Anna Mielnik

Aberrations in Pseudoautosomal Regions (PARs) Found in Infertile Men with Y-Chromosome Microdeletions

CONTEXT The pseudoautosomal regions (PARs) of the Y-chromosome undergo meiotic recombination with the X-chromosome. PAR mutations are associated with infertility and mental and stature disorders. OBJECTIVE The aim of the study was to determine whether men with Y-chromosome microdeletions have structural defects in PARs. DESIGN AND PARTICIPANTS Eighty-seven infertile men with Y-chromosome microdeletions and 35 controls were evaluated for chromosomal rearrangements using commercial or custom (X- and Y-chromosome) array comparative genomic hybridization or by quantitative PCR of selected PAR genes. Multisoftware-defined chromosomal gains or losses were validated by quantitative PCR and FISH. RESULTS Array comparative genomic hybridization confirmed the AZF deletions identified by multiplex PCR. All men with Y-chromosome microdeletions and an abnormal karyotype displayed PAR abnormalities, as did 10% of men with Y-chromosome microdeletions and a normal karyotype. None of the control subjects or infertile men without Y-chromosome microdeletions had PAR duplications or deletions. SHOX aberrations occurred in 14 men (nine gains and five losses); four were short in stature (<10th percentile), and one was tall (>95th percentile). In contrast, the height of 23 men with Y-chromosome microdeletions and normal PARs was average at 176.8 cm (50th percentile). CONCLUSIONS Y-chromosome microdeletions can include PAR defects causing genomic disorders such as SHOX, which may be transmitted to offspring. Previously unrecognized PAR gains and losses in men with Y-chromosome microdeletions may have consequences for offspring.

Activation of GPER-1 Estradiol Receptor Downregulates Production of Testosterone in Isolated Rat Leydig Cells and Adult Human Testis

Purpose Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. Materials and Methods Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Results GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20–30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. Conclusions Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

Possible germ cell-Sertoli cell interactions are critical for establishing appropriate expression levels for the Sertoli cell-specific MicroRNA, miR-202-5p, in human testis

BackgroundTo examine human microRNA expression in fertile men and subsequently to compare expression patterns of miRNAs in fertile and infertile men, specifically men with Sertoli Cell Only (SCO) histopathology.MethodsTesticular tissues from men with azoospermia and SCO, as well as those of men with normal spermatogenesis, were analyzed. MicroRNA was isolated using the miRCURY™ RNA Purification Kit. A miRCURY LNA™ Universal RT system was used for detection of microRNA by quantitative real-time PCR. MicroRNA localization was performed by in situ hybridizations (ISH) on formalin-fixed paraffin embedded (FFPE) tissue utilizing miRCURY LNA™ microRNA ISH technology. Statistical analysis was performed by GenEx V5.0.ResultsMicroRNA expression was determined for 13 normal fertile men and 5 men with the confirmed diagnosis of diffuse SCO. MiR-202-5p expression was reduced by 17-fold (P < 0.00001) in tissue from SCO men compared to normal. MiR-34c-5p was reduced by 346-fold (P < 0.00001), miR-10b was reduced 18-fold (P < 0.00001), miR-191 was reduced 20-fold (P = 0.001) and miR-126 was reduced 40-fold (P < 0.00001)) in tissues from SCO compared to normal fertile men. Using ISH, miR-202-5p was localized to Sertoli cells of men with normal spermatogenesis, but not in the Sertoli cells of men with SCO.ConclusionNumber of miRNAs are differentially expressed in normal fertile men compared to men with SCO. MicroRNA-202-5p is localized to Sertoli cells and its expression dramatically differs between fertile men and men whose germ cells are depleted, suggesting a novel interaction for regulating microRNA expression between the somatic and germ cell components of the seminiferous epithelium.AbstractObjectifsEvaluer l’expression des microARN chez des hommes féconds puis comparer les profils d’expression de ces miRNAs chez des hommes féconds et des inféconds qui présentent plus particulièrement un syndrome de Sertoli seules (SCO) à l’histologie testiculaire.Matériel et MéthodesOnt été analysés des tissues testiculaires d’hommes avec azoospermie et SCO ainsi que ceux d’hommes avec spermatogenèse normale. Les miRNAs ont été isolés avec la trousse de Purification miRCURY™ RNA. Le système miRCURY LNA™ Universal RT a été utilisé pour la détection quantitative de miARNs par PCR en temps réel. La localisation des miARNs a été réalisée par hybridation in situ (HIS) sur des tissus fixés au formol et inclus en paraffine en utilisant la technologie miRCURY LNA™ microRNA ISH. Les analyses statistiques ont été faites avec GenEx V5.0.RésultatsL’expression des microARNs a été faite chez 13 hommes féconds et 5 hommes avec un diagnostic confirmé de SCO diffus. L’expression de miR-202-5p est réduite d’un facteur 17 (P < 0.00001) dans le tissu des hommes SCO par rapport au tissu des hommes à spermatogenèse normale. L’expression de miR-34c-5p est réduite d’un facteur 346 (P < 0.00001), celle de miR-10b d’un facteur 18 (P < 0.00001), celle de miR-191 d’un facteur 20 (P = 0.001) et celle de miR-126 d’un facteur 40 (P < 0.00001) dans les tissus des hommes SCO comparés à ceux des hommes à spermatogenèse normale. MiR-202-5p a été localisé par HIS dans les cellules de Sertoli des hommes à spermatogenèse normale, mais pas dans les cellules de Sertoli des hommes SCO.ConclusionsNombre de miARNs sont exprimés différentiellement chez les hommes féconds par rapport aux hommes SCO. MicroARN-202-5p est localisé dans les cellules de Sertoli et son expression diffère de façon marquée entre les hommes féconds et ceux dont les cellules germinales sont absentes; ceci suggère une nouvelle interaction – entre les cellules somatiques et germinales constitutives de l’épithélium séminifère – impliquée dans la régulation de l’expression des microARNs.

