Endocrine and molecular investigations in a cohort of 25 adolescent males with prominent/persistent pubertal gynecomastia.

Pubertal gynecomastia is a common condition observed in up to 65% of adolescent males. It is usually idiopathic and tends to regress within 1-2 years. In this descriptive cross-sectional study, we investigated 25 adolescent males with prominent (>B3) and/or persistent (>2 years) pubertal gynecomastia (P/PPG) to determine whether a hormonal/genetic defect might underline this condition. Endocrine investigation revealed the absence of hormonal disturbance for 18 boys (72%). Three patients presented Klinefelter syndrome and three a partial androgen insensitivity syndrome (PAIS) as a result of p.Ala646Asp and p.Ala45Gly mutations of the androgen receptor gene. The last patient showed a 17α-hydroxylase/17,20-lyase deficiency as a result of a compound heterozygous mutation of the CYP17A1 gene leading to p.Pro35Thr(P35T) and p.Arg239Stop(R239X) in the P450c17 protein. Enzymatic activity was analyzed: the mutant protein bearing the premature stop codon R239X showed a complete loss of 17α-hydroxylase and 17,20-lyase activity. The mutant P35T seemed to retain 15-20% of 17α-hydroxylase and about 8-10% of 17,20-lyase activity. This work demonstrates that P/PPG had an endocrine/genetic cause in 28% of our cases. PAIS may be expressed only by isolated gynecomastia as well as by 17α-hydroxylase/17,20-lyase deficiency. Isolated P/PPG is not always a 'physiological' condition and should thus be investigated through adequate endocrine and genetic investigations, even though larger studies are needed to better determine the real prevalence of genetic defects in such patients.

High prevalence of micropenis in 2710 male newborns from an intensive-use pesticide area of Northeastern Brazil.

Exposure to endocrine-disrupting chemicals (EDCs) has been suggested to contribute to the increasing trends of external genital malformation in male newborns. In Northeastern Brazil, the poor sanitary conditions found in the favelas encourage the widespread use of pesticides. This 2-year study of a total birth cohort of full-term male newborns in the regional hospitals of Campina Grande (Paraíba, Brazil) sought to (1) accurately establish for the first time the incidences of neonatal male genital malformations, (2) investigate the endocrine and genetic aetiologies of these malformations, and (3) evaluate their associations with possible prenatal exposure to EDCs. A total of 2710 male newborns were explored for cryptorchidism, hypospadias and micropenis. Cases were referred to the Pediatric Endocrine Clinic for endocrine and genetic investigations, and all parents were interviewed about their environmental/occupational exposure to EDCs before/during pregnancy by paediatric endocrinologists using a detailed questionnaire. We observed 56 cases of genital malformation (2.07%), including 23 cryptorchidism (0.85%), 15 hypospadias (0.55%), and 18 micropenis (0.66%). All cases exhibited normal/subnormal testosterone production and none presented androgen receptor or 5α-reductase gene mutation. More than 92% of these newborns presented foetal contamination by EDCs, as their mothers reported daily domestic use of pesticides (i.e., DDT) and other EDCs. Most of these undervirilized male newborns presented additional EDC contamination, as 80.36% of the mothers and 58.63% of the fathers reported paid or unpaid work that entailed the use of pesticides and other EDCs before/during pregnancy for the mothers and around the time of fertilization for the fathers. The high rate of micropenis in our population associated with an elevated percentage of parental environmental/occupational exposure to EDCs before/during pregnancy indicates that foetal contamination may be a risk factor for the development of male external genital malformation.

Polymorphisms of MAMLD1 gene in hypospadias.

PURPOSE: Mastermind-like domain containing 1 (MAMLD1) is a causative gene for the fetal development of male external genitalia. Almost 10% of patients with both severe and non-severe hypospadias exhibit mutations of MAMLD1. The aim of this work was to determine whether polymorphisms of MAMLD1 are a genetic risk factor for hypospadias. MATERIAL AND METHODS: This study included 150 hypospadias with a range of severities and 150 controls. Direct sequencing of the MAMLD1 coding exons and their flanking splice sites was performed. In silico secondary and tertiary structure prediction and accessibility of changed amino acids were evaluated using JPred, Netsurf and PHYRE software. Functional studies of the transactivation of haplotypes on Hes3 promoter were performed in vitro using cDNAs of missense variants of MAMLD1. RESULTS: The p.P286S polymorphism was identified in 17/150 patients and 12/150 controls (11.3% vs. 8.0%, p = 0.32). The p.N589S polymorphism was identified in 22/150 patients and 12/150 controls (14.6% vs. 8.0%, p = 0.068). The double polymorphism (S-S haplotype) was present in 16/150 patients and 6/150 controls (10.6% vs. 4.0%, p = 0.044, OR = 2.87, CI from 1.09 to 7.55). The association of polymorphisms consistently revealed a modification in the structure prediction or amino acid accessibility in all three in silico models. The P286S, N589S and P286S + N589S proteins did not exhibit reduced transactivating activity on Hes3 promoter. CONCLUSION: Polymorphisms of MAMLD1 gene are frequent in patients with hypospadias. Although no change in transactivation was noted on Hes3 promoter, the in silico studies and the significantly increased incidence of the S-S haplotype in hypospadiac patients raise the hypothesis of a particular susceptibility conferred by these variants.

