Study Objective: To determine the metabolic effects of the subcutaneous etonogestrel implant compared with an oral contraceptive in adolescents and young adults (AYAs) with type 1 diabetes (T1D) on body weight, body composition, glucose, lipids, and C-reactive protein levels. Methods: This was a non-randomized, interventional, prospective study. Thirty-nine AYAs with T1D participated; 20 used the implant (Implant-T1D), and 19 used an oral combined contraceptive (OC-T1D). Body composition, HbA1c, intermittent continuous glucose monitoring, lipids, and high-sensitivity C-reactive protein (hsCRP) levels were evaluated. Results: All participants were followed for at least 12 months, and 26 completed the 24-month follow-up. No women discontinued the intervention due to adverse effects. Body weight increased by 0.8 +/- 3.5 and 1 +/- 2.9 kg in the OC-T1D and the Implant-T1D group at 12 months and by 2.6 +/- 3.9 and 3.3 +/- 3.6 kg at 24 months, respectively. OC-T1D and Implant-T1D had similar HbA1c, mean interstitial glucose levels, and time in range throughout the study; no significant difference over time was observed. hsCRP levels increased in both groups and were associated with BMI and HbA1c (P < .001 for both variables). Women in the OC-T1D group had higher total cholesterol, HDL-C, and triglyceride levels compared with the Implant-T1D. Conclusion: Glucose levels were similar in youth using the subdermal progestin implant and an OC. However, both AYA groups showed increased BMI, fat mass, and subclinical inflammation. Changes in lipid levels were associated with the OC method. These data highlight the importance of weight gain prevention in young women with T1D using hormonal contraception.

BACKGROUND Adrenarche reflects the developmental growth of the adrenal zona reticularis, which produces increasing adrenal androgen secretion (e.g., DHEA/DHEAS) from ages ~5-15 years. We hypothesized that the study of the genetic determinants associated with variations in serum DHEAS during adrenarche might detect genetic variants influencing the rate or timing of this process. SUBJECTS AND METHODS Genome-wide genotyping was performed in participants of the Chilean pediatric GOCS cohort (n=788). We evaluated the genetic determinants of DHEAS levels at genome-wide level and in targeted genes associated with steroidogenesis. To corroborate our findings, we evaluated a polygenic risk score for age at pubarche, based upon the discovered variants, in children from the same cohort. RESULTS We identified one significant variant at the genome-wide level in the full cohort, close to the GALR1 gene (P = 3.81x10 -8). In addition, variants suggestive of association (P <1x10 -5) were observed in PRLR, PITX1, PTPRD, NR1H4, and BCL11B. Stratifying by sex, we found variants suggestive of association in SERBP1 and CAMTA1/VAMP3 for boys and near ZNF98, TRPC6, and SULT2A1 for girls. We also found significant reductions in age at pubarche in those children with higher polygenic risk scores for greater DHEAS based on these newly identified variants. CONCLUSIONS Our results disclose one variant associated with DHEAS concentrations at the level of GWAS significance, and several variants with suggestive association, which may be involved in the genetic regulation of adrenarche.

Context: Voice break, as a landmark of advanced male puberty in genome-wide association studies (GWAS), has revealed that pubertal timing is a highly polygenic trait. Although voice break is easily recorded in large cohorts, it holds quite low precision as a marker of puberty. In contrast, gonadarche and pubarche are early and clinically well-defined measures of puberty onset. Objective: To determine whether a polygenic risk score (PRS) of alleles that confer risk for voice break associates with age at gonadarche (AAG) and age at pubarche (AAP) in Chilean boys. Experimental Design: Longitudinal study. Subjects and Methods: 401 boys from the Growth and Obesity Chilean Cohort Study (n = 1194; 49.2% boys). Main Outcome Measures: Biannual clinical pubertal staging including orchidometry. AAG and AAP were estimated by censoring methods. Genotyping was performed using the Multi-Ethnic Global Array (Illumina). Using GWAS summary statistics from the UK-Biobank, 29 significant and independent single nucleotide polymorphisms associated with age at voice break were extracted. Individual PRS were computed as the sum of risk alleles weighted by the effect size. Results: The PRS was associated with AAG (beta=0.01, P = 0.04) and AAP (beta=0.185, P = 0.0004). In addition, boys within the 20% highest PRS experienced gonadarche and pubarche 0.55 and 0.67 years later than those in the lowest 20%, respectively (P = 0.013 and P = 0.007). Conclusions: Genetic variants identified in large GWAS on age at VB significantly associate with age at testicular growth and pubic hair development, suggesting that these events share a genetic architecture across ethnically distinct populations.