Identification of polymorphisms and balancing selection in the male infertility candidate gene, ornithine decarboxylase antizyme 3

BackgroundThe antizyme family is a group of small proteins that play a role in cell growth and division by regulating the biosynthesis of polyamines (putrescine, spermidine, spermine). Antizymes regulate polyamine levels primarily through binding ornithine decarboxylase (ODC), an enzyme key to polyamine production, and targeting ODC for destruction by the 26S proteosome. Ornithine decarboxylase antizyme 3 (OAZ3) is a testis-specific antizyme paralog and the only antizyme expressed in the mid to late stages of spermatogenesis.MethodsTo see if mutations in the OAZ3 gene are responsible for some cases of male infertility, we sequenced and evaluated the genomic DNA of 192 infertile men, 48 men of known paternity, and 34 African aborigines from the Mbuti tribe in the Democratic Republic of the Congo. The coding sequence of OAZ3 was further screened for polymorphisms by SSCP analysis in the infertile group and an additional 250 general population controls. Identified polymorphisms in the OAZ3 gene were further subjected to a haplotype analysis using PHASE 2.02 and Arlequin 2.0 software programs.ResultsA total of 23 polymorphisms were identified in the promoter, exons or intronic regions of OAZ3. The majority of these fell within a region of less than two kilobases. Two of the polymorphisms, -239 A/G in the promoter and 4280 C/T, a missense polymorphism in exon 5, may show evidence of association with male infertility. Haplotype analysis identified 15 different haplotypes, which can be separated into two divergent clusters.ConclusionMutations in the OAZ3 gene are not a common cause of male infertility. However, the presence of the two divergent haplotypes at high frequencies in all three of our subsamples (infertile, control, African) suggests that they have been maintained in the genome by balancing selection, which was supported by a test of Tajimas D statistic. Evidence for natural selection in this region implies that these haplotypes may be associated with a trait other than infertility. This trait may be related to another function of OAZ3 or a region in tight linkage disequilibrium to the gene.

Deletion or underexpression of the Y-chromosome genes CDY2 and HSFY is associated with maturation arrest in American men with nonobstructive azoospermia.