Isolated 'idiopathic' micropenis: hidden genetic defects?

Micropenis is defined as a stretched penile length of less than 2-2.5SD for age. Aetiologies include hypogonadotropic hypogonadism, testicular dysgenesis, defects in testosterone synthesis, androgen resistance [5α-reductase (5αR) deficiency or partial androgen insensitivity] and other rare causes like growth hormone GH deficiency. Often, the cause remains unknown. The aim of this study was to determine whether isolated micropenis with normal plasma testosterone could hide a molecular defect in the androgen pathway. Twenty-six boys with isolated micropenis were included in this study. All of them had 46,XY karyotype, normal luteinizing hormone and follicle-stimulating hormone and a normal plasma testosterone response to human chorionic gonadotropin testing. Androgen receptor (AR), 5αR and steroidogenic factor 1 (SF1) genes were sequenced. A mutation in the AR gene was found in two patients, and a new mutation in the SF1 gene was found in one patient who was the only one to have a low level of inhibin B (InhB). This is the first report of isolated micropenis as a revealing symptom of AR and SF1 mutations. Anti-Mullerian hormone and InhB should thus be evaluated in patients with isolated micropenis, even when plasma testosterone is in the normal range. Detection of gene mutations is helpful for diagnosis, treatment and genetic counselling for probands.

Undervirilization in XY newborns may hide a 5α-reductase deficiency: report of three new SRD5A2 gene mutations.

The observation of ambiguous genitalia in the newborn signals a medical, surgical and psychological emergency. The most crucial decision will be the choice of sex assignment. Rapid and precise diagnosis is thus essential. In XY newborns with normal/high plasma testosterone (T), partial androgen insensitivity syndrome (PAIS) is usually the first diagnosis evoked, which implies an androgen receptor (AR) defect. The diagnosis of steroid-5-alpha-reductase deficiency is rarely considered by the paediatrician. We report three new SRD5A2 gene mutations in four newborns from France, Morocco and Turkey. The newborns presented with ambiguous genitalia and normal plasma T values and the initial diagnosis\PAIS. In all four cases, normal sequences of the complete AR gene excluded this diagnosis and raised the hypothesis of 5α-reductase deficiency. The entire coding region (5 exons) of the SRD5A2 gene was assessed by PCR and direct sequencing analysis. For patient 1, we identified a new homozygous 2bp deletion in exon 1 (c.122_123delAG). Patient 2 had a known homozygous mutation, p.G115D, in exon 2. New compound heterozygous mutations in exon 4 (p.A215V) and exon 5 (p.X255Q) were found in patient 3. Patient 4 presented a new substitution in exon 1 (p.S14R) associated with a known polymorphism (p.V89L). Our data confirm our previous experience and clearly demonstrate that a 5-α reductase defect should be considered in all XY newborns with ambiguous genitalia and normal plasma T secretion, whatever their geographic area or ethnic group; moreover, this defect was not linked to specific phenotype. Early molecular diagnosis is indispensable for the crucial decision of the newborn's sex of rearing.

Phenotype, hormonal profile and genotype of subjects with partial androgen insensitivity syndrome: report of a family with four adult males and one child with disorder of sexual differentiation.

There is little information on the molecular basis of intrafamilial and inter-familial phenotypic heterogeneity with the same androgen receptor (AR) mutation in patients with partial androgen insensitivity syndrome. A genetic analysis was performed in a large kindred with ambiguous genitalia and the genotype-phenotype correlations were analysed. The index case was brought for sex assignment. Family history revealed four other affected members who had hypospadias and varying degrees of virilisation. All the affected males had hemizygous mutations in the third exon of the AR gene (A596T). One was also found to have a heterozygous mutation in the fourth exon of the 5 alpha reductase type 2 gene (G196S). This affected male with double mutations was better virilised compared with the other affected members with a single mutation. The degree of virilisation correlated with serum testosterone levels. Gynaecomastia was not present in any of these subjects. It is concluded that the subject with dual gene defects also had higher levels of testosterone and pubertal virilsation. Testosterone levels possibly govern the degree of pubertal virilisation in subjects with A596T gene defects. It is not clear whether the better pubertal virilsation and higher testosterone are in any way causally related to the SRD5A2 gene defect.

