Andrea Castro

Role of nitric oxide and bcl-2 family genes in the regulation of human endometrial apoptosis☆

OBJECTIVE To investigate the role of nitric oxide (NO) and death regulatory genes, bcl-2 and bax, in human endometria apoptosis. DESIGN Expression of bcl-2, bax, NO synthases (NOS), and the apoptotic effect of L-arginine on endometrial explants in vitro. SETTING Prospective study. PATIENT(S) Thirty-seven eumenorrheic women. INTERVENTION(S) Endometrial samples were obtained with Pipelle suction curette after women signed institutional informed consent forms. MAIN OUTCOME MEASURE(S) DNA fragmentation (TUNEL), immunohistochemistry, and reverse transcription polymerase chain reaction. RESULT(S) Apoptosis was detected in mid and late secretory endometria. L-arginine induced an increase in apoptosis in stroma (threefold), glands (eightfold), and surface epithelia (fourfold) in proliferative but not secretory endometria explants. Immunostaining of Bcl-2 was almost absent in the secretory endometria, whereas Bax increased in the stroma at the end of the menstrual cycle, coincident to the decrease in the bcl-2/bax mRNA relative ratio (P<.05) observed in secretory endometria. CONCLUSION(S) The induction of DNA fragmentation by L-arginine on proliferative endometria suggests that NO may be involved in the endometrial apoptotic process, whose control may be related predominantly to the changes of Bcl-2 expression.

P450-aromatase activity and expression in human testicular tissues with severe spermatogenic failure

There is evidence that impaired spermatogenesis is associated with an imbalance in the oestradiol/testosterone ratio and with Leydig cell (LC) dysfunction. In testis, P450-aromatase, encoded by CYP19, is responsible for the conversion of testosterone to oestradiol. The aims of this study were to quantify CYP19 mRNA expression, aromatase activity and protein localization, and to measure the oestradiol to testosterone ratio in testicular tissues of men with spermatogenic impairment. Twenty-four men with complete Sertoli cell-only syndrome (SCOS), 14 with focal SCOS, 14 with maturation arrest (MA), 8 with mixed atrophy and 30 controls with normal spermatogenesis were subjected to testicular biopsy. All subjects underwent a physical examination, cytogenetic and serum hormonal studies. Testicular CYP19 mRNA was quantified using real time RT-PCR. Testicular aromatase activity was measured using the (3)H(2)0 assay and protein expression was evaluated using immunohistochemistry. In cases, serum testosterone and oestradiol were normal, but the testosterone/LH ratio was lower compared with controls (p < 0.05). Aromatase was localized in the Leydig, Sertoli and germ cells of all tissues, although stronger intensity was observed in LC. Aromatase mRNA and activity were not altered in cases and correlated positively with LC number (r = 0.516 and r = 0.369; p < 0.008). The intratesticular oestradiol/testosterone ratio was elevated (p = 0.005) in complete SCOS patients compared with controls. In conclusion, testicular aromatase seems to be normal in most subjects with impaired spermatogenesis. However, an altered intratesticular oestradiol/testosterone ratio in some patients with complete SCOS suggests that aromatase is increased, which might contribute to Leydig cell dysfunction.

Androgen Receptor Gene CAG and GGN Repeat Polymorphisms in Chilean Men With Primary Severe Spermatogenic Failure

There is ample documentation supporting the fact that androgens are required for normal spermatogenesis. A minority of infertile men have abnormal testosterone blood levels or mild androgen receptor mutations. We investigated the androgen receptor CAG and GGN repeat lengths in Chilean men with spermatogenic impairment. We studied 117 secretory azoospermic/oligozoospermic men (93 idiopathic and 24 excryptorchidic), without Y-chromosome microdeletions, and 121 controls with normal spermatogenesis (42 obstructive and 79 normozoospermic men). Peripheral blood was drawn to obtain genomic DNA for polymerase chain reaction and automated sequencing of CAG and GGN repeats. Testicular characterization included hormonal studies, physical evaluation, and seminal and biopsy analysis. The CAG and GGN polymorphism distributions were similar among idiopathic men, excryptorchidic men, and controls and among the different types of spermatogenic impairment. However, the proportion of the CAG 21 allele was significantly increased in idiopathic cases compared to controls (P = .012 by Bonferroni test, odds ratio = 2.99, 95% confidence interval, 1.27-7.0) and the CAG 32 allele only was observed in excryptorchidic patients (P < .0002, Bonferroni test). Idiopathic cases with Sertoli cell-only syndrome showed the highest proportion of the CAG 21 allele (P = .024, χ(2) test). On the other hand, in idiopathic cases and controls the most common GGN allele was 23, followed by 24, but an inverse relation was found among excryptorchidic cases. The joint distribution of CAG and GGN in control, idiopathic, and excryptorchidic groups did not show an association between the 2 allele repeat polymorphisms (P > 0.05, χ(2) test). Our results suggest that the CAG 21 allele seems to increase the risk of idiopathic Sertoli cell-only syndrome. Moreover, the GGN 24 allele could be contributing to deranged androgen receptor function, associated with cryptorchidism and spermatogenic failure.

