The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 6 2811-2816
Copyright © 2004 by The Endocrine Society
Lack of Defects in Androgen Production in Children with Hypospadias
Nicholas M. Holmes,
Walter L. Miller and
Laurence S. Baskin
Departments of Urology (N.M.H., L.S.B.) and Pediatrics (W.L.M., L.S.B.), University of California, San Francisco, California 94143
Address all correspondence and requests for reprints to: Laurence Baskin, M.D., Department of Urology, University of California, 400 Parnassus Avenue, A610, Box 0738, San Francisco, California 94143. E-mail: lbaskin{at}urol.ucsf.edu.
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Abstract
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Formation of the male urethra requires the synthesis of testosterone, its activation to dihydrotestosterone (DHT) in genital skin, and binding of DHT to the androgen receptor. Defects in any of those steps can cause hypospadias. To determine whether defects exist in the production of androgens in individuals with hypospadias, we examined enzymatic function of 3ß-hydroxysteroid dehydrogenase (3ßHSD), P450c17 (17
-hydroxylase and 17,20 lyase activity), and type 3 17ßHSD. Sixty-eight subjects participated in the study: 48 patients had hypospadias, and 20 had normal male genitalia. Subjects were stratified into groups based on age and severity of hypospadias, as defined by location of the urethral meatus after correction of penile curvature. Hormonal values in boys with hypospadias were compared by nonparametric analysis with those in age-matched controls. Controls excluded individuals with cryptorchidism, micropenis, known endocrine defects, or receiving steroid supplementation. Morning fasting serum levels of pregnenolone, progesterone, 11-deoxycorticosterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione, androstenediol, testosterone, and DHT were determined. To focus on the proximal steps in androgen biosynthesis, 12 individuals with hypospadias underwent standard ACTH stimulation. No significant differences in the androgen precursors and metabolites were found between controls and individuals with hypospadias. The response to ACTH was variable without a significant difference between the patients with different degrees of hypospadias and/or published controls. These data indicate that enzymatic defects in the steroidogenic steps from cholesterol to DHT are not a common etiology of hypospadias. Routine abnormalities in the androgen biosynthetic pathway are an unlikely cause of any degree of hypospadias in boys without accompanying cryptorchidism or genital malformations.
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Introduction
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HYPOSPADIAS AFFECTS APPROXIMATELY one in 250 live male births (1, 2). Roughly 6000 such individuals are born in the United States each year (1, 2), and recent reports suggest an increasing incidence of hypospadias (3). A small percentage of severe hypospadias can be attributed to genetic syndromes or defects involving the androgen receptor (4). Assisted reproductive techniques have also been associated with an increased risk for hypospadias; however, the etiology in the majority of cases of hypospadias remains unknown (5).
Normal male phallic urethral development is directed by testosterone produced by the fetal testis. Testosterone is subsequently converted to dihydrotestosterone (DHT) in genital skin and binds to the androgen receptor in the same cells to facilitate masculinization of the phallus. Thus, defects anywhere along the pathway of androgen production could potentially account for the occurrence of hypospadias (see Fig. 1
) (6). The pathway from cholesterol to androstenedione is the same in the testis and adrenal and uses the same enzymes encoded by single genes (7). The enzyme of 3ß-hydroxysteroid dehydrogenase (3ßHSD) converts pregnenolone to progesterone, 17-hydroxypregnenolone (17OH-pregnenolone) to 17-hydroxyprogesterone (17OH-progesterone), dehydroepiandrosterone (DHEA) to androstenedione, and androstenediol to testosterone. Defects in 3ßHSD would lead to an increase in pregnenolone, 17OH-pregnenolone, DHEA, and androstenediol (see Fig. 1). P450c17 is also necessary in the proximal stages of testosterone synthesis. P450c17 catalyzes the 17-hydroxylation of pregnenolone to 17OH-pregnenolone and progesterone to 17OH-progesterone. The 17,20 lyase activity of P450c17 coverts 17OH-pregnenolone to DHEA, but very little 17OH-progesterone is converted to androstenedione, so that 17OH-progesterone is not a precursor of human androgen synthesis (8, 9). Defects in either 17-hydroxylation or 17,20 lyase activity of P450c17 will lead to an increase in respective steroid hormone precursors (see Fig. 1). The conversion of androstenedione to testosterone in the testis is catalyzed by type 3 17ßHSD (17ßHSDIII), but this reaction can also be catalyzed by 17ßHSD type V, which is widely expressed in extraglandular tissue (10, 11).