Searching for somatic mutations in McCune-Albright syndrome: a comparative study of the peptidic nucleic acid versus the nested PCR method based on 148 DNA samples.

BACKGROUND: Activating mutations of the Gsalpha gene (GNAS), which encodes for the alpha-subunit of the stimulatory G protein, have been identified in patients with McCune-Albright syndrome (MAS). Accuracy and sensitivity in the molecular diagnosis of MAS is mandatory for optimal therapeutic strategy and adapted follow-up, especially for incomplete clinical forms of MAS. To date, the highly sensitive nested PCR method with intermediary digestion by a restriction enzyme at the mutation site is one of the most widely used techniques. This study evaluated a new diagnostic method using a peptidic nucleic acid (PNA) and compared it with the nested PCR method. MATERIAL AND METHODS: One hundred and forty-eight DNA samples from eighty-eight patients presenting clinical symptoms compatible with MAS were included. The DNA samples were mainly obtained from peripheral blood, ovarian tissue or cyst liquid, and bone lesions. The nested PCR method required 4 days. PNA clamping required 1.5 days and utilized the higher thermal stability and specificity of PNA-DNA coupling to inhibit PCR product formation. Direct sequencing was subsequently performed in all cases. RESULTS: The sensitivity of mutation detection was 54% (n = 80) for nested PCR and 46.6% (n = 69) for PNA (P > 0.05). The 11 cases where PNA failed to detect the mutation were mainly incomplete and atypical clinical forms of MAS (n = 10/11). The cost per sample was 50 Euros for PNA clamping versus 136 Euros for nested PCR. CONCLUSION: PNA clamping is a rapid, reliable, and economical method to diagnose MAS. It should be the first-line diagnostic method, although negative results, especially for incomplete clinical forms of MAS, should be confirmed by nested PCR.

A new mutation of the androgen receptor, P817A, causing partial androgen insensitivity syndrome: in vitro and structural analysis.

Androgen insensitivity syndrome (AIS) is an X-linked disease caused by mutations in the androgen receptor (AR) resulting in various degrees of defective masculinization in 46,XY individuals. In the present study, we describe a novel mutation in exon 7 of the AR gene in an Egyptian patient with partial AIS (PAIS). Sequencing analysis of the AR gene revealed a novel missense mutation, P817A, within the ligand-binding domain (LBD). This is the first report of a mutation within the short amino acid motif (codons 815-817) of the beta-strand lying between helices H8 and H9 of the AR LBD. The functional defects of the mutated protein were characterized by in vitro study and included significantly decreased ligand-binding affinity and impaired transactivation potential. Limited proteolysis assays performed with the wild-type and mutant AR receptors incubated with the synthetic agonist R1881 revealed that the P817A mutation resulted in a reduced stabilization of the AR active conformation. Structural analyses showed that this mutation is likely to perturb the beta-sheet interaction between residues 815-817 and 911-913. This structural alteration destabilizes the position of the C-terminal extension, which contains residues critical for androgen function.

Disorders of androgen action.

Disorders of androgen action are the main cause of male pseudohermaphroditism and include 5alphaR deficiency and androgen receptor defects. 5alphaR deficiency is characterized by female genitalia with some degree of masculinization, clitoromegaly, and severely bifid scrotum corresponding to the so-called pseudovaginal perineoscrotal hypospadias. At the onset of puberty, increased muscle mass, development of pubic hair, and phallic growth are associated with the acquisition of male gender identity. Normal or increased levels of testosterone and an elevated testosterone-to-dihydrotestosterone ratio after human chorionic gonadotropin stimulation testing suggest 5alphareductase deficiency, and the diagnosis can be ascertained by identifying the mutation in the 5alphaR-2 gene. Whatever the patient's age at diagnosis, psychological evaluation with 5alphaRD is vital. Androgen receptor defects encompass two clinical expressions: the complete and partial androgen insensitivity syndromes. Complete androgen insensitivity syndrome should be suspected at birth in the presence of inguinal hernia in a girl without genital ambiguity. At puberty, the sign of alert is primary amenorrhea with normal female phenotype and harmonious mammary development but no pubic hair growth. Partial androgen insensitivity syndrome covers a wide spectrum of undervirilized phenotypes ranging from clitoromegaly at birth to infertile men. In all cases, complementary investigations should include plasma testosterone and luteinizing hormone as well as androgen-binding capacity in cultured genital skin fibroblasts. Diagnosis is confirmed by identification of the androgen receptor gene mutation. Although patients with complete androgen insensitivity syndrome are raised as females, patients with partial androgen insensitivity syndrome should be managed according to age at diagnosis, response to treatment with exogenous androgens, and the presence of an androgen gene mutation. Gonadectomy in complete androgen insensitivity syndrome should be performed before puberty, and androgen substitution may improve the development of external genitalia in some patients with partial androgen insensitivity syndrome. Psychological follow-up is necessary.