Decreased testosterone (T) to LH ratio and increased 17β‐estradiol (E2) serum concentrations represent a common finding among patients with severe spermatogenic failure, suggesting a concurrent Leydig cell steroidogenic dysfunction. Aromatase overexpression has been associated with increased serum and intratesticular E2 in these patients. However, it is unknown whether the sulfatase pathway contributes to the increased availability of active estrogens in patients with primary spermatogenic failure.

To investigate the association of partial-AZFc deletions in Chilean men with primary spermatogenic failure and their testicular histopathological phenotypes, analyzing the contribution of DAZ dosage, CDY1 copies, and Y-chromosome haplogroups. We studied 479 Chilean men: 334 infertile patients with histological examination (233 cases with spermatogenic defects and 101 normal spermatogenesis, obstructive controls, OC), and 145 normozoospermic controls (NC). AZFc subdeletions were detected by single-tagged sequences and single nucleotide variants analysis. DAZ-copy number was quantified by real-time qPCR. Y-chromosome haplogroups (Y-hg) were hierarchically genotyped through 16 biallelic-markers. The prevalence of AZFc-partial deletions was increased in cases (6%) compared with NC (1.4%) (P = 0.035). There was no difference between 143 Sertoli-cell only syndrome, 35 maturation arrest, or 35 mix atrophy patients and controls. However, gr/gr deletions were more frequent in 16 subjects with hypospermatogenesis compared with NC (P = 0.003) and OC (P = 0.013). Y-hg R was the most prevalent (~ 50%), but decreased among gr/gr deletions (21%, P = 0.03). The prevalence of Y-hg M increased in cases versus controls, both in total and non-deleted men (3.9 and 3.7% versus 0.4%, P = 0.009 and P = 0.016, respectively). Among gr/gr deletions, Y-hg H increased compared with non-deleted men (14.3% versus 0.4%, P = 0.0047). Partial-AZFc deletions in a Chilean admixed population are associated with secretory azo/oligozoospermia and might have a role in the development of hypospermatogenesis. Low represented haplogroups, Y-hg M and Y-hg H, show an association with the occurrence of spermatogenic failure and gr/gr deletions respectively; however, additional studies are required.

Study Objective: The androgen receptor (AR) harbors a variable repeat number of glutamine residues codified by (CAG)n, which seems to inversely affect AR transcriptional activity. We assessed whether (CAG)n affects the sequence of the androgen-sensitive pubertal events and body composition in prepubertal girls. Design, Setting, Participants, and Interventions: Nested case-control study within the Growth and Obesity Cohort Study of 1196 low-middle income children (approximately 50% girls) from a university clinic in Santiago, Chile. Cases were girls with high dehydroepiandrosterone sulfate (DHEAS; >42 mu g/dL; HD) at age 7.0 (+/- 0.4) years (n = 58). On follow-up, 32 of them had thelarche (TB2) before the age of pubarche (PH2) and 26 had PH2 before the age of TB2. As controls, 107 age-matched girls with normal DHEAS (<= 42 mu g/dL; ND) were selected. Main Outcome Measures: Methylation-weighted mean (CAG)n (mw[CAG]n) was calculated through X-chromosome methylation-sensitive enzyme restriction and polymerase chain reaction followed by automated capillary electrophoresis in peripheral blood DNA. Results: Girls with HD and PH2 before the age of TB2 showed a trend to higher frequency (7/26, 26.9%) of mw(CAG)n <20 compared with ND girls (12/107; 11.2%; P = .087). Accordingly, a direct correlation between age of PH2 and mw(CAG)n was observed in HD (r = 0.352; P = .007) and in ND girls (r = 0.207; P = 033). Moreover, HD girls with mw(CAG)n less than 20 had lower waist circumference and waist/height ratio than HD girls with mw(CAG)n from 20 to less than 25 (P = .027 and P = .012, respectively) at age of DHEAS determination. Conclusion: Our results suggest that a greater transcriptional activity of the AR, given by short number of CAG repeats, might favor the onset of pubarche and reduce central adiposity in prepubertal girls with HD.

