a homozygous missense mutation c.586G>A (p.G196R) in SLC34A3/NaPi-IIc. Ultrasonographic examinations prior to treatment showed grade I nephrocalcinosis for II-4, while II-6 had grade I-II nephrocalcinosis in her remaining kidney. Four siblings and the mother were heterozygous carriers of the mutation, but showed no biochemical abnormalities. With oral phosphate supplements, hypophosphatemia and hypercalciuria improved in both homozygous individuals. Renal calcifications that are presumably due to increased urinary calcium excretion can be the presenting finding in homozygous carriers of G196R in SLC34A3/NaPi-IIc, and some or all laboratory features of HHRH may be masked by vitamin D deficiency.

Anabolic steroids, testosterone-precursors and virilizing androgens induce distinct activation profiles of androgen responsive promoter constructs.

Different androgens, e.g. virilizing androgens such as testosterone and its precursors as well as synthetic anabolic steroids, respectively, induce diverse biological effects. The molecular basis for this variety in biological actions, however, is not well understood. We hypothesized that this variability of actions may be due to steroid-specific target gene expression profiles following androgen receptor (AR)-activation. Therefore, we investigated androgen receptor dependent transactivation of three structurally different androgen responsive promoter constructs ((ARE)(2)TATA-luc, MMTV-luc, GRE-OCT-luc) in co-transfected Chinese hamster ovary (CHO)-cells as an artificial model simulating different natural target genes. Three virilizing androgens (dihydrotestosterone, testosterone, methyltrienolone), three anabolic steroids (oxandrolone, stanozolol, nandrolone) and two testosterone-precursors of gonadal and adrenal origin (dehydroepiandrosterone, androstenedione) were used as ligands (0.001-100 nM). All steroids proved to be potent activators of the AR. Remarkably, anabolic steroids and testosterone-precursors showed characteristic promoter activation profiles distinct from virilizing androgens with significantly lower (ARE)(2)TATA-luc activation. Hierarchical clustering based on similarity of activation profiles lead to a dendrogram with two major branches: first virilizing androgens, and second anabolics/testosterone-precursors. We conclude that steroid-specific differences in gene transcription profiles due to androgen receptor activation could contribute to differences in biological actions of androgens.

Functional assessment and clinical classification of androgen sensitivity in patients with mutations of the androgen receptor gene. German Collaborative Intersex Study Group.

UNLABELLED: In the genetic male, mutations of the androgen receptor (AR) gene cause phenotypes ranging from female to subfertile male. Binding assays on genital skin fibroblasts and DNA analysis alone provide incomplete information about receptor function. We used the sex hormone-binding globulin (SHBG) response to stanozolol as a measure of AR function and correlated the results with phenotypes which were classified according to the degree of defective masculinization. Of the 34 patients investigated, 9 had complete, and 14 had partial androgen insensitivity syndrome (AIS) with predominantly female, ambiguous, or predominantly male phenotype. Eleven subjects served as controls. Mutations were characterized using polymerase chain reaction-single strand conformation polymorphism analysis and direct DNA sequencing. DNA analysis revealed two major deletions, two minor defects leading to premature stop codons in exon 1, and 19 point mutations in the DNA- and hormone-binding domains of the AR gene. After stanozolol, SHBG remained unchanged in patients with complete AIS (102.0 +/- 3.8 [SE]%; range 92.4%-129% of the initial value). The SHBG decrease was diminished in partial AIS with predominantly female (83.8% +/- 1.7%; range 81.3%-87.0%), ambiguous (80.4% +/- 4.4%, range 68.4%-89.1%), and predominantly male (mean 65.9% +/- 4.9%, range 48.6%-80.8%) phenotypes, and normal in controls (51.4% +/- 2.1%, range 35.6%-62.1%). Differences between controls and each AIS group were statistically significant (P < 0.05 - < 0.0001). A close correlation was found between the degree of undermasculinization (AIS phenotype) and the SHBG response. CONCLUSIONS: The SHBG test provides functional information about the severity of the receptor defect in vivo and hence adds to the structural information provided by DNA analysis. It detects receptor defects due to mutations within the entire gene, including the DNA-binding domain, and is a rapid, simple, and cost effective procedure. It may provide useful information for the diagnosis and management of affected children.

Hormonally induced changes in apocrine secretion of transglutaminase in the rat dorsal prostate and coagulating gland.

