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Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

Congenital adrenal hyperplasia causes genital ambiguity in females affected with the severe form of the disease; yet the abnormality is preventable with prenatal dexamethasone treatment that must be given to the mother before the ninth week of gestation. In the period from 1978 to March 2011 we have made prenatal diagnosis in 719 pregnancies. Our results indicate that the average Prader score of those fetuses treated with dexamethasone was 1.7, which is much lower than the average Prader score of 3.73 in those not treated. While our data demonstrate no significant abnormalities in the long-range medical and cognitive outcomes in patients prenatally treated with dexamethasone, the current protocol involves invasive procedures such as chorionic villus sampling or amniocentesis, and all fetuses are treated unnecessarily for several weeks before the sex and the affection status of the fetus is known. We are collaborating with Dr. Dennis Lo in Hong Kong to develop a noninvasive protocol, whereby at the sixth to seventh week of gestation we can determine the sex and the affection status of the fetus by harvesting fetal DNA from the maternal plasma. The method will eliminate invasive procedures and unnecessary prenatal treatment and bring noninvasive prenatal diagnosis to underdeveloped areas where amniocentesis and chorionic villus sampling are not available., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Prenatal treatment of congenital adrenal hyperplasia (CAH) with dexamethasone (DEX) has been in use since the mid- 1980s. Its effectiveness for reducing virilization of external genitalia is well established. DEX treatment has to be started in the 6th-7th postmenstrual week and continued until the results of the prenatal diagnosis are available. Hence, the dilemma is that 7 out of 8 fetuses (boys and unaffected girls) are treated unnecessarily. Girls with CAH are treated until term. Accumulating evidence from animal studies and follow-up data has raised concerns regarding the long-term consequences of this controversial treatment. We have previously reported that direct neuropsychological assessment of children exposed to DEX and controls show normal full-scale IQ, learning and longterm memory. However, the children exposed to DEX during the first trimester had an impaired verbal working memory which was significantly associated with low self-perceived scholastic competence. In addition, the children showed increased self-rated social anxiety. The same cohort of children answered questions concerning friends, activities and gender-related behaviors. The results indicate less masculine and more neutral behavior in short-term DEX-exposed boys. These findings indicate that long-term follow-ups of this group of patients are of extreme importance and that future DEX treatment of CAH may be questioned. We therefore encourage additional studies on larger cohorts in order to draw more decisive conclusions about the safety of the treatment. Until then, it is important that the parents are thoroughly informed about the potential risks and uncertainties, as well as the benefits, of this treatment., (Copyright © 2011 S. Karger AG, Basel.)

Otoacoustic emissions (OAEs) differ between the sexes in humans, rhesus and marmoset monkeys, and sheep. OAEs also are different in a number of special populations of humans. Those basic findings are reviewed and discussed in the context of possible prenatal-androgen effects on the auditory system. A parsimonious explanation for several outcomes is that prenatal exposure to high levels of androgens can weaken the cochlear amplifiers and thereby weaken otoacoustic emissions (OAEs). Prenatal androgen exposure apparently also can alter auditory evoked potentials (AEPs). Some non-hormonal factors possibly capable of producing sex and group differences are discussed, and some speculations are offered about specific cochlear structures that might differ between the two sexes.