STUDY QUESTION:Is the expression of steroidogenic enzyme 17α-Hydroxylase/17,20-Lyase (CYP17A1) down-regulated in Leydig cells (LCs) of men with spermatogenic failure and compensated impairment of LC function, i.e. a low testosterone to LH (T/LH) ratio?SUMMARY ANSWER:Although the transcriptional expression of CYP17A1 is increased, its protein expression is decreased, in isolated LCs of men with spermatogenic failure and reduced serum T/LH.WHAT IS KNOWN ALREADY:Primary spermatogenic defects have been associated with functional and morphological abnormalities of LCs, characterized by decreased serum testosterone (T) levels, decreased T/LH, increased 17β-estradiol (E2) and E2/T ratio, and larger clusters of LCs (LC hyperplasia). CYP17A1 is a key enzyme in the testosterone pathway and has been implicated in the steroidogenic lesion produced by E2 stimulation.STUDY DESIGN, SIZE, DURATION:We studied 18 azoospermic patients with Sertoli cell-only syndrome (SCOS) and signs of LC dysfunction (cases) and 10 obstructive azoospermic/oligozoospermic men with normal spermatogenesis (controls). The SCOS patients were sub-grouped into 9 cases with T/LH <2 and 9 cases with T/LH ≥2. All of the men underwent testicular biopsy for sperm retrieval at the Reproductive Unit of a University Hospital.PARTICIPANTS/MATERIALS, SETTING, METHODS:The transcriptional expression of CYP17A1 and SF-1 (steroidogenic factor 1) was quantified by SYBR®Green-based qPCR in LCs isolated by laser capture microdissection (LCM), and relative expression to the control pool was assessed. CYP17A1 protein expression was semi-quantified by indirect immunofluorescence (IFI) using Image-Pro Plus v7.0 (Media Cybernetics) in testicular tissue. FSH and LH serum concentrations, and serum and intratesticular T (ITT) and E2 (ITE2) were measured by IRMA and RIA, respectively.MAIN RESULTS AND THE ROLE OF CHANCE:Relative CYP17A1 mRNA expression was increased in cases with T/LH <2 compared to cases with T/LH ≥2, by a mean of 3.3-fold (P = 0.002). No corresponding increase in protein expression was found; in fact, CYP17A1 immunostaining intensity assessed by the Integrated Optical Density (IOD) parameter was lower in the cases with T/LH <2 compared to controls (P = 0.008). Relative SF-1 mRNA expression was similar in both case subgroups. CYP17A1 mRNA expression correlated with ITE2 and intratesticular E2/T (r = 0.536; P = 0.026 and r = 0.542; P = 0.016, respectively), while an inverse association was observed for ITE2 and protein level expression (r = -0.421; P = 0.05).LARGE SCALE DATA:Not applicable.LIMITATIONS REASONS FOR CAUTION:We should interpret the results of the semi-quantification of immunofluorescent staining by Image-Pro Plus software with caution, because it is a semi-quantitative method that may have certain difficulties regarding the disposition of protein in the cells. However, it is not influenced by variations in the number of cells that express the protein, as could be the case of western blot analysis in testicular tissue.WIDER IMPLICATIONS OF THE FINDINGS:Dysfunctional LCs of men with SCOS show post-transcriptional deregulation of CYP17A1, with increased mRNA and decreased protein expression, which may be modulated by increased ITE2 levels. In addition, transcriptional expression of CYP17A1 was not associated with changes in SF-1 mRNA expression.STUDY FUNDING/COMPETING INTEREST(S):This work was supported by the National Fund for Scientific and Technological Development (FONDECYT) of Chile to A.C. [grant number 1120176]. The authors declare no conflict of interest.