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FIG. 1. Principal pathways of steroid hormone synthesis. Reaction 1, Mitochondrial cytochrome P450scc catalyzes 20-hydroxylation, 22-hydroxylation, and scission of the C20–22 carbon bond, resulting in the conversion of cholesterol to pregnenolone. Reaction 2, 3ßHSD type II, a short-chain dehydrogenase in the endoplasmic reticulum, catalyzes 3ß-hydroxysteroid dehydrogenase and isomerase activities. Reaction 3, P450c17 catalyzes the 17-hydroxylation of pregnenolone to 17OH-pregnenolone and that of progesterone to 17OH-progesterone. Reaction 4, The 17,20 lyase activity of P450c17 converts 17OH-pregnenolone to DHEA, but very little 17OH-progesterone is converted to  4-androstenedione. Reaction 5, P450c21 catalyzes the 21-hydroxylation of progesterone to deoxycorticosterone (DOC) and that of 17OH-progesterone to 11-deoxycortosol. Reaction 7, P450c11ß converts 11-deoxycortisol to cortisol. Reactions 6, 8, and 9, In the adrenal zona glomerulosa, DOC is converted to corticosterone and then to 18OH-corticosterone by P450c11ß in the zona fasiculata. Reactions 10 and 11 are found in the testes, ovaries, and some peripheral, nonglandular tissues. Reaction 10, Several forms of 17ß-HSD, a reversible non-P450 enzyme of the endoplasmic reticulum, mediate 17ß-hydroxysteroid dehydrogenase activities, converting DHEA to androstenediol (type III), androstenedione to testosterone (type III), and estrone to estradiol (type I). The reverse 17-ketosteroid reductase activities are catalyzed by the type II and IV enzymes. Reaction 11, Testosterone is converted to estradiol by P450aro (aromatase). Reaction 12, Testosterone is converted to DHT by 5-reductase type II (modified from Ref.7 with permission).
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Others have suggested that up to 50% of boys with hypospadias have a disorder in 3ßHSD and the 17,20 lyase activity of P450c17 affecting androgen biosynthesis (12). However, studies of the genes for 3ßHSD and P450c17 have found mutations in only a few patients (13). Only five patients with genetically proven 17,20 lyase deficiency have been described (14, 15, 16, 17), and mutations in the 3ßHSD enzyme are not found until the precursor/product ratio exceeds 8 SD above the mean (18, 19). These observations suggest that defects in 3ßHSD and P450c17 should be rare causes of hypospadias. To determine whether defects in the proximal steps of steroidogenesis are a common cause of hypospadias (12), we examined the principal steroidal precursors and products of 3ßHSD, P450c17, and 17ßHSDIII in patients with hypospadias and age-matched normal controls.
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Subjects and Methods
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The study was approved by the committee on human research at University of California-San Francisco (UCSF). After obtaining informed consent from the patient’s parent/guardian, children with isolated hypospadias were enrolled through the UCSF Pediatric Clinical Research Center. Subjects with hypospadias were excluded if they had cryptorchidism or known genetic defects. Subjects were grouped according to age (<1 yr; prepubertal patients, >1 yr) and according to the location of the urethral meatus after the correction of penile curvature: distal shaft, midshaft, and proximal (which included proximal shaft hypospadias, and penoscrotal and scrotal hypospadias). Controls were enrolled from individuals undergoing elective circumcision, excluding those with cryptorchidism, micropenis, known endocrine defects, or a history of steroid supplementation. Morning (0700–0900 h) fasting blood was obtained for measurements of progesterone, pregnenolone, 17OH-progesterone, 17OH-pregnenolone, DHEA, androstenedione, cortisol, 11-deoxycortisol, 11-deoxycorticosterone, androstenediol, DHT, and testosterone. In the majority of patients, blood samples were obtained after the induction of general anesthesia using either halothane or sevoflurance. Phlebotomy was performed at the time of iv catheter placement. Reconstructive surgery was then performed. In 12 patients, these hormones were assayed before and 60 min after iv administration of 0.25 mg ACTH. In this case, fasting patients were admitted to the Pediatric Clinical Research Center, and a nurse experienced in the ACTH stimulation test placed an iv catheter after the application of EMLA. The tests were performed between 0700 and 0900 h.