Objectives: To investigate the long-term effects of prenatal treatment of congenital adrenal hyperplasia (CAH) with emphasis on behavioural problems and temperament., Design: A population-based long-term follow-up study of Swedish children at risk for virilising CAH, who had received treatment prenatally with dexamethasone (DEX). The questionnaire-based follow-up was performed when the children had reached school age., Methods: Standardised parent-completed questionnaires were used to evaluate adaptive functioning, behavioural/emotional problems and psychopathology, social anxiety and temperament in DEX-exposed school-aged children (n=26) and matched controls (n=35). In addition, the association between parental questionnaires and children's self-ratings was investigated., Results: There were no statistically significant differences between DEX-exposed children and controls in measures of psychopathology, behavioural problems and adaptive functioning. In a questionnaire on temperamental traits, DEX-exposed children were described by their parents as being more sociable than controls (P=0.042). The correlation analysis showed only modest parent-child agreement on social anxiety, i.e. the increased social anxiety in children's self-ratings was not confirmed by their parents., Conclusions: DEX-treated children showed good overall adjustment. The parent-child agreement with respect to social anxiety was modest, highlighting the importance of multiple information sources and assessment methods. The clinical significance of the observed difference in sociability cannot be determined within the frameworks of this study. Additional studies of larger cohorts are essential to make more decisive conclusions on the safety of the treatment. Until then, it is important that parents are thoroughly informed about the benefits and potential risks and uncertainties of this controversial treatment.

Normal male sex development requires the SRY gene on the Y chromosome, the regression of Müllerian structures via anti-Müllerian hormone (AMH) signalling, the development of the Wolffian duct system into normal male internal genital structures consequent to testosterone secretion by the testicular Leydig cells, and finally, sufficient activation of testosterone to dihydrotestosterone by 5alpha-reductase. All these events take place during weeks 8-12 of gestation, a narrow window of sexual differentiation. Recent studies in human fetal development have demonstrated the early fetal expression of the adrenocorticotrophic hormone (ACTH) receptor and all steroidogenic components necessary for the biosynthesis of cortisol. These findings provide compelling evidence for the assumed pathogenesis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency, diminished feedback to the pituitary due to glucocorticoid deficiency, subsequent ACTH excess, and up-regulation of adrenal androgen production with subsequent virilization. Another CAH variant, P450 oxidoreductase deficiency, manifests with 46,XX disorder of sex development (DSD), i.e., virilized female genitalia, despite concurrently low circulating androgens. This CAH variant illustrates the existence of an alternative pathway toward the biosynthesis of active androgens in humans which is active in human fetal life only. Thus CAH teaches important lessons from nature, providing privileged insights into the window of human sexual differentiation, and particularly highlighting the importance of steroidogenesis in the process of human sexual differentiation.

Congenital genital anomalies are a very complex pathology. In order to clarify its causes it is important to revert to the genetic conditions and regularities of embriological development. The genital disturbances are mostly determined by chromosomal or endocrinic disorders or by impaired biochemical processes. Clinical problems arise when the genetical sex is in discrepancy with ambiguous genitalia. True hermaphroditism, congenital adrenal hyperplasia, testicular feminization and gonadal dysgenesis are the most common syndromes. Diagnostic criteria applied are similar for all (establishment of karyotype, investigation of hormones and their derivates, genital ultrasound and endoscopy, if needed - radiological examination), but medical and surgical treatment is applied to each patient individually.

The ratio of the length of the second digit (2D) to the length of the fourth digit (4D) is greater in women than in men. Since androgens are involved in most somatic sex differences and since the sexual dimorphism in 2D:4D is stable from 2 years of age in humans, it was hypothesized that finger length pattern development might be affected by early androgen exposure. Human females with congenital adrenal hyperplasia (CAH) are exposed prenatally to higher than normal levels of adrenal androgens, providing an opportunity to test the effects of early androgen exposure on digit ratios. The 2D:4D was calculated for females with CAH, females without CAH, males with CAH, and males without CAH. Females with CAH had a significantly smaller 2D:4D on the right hand than did females without CAH. Males with CAH had a significantly smaller 2D:4D on the left hand than did males without CAH. A subset of six males with CAH had a significantly smaller 2D:4D on both hands compared with their male relatives without CAH. These results are consistent with the idea that prenatal androgen exposure reduces the 2D:4D and plays a role in the establishment of the sex difference in human finger length patterns. Finger lengths may therefore offer a retrospective marker of perinatal androgen exposure in humans.