Estradiol (E2 ) is normally metabolized to hydroxyestradiols and methoxyestradiols by CYP1A1, CYP1B1 and COMT. However, an altered production of these metabolites by a disturbed expression of these enzymes is associated with reproductive and non-reproductive pathologies. In vitro studies suggest that increased hydroxyestradiols and methoxyestradiols intratesticular generation is related to male infertility, but no studies have explored whether infertile men have a disturbed testicular expression of the enzymes that generate these E2 metabolites. The aim of this study was to assess CYP1A1, CYP1B1 and COMT testicular expression at mRNA and protein level in men with spermatogenic impairment. Seventeen men with primary spermatogenic failure (13 with Sertoli cell-only syndrome and four with maturation arrest) and nine controls with normal spermatogenesis were subjected to testicular biopsy. mRNA was quantified using real-time RT-PCR and protein expression was evaluated using western blot and immunohistochemistry followed by integrated optic density analysis. Besides, the effects of hydroxyestradiols and methoxyestradiols on testosterone-induced transcriptional activity were evaluated in TM4 cells using a luciferase reporter assay system. Our results show that patients with Sertoli cell-only syndrome had significantly elevated COMT expression at the mRNA level, higher COMT immunoreactivity in their seminiferous tubules and increased protein expression of the soluble COMT isoform (S-COMT), whereas patients with maturation arrest had significantly elevated CYP1A1 mRNA levels and higher CYP1A1 immunoreactivity in interstitial space. Finally, 2-hydroxyestradiol decreased testosterone-induced transcriptional activity in Sertoli cells in vitro. In conclusion, male infertility is related to disturbed testicular expression of the enzymes responsible for producing hydroxyestradiols and/or methoxyestradiols. If these changes are related with increased intratesticular hydroxyestradiols and methoxyestradiols concentrations, they could elicit an impaired Sertoli cell function. Our results suggest CYP1A1 and COMT as new potential targets in treating male infertility.

AIMS:To assess serum oestrogen levels and oestrogenic activity in adolescents with Type 1 diabetes compared with a healthy control group.METHODS:We conducted a cross-sectional study that evaluated adolescents with Type 1 diabetes (n = 38) and healthy adolescents (control group; n = 32). Serum oestrogens, urinary oestrogen metabolites and serum oestrogenic activity were assessed. Oestrogenic activity was evaluated in an in vitro cell proliferation assay using a modified E-screen assay with MCF-7/BUS cells.RESULTS:Adolescents with Type 1 diabetes had lower oestrogenic activity levels in both phases of the menstrual cycle compared with the control group (follicular phase: 76 vs 94%; luteal phase: 97 vs 131%; P < 0.01), even after adjusting for BMI, oestradiol and oestrone levels. Postmenarcheal adolescents with Type 1 diabetes had lower oestradiol levels compared with control subjects in the follicular phase (63.3 pmol/l vs 89.4 pmol/l; P < 0.01) and higher oestrone levels compared with controls in the luteal phase (196 vs 151.9 pmol/l; P < 0.05).CONCLUSIONS:Adolescents with Type 1 diabetes had lower levels of serum oestrogenic activity, and these were lower than expected based on their serum oestradiol levels. We postulate that changes in the serum milieu of oestrogens in patients with Type 1 diabetes may explain their decreased oestrogenic activity and may play a role in their adverse metabolic profile.

Several observational studies have showed a combination of lower testosterone (T) to LH ratio and higher estradiol (E2 ) to T ratio in secretory infertile men compared to men with normal spermatogenesis, suggesting a steroidogenic dysfunction of Leydig cells (Lc) that may involve increased aromatase activity. Low T/LH ratio is associated with Lc hyperplasia, which together with LH hyperstimulation may represent compensation for impaired T production. Aromatase expression and oestrogen production are mainly detected in Lc of the testis, although Sertoli and germ cells also contribute to testicular aromatase activity. The aim of this study was to assess the transcriptional expression of CYP19A1 (aromatase) in isolated Lc of subjects with Sertoli cell-only syndrome (SCOS) and signs of Lc impairment. Nineteen patients with SCOS and 10 controls with normal spermatogenesis who had medical indication of testicular biopsy for sperm retrieval were studied. Leydig cells were isolated by Laser Capture Microdissection (LCM) and CYP19A1 mRNA expression was quantified by SYBR® Green-based qPCR. In addition, testicular T and E2 and serum hormonal levels were measured. Relative to control group, CYP19A1 was overexpressed more than twofold in 10/19 cases (2.3-12.2-fold increase), showing a significant increment in cases with low T/LH ratio (T/LH < 2) compared to cases with T/LH ≥ 2 (p = 0.038, REST® ). Moreover, Rq data for CYP19A1 had a direct correlation with testicular levels of E2 and the E2 /T ratio (r = 0.869; p < 0.001 and r = 0.633; p = 0.005). In summary, Lc from infertile patients with signs of Lc dysfunction overexpressed aromatase and showed an increment of testicular E2 . Our results suggest that increased expression of aromatase in Lc leads to higher E2 production and may account for the functional impairment of the Lc in patients with SCOS.