Statistical analysis of the mean plasma concentrations was performed using unpaired t test. All assays, except cortisol, were performed by Esoterix, Inc. (Calabasas, CA); cortisol was measured by the UCSF clinical chemistry laboratory by immunoassay.
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Results
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Sixty-eight subjects participated in the study: 48 patients with hypospadias and 20 controls with normal male genitalia. All subjects with hypospadias were offered ACTH stimulation testing, and 12 participated in this portion of the study. The median age of the 48 patients in the study was 1.0 yr (range, 4.1 months to 9.8 yr). Due to the age-dependent variability in serum concentrations of androgens and their precursors, results were analyzed according to age, with the patients split into two groups: less than 1 yr of age and more than 1 yr of age (Table 1). Patients were categorized further based on the location of the hypospadiac urethral meatus: distal; midshaft, or proximal. Age-matched controls were used for comparison. No untoward complications occurred as a result of ACTH stimulation.
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TABLE 1. Mean serum concentrations (nanograms per deciliter; except cortisol, micrograms per deciliter), range, and SD of steroids in controls, distal, mid, and proximal shaft hypospadias
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Table 1
compares the mean concentration, range, and SD of steroid hormones as a function of age and degree of hypospadias. The individual morning fasting levels (not shown) and the mean levels for the patients with hypospadias fall within the range of the control values. There was no evidence of an increased precursor to product ratio for the steroidal conversions catalyzed by 3ßHSD, P450c17 (17-hydroxylase or 17,20 lyase activity), or 17ßHSDIII. Note the paucity of significant values. Three comparisons had a P value just under 0.05: 17OH-progesterone, comparing distal hypospadias to controls less than 1 yr of age; and deoxycorticosterone and 11-deoxycortisol, comparing midshaft hypospadias to controls less than 1 yr of age. These values appear to be random variations; none of these steroids is in the androgen biosynthetic pathways, and most notably, the values for testosterone and DHT did not differ from the controls for any group studied. Cortisol was clearly lower in control children less than 1 yr of age than in subjects with hypospadias (P = 0.004), most likely due to random variation of the small numbers studied.
As tropic hormone stimulation is a more sensitive test for mild defects in steroidogenesis, we offered ACTH testing to all 48 patients; the parent/guardian consented for 12 children (age, 0.7–4.1 yr; mean, 1.9 yr) to participate in this study. Table 2 shows the pooled steroid hormone data before and after ACTH testing for the 12 patients with hypospadias. None of the pooled values nor any of the individual values (not shown) was significantly different form the published control data for ACTH responses from the reference laboratory. Any apparent differences are probably a product of stochastic variation, given the small numbers of patients who could be tested, and none was clinically significant. Thus, we found no evidence of an increase in precursor to products ratios for reactions catalyzed by 3ßHSD, P450c17, or 17ßHSDIII.