To introduce we report the embryological origin of the male genitals and we present the endocrine control of the sex phenotypic differentiation and the most important features of the sexual differentiation abnormalities (ambiguous genitalia of newborn, gonadal dysgenesis, chromosome anomalies). Subsequently we report the classification of cryptorchidism and its correlation with hormonal and histological abnormalities. Furthermore we tried to expose the various different thoughts about testicular descent, anomalies associated with cryptorchidism and implications with malignant degeneration and infertility. Finally we describe the Magenta hospital experience from 1972 to 1998 reporting the surgical procedures for undescended testicle and for scrotal anomalies.

We report on the prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase in 20 at-risk pregnancies (16 salt-wasting and 4 simple virilizing families). We have diagnosed 3 affected fetuses (2 males and 1 female), 3 healthy homozygotes (2 males and 1 female), and 14 healthy heterozygotes (7 females and 7 males). These data were collected over 4 years. In 16 fetuses, the diagnosis was made with measurements of 17-hydroxyprogesterone (17-OHP) and delta-4-androstenedione (delta) in amniotic fluid (AF), human leukocyte antigen (HLA) typing of amniotic cells, as well as karyotypes between the 16th and 18th weeks of gestation. In 4 fetuses, DNA analysis of amniotic cells was also performed. In 3 pregnancies in which affected fetuses were suspected (on the basis of HLA typing and measurements of 17-OHP and delta concentrations in AF), the fetuses were electively aborted between the 17th to 19th weeks of gestation by parental decision. In all aborted fetuses, diagnosis was confirmed with HLA typing, autopsy findings of hyperplastic adrenal glands, and ambiguous genitalia in female fetuses. Postnatal diagnosis was confirmed in healthy fetuses with HLA typing and serum measurements of 17-OHP concentrations, and in 4 of them with DNA analysis. In 3 of the 4 families, DNA analyses revealed the following mutations: in Family 1, the index case mutation was Intron 2, Exon 3/Exon 6, and the fetus was Normal/Exon 6; in Family 2, the index case mutation was Ex1 Int2 Ex3/ Int2, and the fetus was Ex1 Int2 Ex3/Normal; and in Family 3, the index case mutation was Ex8(356)/Ex8(356), and the fetus was Ex8(356)/ Normal. We also report one case of prenatal diagnosis and treatment. Dexamethasone 0.5 mg BID (20 micrograms/kg/d) was given starting at 6th week of gestation. Prenatal diagnosis suggested, but did not prove, that the female fetus was a heterozygote as the fetus lacked the paternal mutation Ex8(318). No mutation was found in the mother. The fetus, the mother, and the affected sib shared a haplotype, further suggesting heterozygosity. The unaffected status was confirmed postnatally.

A case report is presented herein of a 33-year-old woman with a history of congenital adrenal hyperplasia in 2 prior births. At 30 weeks of gestation, a scan of the fetal perineum demonstrated ambiguous genitalia which was confirmed at birth. This case demonstrates that when the fetal perineum is well visualized, the diagnosis of normal and abnormal genital development can be made sonographically. This can assist in perinatal/neonatal management, planning and in some cases, can also serve as an additional tool to monitor the success of prenatal steroid therapy of fetal congenital adrenal hyperplasia.