STUDY QUESTION: Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders?SUMMARY ANSWER: Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45, X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders.WHAT IS KNOWN ALREADY: The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to three AZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function.STUDY DESIGN, SIZE, DURATION: We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function.PARTICIPANTS/MATERIALS, SETTING, METHODS: All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs.MAIN RESULTS AND THE ROLE OF CHANCE: We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45, X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: -2.75 and -2.62), while a man with bipolar disorder was very tall (Z score: +2.56).LARGE SCALE DATA: N/A.LIMITATIONS, REASONS FOR CAUTION: The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45, X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues.WIDER IMPLICATIONS OF THE FINDINGS: This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45, X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions.

Objective: To determine whether decreased testicular levels of enzymes necessary for retinoic acid biosynthesis were associated with male infertility, as retinoic acid is known to be necessary for spermatogenesis.Design: Observational analysis of testicular tissue samples, sperm indices, and serum hormone concentrations.Setting: Two infertility centers in Chile.Patient(s): 32 infertile men and 11 control men.Intervention(s): Measurement of the three enzymes necessary for retinoic acid biosynthesis, aldehyde dehydrogenase (ALDH) 1A1, 1A2, and 1A3, in testicular tissue by a novel liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) peptide assay.Main Outcome Measure(s): ALDH isozyme levels compared by type of infertility and correlated with testicular germ cell numbers, sperm parameters, and serum and intratesticular hormone concentrations.Result(s): Men with infertility had statistically significantly reduced levels of ALDH1A2 but not ALDH1A1 or ALDH1A3 in their testicular tissue compared with men with normal spermatogenesis. The ALDH1A2 protein levels were strongly correlated with the number of germ cells found via testicular biopsy.Conclusion(s): These findings suggest that ALDH1A2 is the enzyme involved in retinoic acid biosynthesis in human germ cells. Further study of the relationship between intratesticular ALDH1A2 and male infertility is warranted to determine whether men with infertility have a reduced ability to synthesize retinoic acid within their germ cells that could impair spermatogenesis. (C) 2014 by American Society for Reproductive Medicine.

Purpose To determine the prevalence of South Amerindian Y chromosome in Chilean patients with spermatogenic failure and their association with classical and/or AZFc-partial Y chromosome deletions. Methods We studied 400 men, 218 with secretory azo/oligozoospermia (cases) and 182 controls (116 fertile and/or normozoospermic, and 66 azoospermic with normal spermatogenesis). After a complete testicular characterization (physical evaluation, hormonal and/or biopsy) peripheral blood was drawn to obtain DNA for Y chromosome microdeletions, AZFc-partial deletions and biallelic analysis by allele specific polymerase chain reaction (PCR) of the M3 (rs3894) single nucleotide polymorphism (SNP). Results Classical AZF microdeletions were found in 23 cases (Y-microdeleted). AZFc-partial deletions were observed in 10 cases (6 “gr/gr”, 3 “b2/b3” and 1 “b1/b3”) and 4 controls (4 “gr/gr”). The AZFc-partial deletions were mainly associated with the absence of DAZ1/DAZ2 (64 %). No significant differences in the prevalence of AZFc-partial deletions were observed between cases and controls. We observed a significant higher proportion of the Q1a3a haplogroup in Y-microdeleted men compared to patients with spermatogenic failure without deletions and control men ( P < 0.01 and P < 0.05, respectively by Bonferroni test). Among them, patients with AZFb deletions had an increased prevalence of the Q1a3a haplogroup compared to controls, cases without deletions and to those with complete or partial-AZFc deletions ( P < 0.01, Bonferroni test). Conclusions The Q1a3a South Amerindian lineage seems to increase the susceptibility to non AZFc microdeletions. On the other hand, in Chilean population the AZFc-partial deletions (“gr/gr”, “b1/b3” and/or “b2/b3”) does not seem to predispose to severe spermatogenic impairment.