Coagulating gland and dorsal prostate of the rat are peculiar in secreting transglutaminase, a protein-cross linking enzyme that is released in an apocrine fashion. To elucidate whether or not the intracellular pathway and the unusual extrusion mechanism proceed constitutively or were differentially regulated, transglutaminase immunoreactivity was studied both at the light and electron microscopic levels. In addition, ultrastructural morphometry and scanning densitometry were applied to quantitate hormone-dependent distribution of transglutaminase. Coagulating glands and dorsal prostate, respectively, from sexually active rats were compared to those from sexually inactive, castrated, estradiol-treated or testosterone-substituted castrated animals. In intact, sexually active animals, no labeling of the cisternae of rough endoplasmic reticulum was seen, but instead the hyaloplasm was labeled. In the supranuclear portions of the cells an increase in labeling density of the hyaloplasm subjacent to the plasma membrane was found, whereas no labeling of either Golgi stacks or vesicles was observed. Apical blebs projecting into the acinar lumen were densely labeled. In castrated animals, epithelium showed a reduction of rough endoplasmic reticulum, loss of secretory blebs, and a decrease in cell size. Morphometric analysis of immunolabeling of coagulating gland epithelium from experimental animals resulted in a highly significant reduction of labeling of the hyaloplasm and apical blebs which was reversed by testosterone supplementation of castrated animals. After estrogen treatment, the reduction in immunolabeling was less pronounced, but morphology of apical blebs was obviously changed. Results from scanning densitometry of Western blots correlated with quantitative immunoelectron microscopical findings. Northern blot analysis using a secretory transglutaminase cDNA probe showed characteristic changes at the RNA levels. Our results indicate that apocrine secretion of transglutaminase in rat coagulating gland and dorsal prostate is a hormonally controlled process, where androgen deprivation results in impaired biosynthesis and release of transglutaminase, whereas estradiol treatment only partially inhibits secretion, but changes morphological features of the glandular epithelium, especially apocrine bleb formation.

Effects of androgen deprivation and estrogen treatment on the structure and protein expression of the rat coagulating gland.

The effects of androgen deprivation and estrogen stimulation on rat coagulating gland were determined by immunohistochemistry and morphometric quantification of different tissue compartments. In castrated or estrogen-treated or estrogen-treated castrated animals, the reduction of the glandular lumen is the most obvious morphological alteration, which is accompanied by an increase in stromal tissue, especially within the lamina propria. Regressive changes occur most rapidly in castrated animals (already by the end of the first week), slower in estrogen-treated castrated animals, and still slower in estrogen-treated normal animals. In castrated animals, epithelium shows a reduction of rough endoplasmic reticulum, loss of secretory blebs, and a decrease of cell size and immunoreactivity for secretory transglutaminase. The reduction of glandular lumen results from an impressive increase in connective tissue of the lamina propria. Smooth muscle cells become atrophic in castrated animals, less so in estrogen-treated animals and in castrated estrogen-treated animals. A relative increase in thickness of the smooth muscle cell layer occurs in all experimental groups and is most obvious in estrogen-treated normal animals. The proportion of myofilament and intermediate filament proteins (smooth muscle-specific actin and desmin immunoreactivities) remains nearly unaltered in these cells after hormonal challenge. A redistribution of intermediate filaments occurs forming thicker bundles within the cells. No indication for increased mitotic activity of estrogenized smooth muscle cells has been found. After castration, and after estrogen treatment, the fibroblasts and the smooth muscle cells, respectively, appear responsible for the architectural changes within the coagulating gland. Reactions of the stroma are differentially regulated after estrogen treatment and androgen deprivation. No indication for increased biosynthetic activities of smooth muscle cells has been observed in any of the experimental conditions.

Estrogen-induced morphological and immunohistochemical changes in stroma and epithelium of rat ventral prostate.

Prostatic smooth muscle cells have been regarded to play a major pathogenetic role during the development of benign prostatic hyperplasia (BPH) in elderly men. Altered hormonal signals (increased estrogen) have been made responsible for the "metabolic" transformation of prostatic smooth muscle cells, which were thought to produce increased amounts of connective tissue fibers observed in BPH. In order to find out the role of metabolically "activated" smooth muscle cells, hormone stimulation experiments were performed in male rats. The effects of androgen deprivation and estrogen stimulation were recorded by semiquantitative analysis of intermediate and myofilament proteins in stromal smooth muscle cells. In castrated or estrogen-treated or estrogen-treated and castrated animals, the reduction of the glandular lumen is the most obvious morphological alteration, accompanied by an increase in connective tissue. Regressive changes occurred most rapidly in castrated animals (already within the first week), slower in castrated estrogen-treated animals and still slower in normal estrogen-treated animals. Regression of the epithelium was accompanied by a marked decrease in immunoreactivity for prostatic binding protein (PBP) in castrated animals, while PBP immunoreactivity in estrogenized animals was retained for up to 6 weeks. Smooth muscle cells became atrophic in castrated animals. This effect was attenuated in estrogen-treated animals. There was no indication for enhanced collagen synthesis by smooth muscle cells. Actin and desmin-immunoreactivity were only slightly altered in experimental animals and showed a changed distribution pattern. Prostatic smooth muscle cells respond less markedly to hormonal alterations than do the fibroblasts.