Since 1986, prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OHD) have been carried out in 239 pregnancies. In 145, diagnoses were made by amniocentesis, whereas 77 were diagnosed using chorionic villus sampling. A newly developed, rapid allele-specific polymerase chain reaction was used for DNA analysis in some cases. Of 239 pregnancies evaluated, 37 babies were affected with classical 21-OHD. Of these, 21 were females, 13 of whom were treated prenatally with dexamethasone. Dexamethasone administered at or before 10 weeks gestation (9 affected female fetuses) was effective in reducing virilization. Seven fetuses had affected female siblings (Prader stages 1-5); 3 of these were born with entirely normal female genitalia, whereas the other 4 were significantly less virilized (Prader stages 1-2) than their siblings. The remaining 2 newborns had male siblings; 1 was born with normal genitalia, and the other was Prader stage 1. No significant or enduring side-effects were noted in either the mothers or the fetuses, indicating that dexamethasone treatment is safe. Prenatally treated newborns did not differ in weight, length, or head circumference from untreated unaffected newborns. Based on our experience, proper prenatal diagnosis and treatment of 21-OHD is effective in significantly reducing or eliminating virilization in the affected female. This spares the newborn female the consequences of genital ambiguity, i.e. genital surgery, sex misassignment, and gender confusion.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is associated with hormonal imbalance which predisposes affected females to prenatal development of genital ambiguity. Because the disease is usually not lethal and can be treated with glucocorticoids, affected pregnancies are seldom terminated. Dexamethasone can be administered to the pregnant mother and is effective in correcting the fetus's adrenal hormone imbalance during gestation. Nearly a decade's experience with prenatal treatment of CAH indicates that the risk-benefit ratio is favorable for mother and fetus with careful medical supervision of gestationally administered dexamethasone.

What are the respective places for prenatal treatment and neonatal screening in 21 hydroxylase deficiency, (21OHD) CAH? Current diagnostic procedures for 21OHD CAH do not prevent potential or actual morbidity and mortality from salt wasting and hypoglycaemia, ambiguous genitalia and late diagnosis. Presentation is with life-threatening illness or virilisation with long-term physical, psychological and psychosexual sequelae. Screening the whole newborn population would add only a very small additional unit cost in the screening laboratory already measuring TSH and phenylalanine. There are still few data on which to base an assessment of the efficacy of neonatal screening in reducing morbidity and mortality. Screening should now be adopted on a regional, or national, basis to assess both efficacy and cost-effectiveness. Although more (uncontrolled) data are now available concerning prenatal dexamethasone therapy, the degree of benefit in terms of reduced virilisation in relation to potentially significant maternal side effects is unclear and possible long term childhood side effects have not been studied. At present, therefore, there is insufficient evidence regarding the safety of mother and fetus to recommend the general use of dexamethasone outwith the context of controlled scientific studies. There is an urgent need for prospective controlled studies to be undertaken which would, in time, resolve both questions.

The most common enzymatic defect of steroid synthesis is adrenal steroid 21-hydroxylase deficiency. Inhibited formation of cortisol causes increased pituitary release of ACTH, driving the adrenal cortex to overproduce androgens, whose synthesis does not involve the 21-hydroxylase enzyme. This hormonal setting is established in the embryonic period and affects development of genetic females, misdirecting differentiation of the external genitalia toward male type. At birth, the genitalia are visibly ambiguous (enlarged clitoris, fused labia) or in some cases even male in appearance (phallus with urethral opening, rugated scrotal sac), leading to wrong sex assignment. Adrenal steroid 21-hydroxylase deficiency is the most common basis of female pseudohermaphroditism. These females, however, have normal fertility and potential for gestation (gonads are functional and the internal duct-derived structures are well-formed), thus the sex of rearing should always be female. Management is by life-long hormonal (glucocorticoid) replacement, with surgical correction of the genital ambiguity. Prenatal diagnosis of 21-hydroxylase deficiency, first possible by steroid assay of the amniotic fluid, has utilized HLA typing for identification of loci (antigens B and DR) in close linkage with the 21-hydroxylase gene, and now increasingly relies on DNA analysis for linked HLA or C4 genes or for mutant 21-hydroxylase alleles directly by molecular genetic techniques. The most recent clinical advance is a program of combined prenatal diagnosis with karyotyping and suppression of fetal androgen production in genetic females by steroid administration to the mother. This is the first instance of an inborn metabolic error to be prenatally treated. A series of 85 managed pregnancies is reported on, including accuracy of diagnosis, response of the mother to steroid treatment, and outcome for treated and untreated male and female fetuses (of 77 born by 6/91). Prenatal diagnosis by current techniques is accurate. Normal growth and development patterns postnatally suggest that dexamethasone treatment is safe.