Maturation arrest (MA) refers to failure of germ cell development leading to clinical nonobstructive azoospermia. Although the azoospermic factor (AZF) region of the human Y chromosome is clearly implicated in some cases, thus far very little is known about which individual Y-chromosome genes are important for complete male germ cell development. We sought to identify single genes on the Y chromosome that may be implicated in the pathogenesis of nonobstructive azoospermia associated with MA in the American population. Genotype-phenotype analysis of 132 men with Y-chromosome microdeletions was performed. Protein-coding genes associated with MA were identified by visual analysis of a genotype-phenotype map. Genes associated with MA were selected as those genes within a segment of the Y chromosome that, when completely or partially deleted, were always associated with MA and absence of retrievable testicular sperm. Expression of each identified gene transcript was then measured with quantitative RT-PCR in testicular tissue from separate cohorts of patients with idiopathic MA and obstructive azoospermia. Ten candidate genes for association with MA were identified within an 8.4-Mb segment of the Y chromosome overlapping the AZFb region. CDY2 and HSFY were the only identified genes for which differences in expression were observed between the MA and obstructive azoospermia cohorts. Men with obstructive azoospermia had 12-fold higher relative expression of CDY2 transcript (1.33 ± 0.40 vs. 0.11 ± 0.04; P=0.0003) and 16-fold higher expression of HSFY transcript (0.78 ± 0.32 vs. 0.05 ± 0.02; P=0.0005) compared to men with MA. CDY2 and HSFY were also underexpressed in patients with Sertoli cell only syndrome. These data indicate that CDY2 and HSFY are located within a segment of the Y chromosome that is important for sperm maturation, and are underexpressed in testicular tissue derived from men with MA. These observations suggest that impairments in CDY2 or HSFY expression could be implicated in the pathogenesis of MA.

Diagnosis of the gr/gr Y Chromosome Microdeletion Does Not Help in the Treatment of Infertile American Men

PURPOSE The phenotypic effects of the gr/gr partial azoospermia factor c deletion vary geographically and to our knowledge have not been reported in the American population. We evaluated the clinical characteristics of infertile American men with the gr/gr deletion. MATERIALS AND METHODS We retrospectively reviewed clinical data on 1,410 infertile men tested for the gr/gr deletion. We analyzed sperm concentration and the outcome of microdissection testicular sperm extraction with respect to gr/gr status. RESULTS We identified 73 men with gr/gr deletions, including 43 of 989 (4.3%) with azoospermia, 18 of 317 (5.7%) with severe oligospermia (less than 5 million sperm per ml), 6 of 61 (9.8%) with oligospermia (5 to less than 20 million sperm per ml) and 6 of 43 (14%) infertile men with normospermia (greater than 20 million sperm per ml). A gr/gr deletion correlated with higher sperm production. The gr/gr deletion rate was higher in men with normospermia than in those with a sperm concentration of less than 20 million and less than 5 million per ml (p = 0.021 and 0.006, respectively). Microdissection testicular sperm extraction was done in 22 azoospermic men with gr/gr deletions and sperm were retrieved in 14 (64%). This retrieval rate was similar to that at our center in men with idiopathic nonobstructive azoospermia (p = 0.13). CONCLUSIONS Diagnosis of the gr/gr deletion did not predict impaired sperm production in our patient population and did not appear to alter the prognosis for surgical sperm retrieval. Despite the established modulatory impact of the gr/gr deletion on sperm production in some populations at this time the clinical value of testing infertile American men for the gr/gr deletion is not clear.

Heat shock factor Y chromosome (HSFY) mRNA level predicts the presence of retrievable testicular sperm in men with nonobstructive azoospermia

OBJECTIVE To evaluate heat shock factor Y chromosome (HSFY) mRNA as a biomarker for the presence of retrievable testicular sperm. DESIGN Case-control study. SETTING Academic medical center. PATIENT(S) Men with nonobstructive azoospermia (NOA). INTERVENTION(S) Testicular tissue from men with successful or failed testicular sperm extraction was evaluated with qunatitative real-time polymerase chain reaction (qRT-PCR) for expression of HSFY mRNA. MAIN OUTCOME MEASURE(S) Area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and probability of sperm retrieval based on HSFY testing. RESULT(S) We found higher HSFY mRNA expression in testicular tissue from NOA patients in whom sperm were successfully retrieved compared with those in whom sperm were not found, with good discrimination between the groups in all histologic variants of NOA (AUC 0.89 overall, 0.98 for patients with Sertoli cell only [SCO] histology, 0.90 for patients with maturation arrest [MA] histology). Sensitivity and specificity were, respectively, 67% and 93% overall, 92% and 100% for SCO patients, and 67% and 92% for MA patients. The probabilities of sperm retrieval for HSFY-positive and -negative patients were, respectively, 93% and 31% overall, 100% and 7% for SCO patients, and 91% and 32% for MA patients. CONCLUSION(S) Detection of HSFY mRNA expression by qRT-PCR has promising application in the evaluation and counseling of men with NOA before attempted sperm retrieval surgery.