BACKGROUND:The etiology of hypospadias is multifactorial. Abnormal androgenic secretion and/or action during the development of external genitalia may be involved in the etiology of this congenital malformation. This study explored CAG and GGN polymorphisms in the androgen receptor (AR) gene, which may affect its transcriptional activity, in patients with isolated hypospadias. METHODS:The length of the CAG/GGN polymorphisms was determined in 44 boys with non-severe (glandular) or severe (penile or penoscrotal) isolated hypospadias and with a normal hormonal evaluation. In addition, 79 healthy men, as controls, were studied. RESULTS:Mean CAG repeats were significantly higher in total and severe cases compared to controls (24.4 +/- 2.8 and 24.7 +/- 3.1 vs. 22.7 +/- 3.3, respectively; p<0.05, Student's t and Bonferroni test). In addition, a frequency of CAG alleles >23 was significantly different in total and severe cases compared to controls (70.5% and 74.1% vs. 39.2%, respectively, p<0.05, chi2 and Bonferroni test). The median number and the distribution of GGN polymorphisms were similar in cases and controls. CONCLUSION:Boys with isolated hypospadias have longer CAG alleles in their AR, which may be related with the development of this congenital malformation.

We characterised and correlated the histological and hormonal aspects of a cohort of 261 azo/oligozoospermic men, applying a quantitative/qualitative evaluation of testicular tissue and serum and intratesticular hormonal measurements. One hundred and 93 azo⁄oligozoospermic patients were diagnosed as: complete sertoli cell only syndrome (cSCOS), n = 76; focal SCOS, n = 31; maturation arrest, n = 34; hypospermatogenesis, n = 17; mixed atrophy, n = 25; and severe atrophy, n = 10. Normal spermatogenesis was observed in 68 infertile men (controls). Patients with cSCOS, focal SCOS, mixed and severe atrophy had larger LC/clusters (11.5; 11.0; 10.7; 18.9 LC/cluster) than controls (6 LC/cluster; P < 0.001). cSCOS, focal SCOS, mixed and severe atrophy patients had higher FSH, LH and lower T/LH ratio serum levels than the other groups. Intratesticular testosterone concentrations were higher in tissues with complete or focal SCOS (45.6 ng mg(-1) protein) and mixed atrophy (79.0 ng mg(-1) protein) than normal tissues (20.3 ng mg(-1) protein; P = 0.03 and P = 0.007). Considering all subjects, significant correlations were found between T/LH ratio and Leydig cells/cluster (r = 0.510, P < 0.001), FSH levels (r = -0.692, P < 0.001) and with intratesticular testosterone (r = -0.354, P = 0.001); these correlations follow the pattern of severity of spermatogenic damage. By a thorough histological evaluation, we validate the concept that the severity of spermatogenic impairment is associated with major morphological and functional disturbance of the Leydig cell compartment.

Androgens are essential for spermatogenesis. It has been postulated that androgen activity is modulated directly or indirectly by genetic variability in the androgen receptor gene sequence, including CAG/GGN polymorphisms and single-nucleotide polymorphisms (SNP). In this study, the frequency of 6 SNPs that constitute a haplotype in the androgen receptor sequence was determined by enzyme restriction assays and allele-specific polymerase chain reactions in 117 secretory azo/oligozoospermic men (93 idiopathic and 24 excryptorchidic), and in 121 controls with normal spermatogenesis (42 obstructive and 79 normozoospermic men) whose hormonal measurements and length of CAG/GGN polymorphisms were previously determined. The frequency of these 6 SNPs was not different between patients and controls. A total of 10 haplotypes (HAPs 1-10) formed by these 6 SNPs were found, and one of these haplotypes was observed with high frequency in the total population (HAP1, 83.2%; P < .001, χ(2) test). The frequency of the 10 haplotypes was not different between patients and controls, except for HAP5, which was only detected in one patient with a history of bilateral cryptorchidism (P = 0.014, Bonferroni test). On the other hand, no associations were found between the haplotypes studied and shorter or longer CAG or GGN polymorphisms. Interestingly, we found that the CAG 21 allele, which was previously correlated with an increased risk of idiopathic spermatogenic impairment, was more frequently found among the less common haplotypes that have higher follicle-stimulating hormone serum levels. In summary, we did not find an increased frequency of particular haplotypes in infertile men with idiopathic spermatogenic impairment compared with control men; however, we found that the CAG 21 allele, which appears to be associated with male infertility, is observed at a significantly higher proportion among the less common androgen receptor haplotypes.