The mechanisms and disturbances in the differentiation of male and female genitalia are described in relation to a correct radiological diagnosis. The methods of investigations include the flushing technique and the catheter technique. The advantage of the flushing technique (retrograde injection of contrast medium under moderate pressure) is its minimal invasiveness. In the catheter technique the bladder is filled with the help of a second catheter or suprapubic puncture, and a micturating cystourethrogram is conducted. The authors use a combination of both methods, filling the lower section of the urogenital sinus with a catheter and the upper by intravenous pyelography accompanied at times with a suprapubic bladder puncture. Quality of the radiographic picture ensures visualisation of all the important structures, as demonstrated by the accompanying figures.

21-Hydroxylase deficiency results in congenital adrenal hyperplasia and leads to masculinization of the external genitalia of affected females. This complication could be avoided if fetal adrenal gland function were suppressed. A woman with mild 21-hydroxylase deficiency whose previous female child had classic congenital adrenal hyperplasia with masculinization was given dexamethasone beginning at the tenth week of gestation. Maternal estriol and cortisol values indicated rapid and sustained fetal and maternal adrenal gland suppression. At 39 weeks' gestation, the patient was spontaneously delivered of a female neonate with normal external genitalia. Postnatal tests indicated the infant was a single heterozygote for 21-hydroxylase deficiency. This study demonstrates prolonged suppression of the fetal adrenal gland with dexamethasone and suggests it might prevent abnormal masculinization in fetuses with severe congenital adrenal hyperplasia.

A multicentric study of prenatal treatment of congenital adrenal hyperplasia (CAH) resulting from 21-hydroxylase deficiency in 43 pregnancies at risk for CAH is presented. The mothers were given dexamethasone per os, 0.5 mg either 12-hourly or 8-hourly. From the analysis of the results obtained in the present study and review of the literature, it would appear that the first condition for successful prevention of female virilization in utero (a total of 6 cases) is to start treatment as early as possible, no later than the 7th week. The dose of dexamethasone should be related to maternal size: 20 micrograms/kg/day (in 2 or 3 fractioned) doses would seem to be both efficient and safe. Adrenal suppression of both maternal and fetal adrenal function should be controlled by appropriate hormonal determinations. Finally, the advantages of early prenatal diagnosis or no prenatal diagnosis are discussed.

Dermatoglyphic findings were compared in 42 patients (32 females, 10 males) with Congenital Adrenal Hyperplasia (CAH) and 110 normal controls (70 females, 40 males). In CAH males, an excess of whorls (p less than 0.001), an increased total finger ridge count (p less than 0.05), and an increased frequency of patterns in the fourth interdigital area (p less than 0.025) was found. A main line A terminating high in the hypothenar area (p less than 0.05), and a missing c-triradius or an abortive main line C (p less than 0.05) was observed in CAH females. Both sexes displayed an increase in the frequency of small radially directed hypothenar patterns (p less than 0.05) and sydney lines (p less than 0.01).

The hypothesis that testicular descent may be governed by Müllerian inhibiting substance (MIS) was investigated by observing the position of the testes in 13 children with XY karyotype and persistent Müllerian duct syndrome (PMDS). It was found that there was a direct relationship between failure of testicular descent and the degree of development of the Müllerian system. Where the Müllerian system was complete, the testes were in an ovarian position but where only the vagina was present, the testes were sometimes found in the inguinal region. To discover whether excessive androgen activity in females might produce ovarian descent, the position of the ovaries in 15 children with severe adrenogenital syndrome was observed. Despite complete genital masculinisation in three children and almost complete in six, all but one ovary was in the normal position: that ovary had descended in an inguinal hernia sac. These findings suggest that MIS rather than androgens may be responsible for the first or abdominal phase of testicular descent. Even excessive androgen activity failed to cause ovarian descent.