Ubiquitin Specific Protease 26 (USP26) Expression Analysis in Human Testicular and Extragonadal Tissues Indicates Diverse Action of USP26 in Cell Differentiation and Tumorigenesis

Ubiquitin specific protease 26 (USP26), a deubiquitinating enzyme, is highly expressed early during murine spermatogenesis, in round spermatids, and at the blood-testis barrier. USP26 has also been recognized as a regulator of androgen receptor (AR) hormone-induced action involved in spermatogenesis and steroid production in in vitro studies. Prior mutation screening of USP26 demonstrated an association with human male infertility and low testosterone production, but protein localization and expression in the human testis has not been characterized previously. USP26 expression analysis of mRNA and protein was completed using murine and human testis tissue and human tissue arrays. USP26 and AR mRNA levels in human testis were quantitated using multiplex qRT-PCR. Immunofluorescence colocalization studies were performed with formalin-fixed/paraffin-embedded and frozen tissues using primary and secondary antibodies to detect USP26 and AR protein expression. Human microarray dot blots were used to identify protein expression in extra-gonadal tissues. For the first time, expression of USP26 and colocalization of USP26 with androgen receptor in human testis has been confirmed predominantly in Leydig cell nuclei, with less in Leydig cell cytoplasm, spermatogonia, primary spermatocytes, round spermatids, and Sertoli cells. USP26 likely affects regulatory proteins of early spermatogenesis, including androgen receptor with additional activity in round spermatids. This X-linked gene is not testis-specific, with USP26 mRNA and protein expression identified in multiple other human organ tissues (benign and malignant) including androgen-dependent tissues such as breast (myoepithelial cells and secretory luminal cells) and thyroid tissue (follicular cells). USP26/AR expression and interaction in spermatogenesis and androgen-dependent cancer warrants additional study and may prove useful in diagnosis and management of male infertility.

Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome

The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome (KS), based on the detection of supernumerary X-chromosomes (X-chs). DNA was extracted from peripheral blood samples of twenty-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction (MS-qPCR) was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific transcript (XIST-U and XIST-M) gene. X-ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and fluorescent in situ hybridization (FISH) results. Data analysis was performed using the Roche® LightCycler software V. 3.5.3., including determination of crossing points (CPs) by fit-point analysis and melting curve analysis. X-ch disomy was detected in all female controls and KS patients; male controls expressed XIST-M only. CPs ranged from 29.5 to 32.5 (standard deviation (s.d.) 0.8) for XIST-U and from 29 to 31 (s.d. 0.6) for XIST-M. Limit of detection of mosaicism was 1%. Based on XIST-U/XIST-M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism (1.8% and 17.8%) was comparable to FISH results (2.3% and 15%, respectively). Turnaround time from DNA deamination to final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific and rapid test for the detection of X-ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade 47,XXY/46,XY mosaicism.

Bulbocavernosus muscle area measurement: a novel method to assess androgenic activity

Serum testosterone does not correlate with androgen tissue activity, and it is critical to optimize tools to evaluate such activity in males. Ultrasound measurement of bulbocavernosus muscle (BCM) was used to assess the relationship between the number of CAG repeats (CAGn) in the androgen receptor (AR) and the BCM size; the changes in the number of CAGn over age were also evaluated. Transperineal ultrasound measurement of the BCM was also performed. AR CAGn were determined by high performance liquid chromatography, and morning hormone levels were determined using immunoassays. Forty-eight men had CAG repeat analysis. Twenty-five were <30 years of age, mean 23.7 years (s.d. = 3.24) and 23 were >45 years of age, mean 53 years (s.d. = 5.58). The median CAGn was 21 (13–29). BCM area was greater when the number of CAGn were <18 as compared to the number of CAGn >24 (P = 0.04). There was a linear correlation between the number of CAGn and the BCM area R2 = 16% (P = 0.01). In the 45 to 65-years-old group, a much stronger negative correlation (R2 = 29%, P = 0.01) was noticed. In the 19 to 29-years-old group, no such correlation was found (R2 = 4%, P = 0.36). In older men, the number of CAGn increased with age (R2 = 32%, P = 0.01). The number of CAGn in the AR correlates with the area of the BCM. Ultrasound assessment of the BCM is an effective surrogate to evaluate end-organ activity of androgens. The number of CAGn may increase with age.