A novel E153X point mutation in the androgen receptor gene in a patient with complete androgen insensitivity syndrome.

AIM: To study a 46, XY newborn patient with a phenotype suggestive of an androgen insensitivity syndrome to confirm an anomaly in the AR gene. METHODS: Genomic DNA from leukocytes was isolated in order to analyze SRY gene by PCR and sequencing of the eight exons of AR gene. Isolation of human Leydig cell mesenchymal precursors from the testis was performed in order to study testosterone production and response to hCG stimulation in culture. RESULTS: Surgical exploration disclosed two testes, no Wolffian structures and important Müllerian derivatives. The SRY gene was present in peripheral blood leukocytes. Sequencing of the AR gene evidenced a previously unreported G to T transversion in exon 1 that changed the normal glutamine 153 codon to a stop codon. Interstitial cell cultures produced sizable amounts of testosterone and were responsive to hCG stimulation. CONCLUSION: This E153X nonsense point mutation has not been described previously in cases of AIS, and could lead to the synthesis of a short truncated (153 vs 919 residues) non functional AR probably responsible for the phenotype of complete androgen insensitivity syndrome (CAIS).

Increased oestradiol level in seminal plasma in infertile men.

Seminal hormonal patterns in fertile and infertile men were investigated. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, testosterone and oestradiol were assessed by radioimmunoassay, and dehydroepiandrosterone sulphate (DHAS) by bioluminescence assay, on blood and seminal plasma of 23 fertile men and 83 infertile men. For fertile men, mean FSH, LH, testosterone and DHAS concentrations were lower and mean oestradiol was higher in seminal than in blood plasma; prolactin did not differ. For infertile men, mean seminal FSH and LH showed a moderate but significant increase compared with fertile men; testosterone, DHAS and prolactin did not differ but mean seminal oestradiol was significantly increased. Of the infertile men, 53% had seminal oestradiol concentration above the 90th percentile value for fertile men. The meaning of this seminal oestradiol increase is unclear since it is not known whether it is the cause or the consequence of the alteration of spermatogenesis in infertile men. Further studies are required to explore the possible therapeutic implications.

Free and total plasma testosterone in men and women with acne.

Androgens are important in the pathogenesis of acne, and free testosterone in the serum is regarded as the biologically active component. In this study, serum free and total testosterone levels were measured in men (34) and women (14), suffering from acne but otherwise healthy. The plasma values for both groups of patients did not differ significantly from those of the age matched control groups.

[Hormonal evaluation in male infertility]. / Bilan hormonal au cours des stérilités masculines.

Recent progress in the study of the hormonal regulation of spermatogenesis justifies endocrine examination in the case of male sterility. The most important complementary investigation are the assays of FSH, LH and plasmatic testosterone. Radioimmunoassay of FSH is the fundamental examination, since this hormone is considered to be an indicator of germinal function. Thus, in the case of oligospermia, or even azoospermia, FSH assay is decisive. When the FSH levels (in conjunction with LH levels) are high and combined with azoospermia, there is a possibility of testicular dysgenesis linked with a karyotype anomaly (XXY etc.). In some cases the germinal affection is secondary to cryptorchidism, orchitis, torsion, medicinal alteration, or radiotherapy. Decreased testosterone values combined with an insufficient FSH and LH response to stimulation tests indicate a gonadotrophic deficit, which is the best indication for substitution therapy using gonadotrophins or LH-RH. An increase in LH, contrasting with a normal FSH value, evokes the exceptional case of a disturbance of androgen receptivity. Normal FSH (and LH) values suggest excretory sterility. Lastly, when hyperprolactinemia is suspected, an assay of plasmatic prolactin is necessary. A "simple" hormonal evaluation allows a routine etiological approach to the diagnosis of sterility, and is thus an important element in the investigative strategy applied to male sterility, used along with the other complementary and indispensible examinations.