Vol. 181, No. 4, Supplement, Wednesday, April 29, 2009786 THE JOURNAL OF UROLOGY®

identify the unique metabolic signature of spermatogenesis in human testes.

METHODS: After receiving IRB approval, we compared the spectra of testicular tissue biopsies from men with three different histologic patterns: 1) normal spermatogenesis (prior paternity; undergoing vasectomy reversal; n = 10), 2) maturation arrest (MA; infertile men with NOA; n = 8), and 3) Sertoli-cell only (SCO; infertile men with NOA; n = 9). Clinical diagnoses were based on thorough evaluation by a fellowship trained, reproductive urologist. Testis biopsies were flash frozen and stored at -80°C. Quantitative MRS data were acquired using a Varian INOVA spectrometer. For each specimen, spectra of 19 tissue metabolites were identified using the program, QUEST. Concentrations were calculated relative to the peak area of a standardized signal. One-way ANOVA was performed to determine differences in MR spectra between diagnostic groups.

RESULTS: Only phosphocholine (PC) level was significantly different between groups. Mean PC levels were 3 times higher in normal spermatogenesis compared to SCO (1.5 ± 0.3 vs. 5.4 ± 1.4 mmol/kg; p= 0.01). PC concentrations in the MA group ranged between normal and SCO groups. Detailed pathologic review of the MA subjects showed rare spermatids or mature sperm in samples with PC levels in the “normal spermatogenesis” range, whereas none of these were seen in subjects within the “SCO” range.

CONCLUSIONS: PC levels by HR-MAS are significantly higher in ex vivo testicular tissues that demonstrate spermatogenesis. These data suggest a unique chemical signature for spermatogenesis that may be used to develop a novel, non-invasive, diagnostic study for men with NOA.

Source of Funding: None

2166ARRAY-BASED COMPARATIVE GENOMIC HYBRIDIZATION REVEALS 9P24 AS A GENETIC HOTSPOT FOR AMBIGUOUS GENITALIA AND GONADAL DYSGENESIS

Michael H Hsieh*, Shuo Han, Mounia Louet, Dolores J Lamb, Houston, TX

INTRODUCTION AND OBJECTIVE: 9p24 has been postulated to be a genetic hotspot for genital anomalies and infertility, but most studies have utilized routine karyotype for characterization of this chromosomal region. This study was designed to test the hypothesis that submicroscopic chromosomal aberrations (unrecognized by routine karyotype) in 9p24 are associated with congenital genitourinary defects and can be identified through chromosomal microrrays (CMA) .

METHODS: We used a CMA method designed in the Kleberg Cytogenetics Laboratory at Baylor College of Medicine. CMA was developed for the clinical diagnosis of known chromosomal disorders using large insert genomic DNA clones as targets for comparative genomic hybridization (CGH). This allows for detection of duplications or deletions well below the level of discrimination of standard karyotype. Using this targeted CGH-based microarray, we analyzed three cases of ambiguous genitalia. Genomic DNA from test samples and control samples was differentially labeled with fluorescent dyes and hybridized to the array. The analysis was performed using the same normalization and data processing methods successfully used for expression arrays. Findings were validated by site-specific FISH analysis.

RESULTS: Three out of 50 patients with ambiguous genitalia who were screened by CMA featured abnormal findings. Our first case, a 46 XY child with feminized external genitalia had a karyotype demonstrating an inverted duplication of chromosome 9p. CMA revealed a deletion spanning 7 Mb from 9p24.1 to the telomere region confirming and further defining the karyotype analysis. In our second case, a 9 year old 46, XY child diagnosed with Swyer Syndrome was found to have a 9p24.3 deletion on karyotype and CMA, with the latter also identifying a duplication of 0.25 Mb of 7p22.3. In our final case, a child with ambiguous genitalia was found to have a deletion of 9.5 Mb in 9p23p24.3 and a duplication of 25.9 Mb of 9p13.1p23. Parental FISH analysis reveals if these results represent inherited polymorphisms or de novo events likely to be pathogenic.

CONCLUSIONS: CMA is a powerful tool useful for identification of unrecognized genomic alterations that could be responsible for abnormal urogenital development. Our findings reveal several submicroscopic structural chromosomal defects in 9p24, confirming that this is a genetic hotspot for genital abnormalities and associated infertility. This suggests that genes related to genitourinary development may reside in this region.

Source of Funding: 1R01DK078121-01 to DJL and American Urological Association Foundation (MHH)

2167REAL-TIME PCR ANALYSIS DETECTION OF AZF MICRODELETIONS: A RAPID AND ACCURATE METHOD FOR INFERTILITY SCREENING

Anna Mielnik, Elena Gimenez*, Peter N Schlegel, Darius A Paduch, New York, NY

INTRODUCTION AND OBJECTIVE: Y chromosome microdeletion (YCM) testing is important in evaluation of patients with oligospermia and azoospermia, however it is labor intensive and thus often underutilized. In the present study we developed a novel, single step real-time PCR (RT-PCR) approach to YCM screening and compared its performance with the standard PCR method.

METHODS: Ten sequence-tagged sites (STSs) representing all AZFa, AZFb, AZFc and partial deletions of AZFc were selected for RT-PCR analysis. In addition, SRY gene presence was evaluated as a positive control. Thirty-six patients with known AZF deletions detected by traditional PCR were screened using the new RT-PCR approach. For each screen, normal male and female DNAs were simultaneously amplified with patients samples. PCR amplifications were performed in a total volume of 10ul containing 10ng of genomic DNA, 0.2uM of each primer and 1x SYBR Green I Master Mix under following conditions: 10min at 94 C followed by 35 three temperature cycles (10s at 95, 15s at 60 and 25s at 72 C). All RT-PCR and melting curve analyses were performed in duplicate wells in 480 Roche LightCycler (Mannheim, Germany) at least twice on different runs. For each tested STS, mean values of crossing points (Cp) and melting temperatures were calculated using LightCycler software version 1.5.0.

RESULTS: Patients tested for AZF deletions by RT-PCR had high concordance (100 % sensitivity and specificity) for AZFa, AZFb, AZFc, AZFb+c and b1/b3 deletions when compared to detection by traditional PCR. Cp values for deleted STS were either 0 or differed markedly from that of male controls; melting curve analysis showed no single peak as in female controls. For all implemented STS, mean Cp values were specific and reproducible for male controls with intact Y chromosome. For all female controls Cp values for given STS were negative and melting curve analysis showed no single peak as seen in male controls.

CONCLUSIONS: The new RT-PCR with melting curve analysis approach was as accurate and sensitive as standard PCR. This single step method does not involve intensive and laborious post-PCR manipulations, requires a small amount of DNA (10ng v 150 ng), can yield results within 2-3 hours, and virtually eliminates cross-contamination. Our method allows for quantitative assessment of gene dosing that is critical for multiple copy genes located on Y. The new technique combines accurate, reproducible results with convenience of execution and appears to be an improved method for screening infertile patients for YCM.

Source of Funding: None

2168CAN ORCHIDOPEXY IN ADULTHOOD IMPROVE FERTILITY?

Koji Chiba*, Tomomoto Ishikawa, Kohei Yamaguchi, Atsushi Takenaka, Masato Fujisawa, Kobe, Japan

INTRODUCTION AND OBJECTIVE: Undescended testis (UT) is a frequent congenital disease which is often diagnosed and treated during childhood. However, UT sometimes escapes detection until