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TABLE 2. Mean serum concentrations (nanograms per deciliter; cortisol, micrograms per deciliter) and range of steroids in distal, mid, and proximal shaft hypospadias before and 60 min after ACTH stimulation
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Discussion
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Male urethral development requires the action of DHT produced in local tissues between 8 and 16 wk. DHT is produced from circulating testosterone, which is manufactured by the fetal testis under the stimulation of hCG. A defect in any step in steroid biosynthesis is generally indicated by increased serum concentrations of the precursor steroid and low concentrations of the downstream products. The elevated precursor concentrations are easier to identify when steroidogenesis is stimulated, so that more precursors accumulate proximal to the disordered enzyme even when there are alternate pathways for that steroid’s metabolism. Eliciting defects in the distal steps in androgen biosynthesis, such as conversion of androstenedione to testosterone by 17ßHSDIII or conversion of testosterone to dihydrotestosterone by 5-reductase type II requires hCG stimulation of the testis as 17ßHSDIII is expressed only in the Leydig cells and 5-reductase type II in genital tissue. Testicular stimulation requires several im injections of hCG over a period of days or weeks; this was not practical in this study. However, as the enzymatic steps upstream from androstenedione are the same in the adrenal and the gonad, these steps can be studied with short-term stimulation of the adrenal with ACTH. Thus, ACTH testing of adrenal function can be a useful approach for diagnosing defects in the proximal steps in testicular steroidogenesis.
Previous work examined the pituitary-gonadal axis in patients with hypospadias by measuring LH, FSH, DHT, and testosterone before and after the administration of human chorionic gonadotropin (20, 21). Steroid precursor to product ratios must be interpreted with caution, however. Variables such as diurnal variation, episodic secretion, and stress may influence values in baseline samples. For example, one of our patients with hypospadias might have been interpreted as having a mild defect in 3ßHSD activity if only the ratio of the pregnenolone/progesterone had been solely examined, but in this case the ratios of 17OH-pregnenolone/17OH-progesterone and DHEA/androstenedione were normal. Studies examining the ratios of 5/4 steroids show much higher ratios (2–6 SD above the mean) in hirsute women compared with controls, yet none of these individuals had 3ßHSDII gene mutations (18, 19).
As noted in Table 1, the levels of 17OH-progesterone in the subjects less than 1 yr of age with distal hypospadias were statistically higher than control values (P = 0.046). However, the values are within the normal range, and levels of both steroids upstream (progesterone and pregnenolone) and downstream (11-deoxycortisol) were normal, suggesting that this statistical discrepancy is not relevant. Thus, steroid ratios alone may be insufficient to diagnose an enzymatic defect. Any presumed defect identified by hormonal testing must be consistently reproducible and must be confirmed at a molecular genetic level before a diagnosis can be made with confidence.
As development of the urethra occurs between 8 and 16 wk gestation, the hormonal milieu at the time of postnatal testing may not reflect events early in gestation (22, 23). Furthermore, subjects in this study were tested at an age when testosterone secretion is normally minimal. For this reason, analysis of the proximal steps of steroidogenesis is most easily performed by ACTH testing of the adrenal cortex, as the same enzymes are involved. In the 12 subjects with hypospadias whose families consented to preoperative ACTH stimulation, we found no abnormalities. Cortisol levels increased appropriately 60 min after ACTH, indicating that the test was performed appropriately. Furthermore, the precursor/product ratios were normal, and the pre- and post-ACTH steroid levels across the chart fell within the normal ranges.
The cellular, biochemical, and genetic basis of hypospadias remains unknown. Our study has shown that defects in enzymatic steps from cholesterol to androstenedione are unlikely in boys with any degree of hypospadias without accompanying genital anomalies. Studies of 17ßHSDIII and 5-reductase, for which testicular stimulation is required, and of the androgen receptor have been similarly unrewarding (4, 24). All disorders of steroidogenic enzymes are autosomal recessive, yet there is little evidence that routine hypospadias is heritable. A more productive approach may be to focus at the organogenesis of phallic development. Hypospadias may be a result of abnormal mesenchymal-epithelial interactions during phallic urethral development (22, 23). Such studies may implicate a role for endocrine disruptors as an explanation for hypospadias (25).
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Footnotes
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This work was supported by NIH Grants M01-RR-01271 (to University of California-San Francisco Pediatric Clinical Research Center), DK-058105, and HD/DK-41958.
Abbreviations: DHEA, Dehydroepiandrosterone; DHT, dihydrotestosterone; 3ßHSD, 3ß-hydroxysteroid dehydrogenase; 3ßHSDIII, 3ß-hydroxysteroid dehydrogenase type III; 17OH, 17-hydroxy.
Received December 5, 2003.
Accepted March 7, 2004.
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Abstract
Introduction