Testis development in the absence of SRY: chromosomal rearrangements at SOX9 and SOX3

Duplications in the ~2 Mb desert region upstream of SOX9 at 17q24.3 may result in familial 46,XX disorders of sex development (DSD) without any effects on the XY background. A balanced translocation with its breakpoint falling within the same region has also been described in one XX DSD subject. We analyzed, by conventional and molecular cytogenetics, 19 novel SRY-negative unrelated 46,XX subjects both familial and sporadic, with isolated DSD. One of them had a de novo reciprocal t(11;17) translocation. Two cases carried partially overlapping 17q24.3 duplications ~500 kb upstream of SOX9, both inherited from their normal fathers. Breakpoints cloning showed that both duplications were in tandem, whereas the 17q in the reciprocal translocation was broken at ~800 kb upstream of SOX9, which is not only close to a previously described 46,XX DSD translocation, but also to translocations without any effects on the gonadal development. A further XX male, ascertained because of intellectual disability, carried a de novo cryptic duplication at Xq27.1, involving SOX3. CNVs involving SOX3 or its flanking regions have been reported in four XX DSD subjects. Collectively in our cohort of 19 novel cases of SRY-negative 46,XX DSD, the duplications upstream of SOX9 account for ~10.5% of the cases, and are responsible for the disease phenotype, even when inherited from a normal father. Translocations interrupting this region may also affect the gonadal development, possibly depending on the chromatin context of the recipient chromosome. SOX3 duplications may substitute SRY in some XX subjects.

Absence of Y chromosome microdeletions in patients with cryptorchidism and hypospadias.

Microdeletions of the Y chromosome have been observed in some patients with cryptorchidism and severe defects of spermatogenesis. We investigated whether microdeletions of the Y chromosome may be present in patients with cryptorchidism and hypospadias. Peripheral blood was obtained from 20 male patients 5.8 +/- 4.1 years (range: 0.4-14 years) with cryptorchidism and hypospadias for somatic DNA analysis of Y chromosome using multiplex polymerase chain reaction. These patients had no identifiable genetic syndrome, other genitourinary malformations or an abnormal karyotype. We evaluated the presence or absence of amplification using a set of 34 different sequence-tagged sites (STS) in each patient. All patients showed normal length amplifications for each of the regions evaluated, suggesting that microdeletions of the Y chromosome are not a frequent cause of hypospadias associated with cryptorchidism.

Levels of the retinoic acid synthesizing enzyme aldehyde dehydrogenase-1A2 are lower in testicular tissue from men with infertility.

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.

Histological and hormonal testicular function in oligo/azoospermic infertile men

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.

DAX-1 and DAX-1A expression in human testicular tissues with primary spermatogenic failure

DAX-1 [dosage-sensitive sex reversal-adrenal hypoplasia congenital (AHC) critical region on the X chromosome gene 1; NR0B1] is an orphan nuclear receptor that acts as a transcriptional repressor in adrenal/gonadal development, steroidogenesis and probably spermatogenesis. An alternatively spliced form called DAX-1A (NR0B1A) has been described in several tissues including the testis, and in vitro studies have shown an inhibitory effect on DAX-1 transcriptional function. We aimed to study the mRNA and protein expression of DAX-1 in testicular tissues of 65 men with primary spermatogenic failure [complete Sertoli cell only syndrome (SCOS), focal SCOS, maturation arrest and mixed atrophy] compared with 33 controls with normal spermatogenesis. As a novel finding, we observed intense immunostaining, not only in the nucleus of Sertoli cells, but also in pachytene spermatocytes and round spermatids. The quantitative mRNA expression of DAX-1 and DAX-1A was similar between cases and controls and was not associated with the levels of gonadotrophins and steroids. Moreover, DAX-I transcript expression level was ∼750-fold higher than DAX-1A, and there was a strong positive correlation between them (r = 0.52; P< 0.001). We conclude that, in addition to Sertoli cells, DAX-1/DAX-1A is expressed in germ cells from spermatogonia to round spermatids. Besides, the similar mRNA expression of DAX-I and DAX-IA in testicular tissues from cases and controls does not support the involvement of DAX-1 in the etiology of primary spermatogenic failure. Finally, the low level of expression of the alternative transcriptional variant DAX-1A would not support its putative inhibitory function in vivo.

Androgen receptor CAG and GGN polymorphisms in boys with isolated hypospadias.

Abstract 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, χ2 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.

Analysis of 6 single-nucleotide polymorphisms in the androgen receptor gene in Chilean patients with primary spermatogenic failure.

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.