Phil Bach

Patients with Inflammatory Bowel Disease Exhibit Dysregulated Responses to Microbial DNA

Background A critical role for the gut epithelium lies in its ability to discriminate between pathogens and commensals and respond appropriately. Dysfunctional interactions between microbes and epithelia are believed to have a role in inflammatory bowel disease (IBD). In this study, we analyzed microbiota and gene expression in IBD patients and examined responses of mucosal biopsies to bacterial DNA. Methods Biopsies were taken from non-inflamed areas of the colon in healthy controls (HC) and Crohns disease (CD) and ulcerative colitis (UC) patients in remission. Biopsies were snap-frozen or cultured with DNA from Lactobacillus plantarum (LP) or Salmonella dublin (SD). Gene expression was analyzed under basal conditions and in response to DNA. Gene networks were analyzed using Ingenuity Pathways software. Mucosal-associated microbiota was analyzed using terminal restriction fragment length polymorphism. Frequency of single nucleotide polymorphisms in NOD2 and TLR9 was assessed. Results Patients with IBD had altered microbiota, enhanced expression of inflammatory genes, and increased correlations between specific gene expression and microbes. Principle component analysis showed CD and UC patients to cluster independently from healthy controls in both gene expression and microbial analysis. DNA from LP stimulated anti-inflammatory pathways in controls and UC patients, but induced an upregulation of IL17A in CD patients. There were no differences in SNP frequencies of TLR9 or NOD2 in the groups. Conclusions Patients with Crohns disease exhibit altered responses to bacterial DNA. These findings suggest that the gut response to bacterial DNA may depend not only on the specific type of bacterial DNA, but also on the host.

Independently Interpreted Retrograde Urethrography Does Not Accurately Diagnose and Stage Anterior Urethral Stricture: The Importance of Urologist-performed Urethrography

OBJECTIVE To compare the accuracy of retrograde urethrogram (RUG) interpretation between the primary physician performing the procedure and the independent physician interpreting the films to evaluate the suitability of relying on independent physician interpretations for the purposes of preoperative urethral stricture surgery planning. METHODS A retrospective review was performed on a cohort of 397 patients undergoing anterior urethroplasty over a 7-year period at a single center. Preoperative RUG findings as reported at the time by both the urologist performing the urethrography and the independent interpreter (radiologist) were abstracted and compared with stricture location and length as measured intraoperatively. RUG adequacy was defined as a comment on the presence, location, and length of the urethral stricture. RESULTS Only 49% of independently reported RUG studies were deemed adequate, and 87% of independently reported studies correctly diagnosed the presence of a stricture. Forty-nine percent of independently reported studies correctly identified stricture location compared with 96% of primary physician-reported cases (P <.001). The reported stricture lengths were 3.23 ± 2.25, 4.19 ± 2.49, and 4.51 ± 2.65 cm for the independently reported RUGs, primary physician-reported RUGs, and the intraoperative measurements, respectively. Differences between all the groups were statistically significant (P <.001). Independently reported length had a 0.47 R(2) coefficient of correlation to the intraoperative length (P <.001) compared with a 0.93 R(2) coefficient of correlation between primary physician-reported length and intraoperative length (P <.001). CONCLUSION Independently reported RUGs are not as accurate as primary physician-reported RUGs, and caution should be used when they are used for preoperative planning.

Microdissection testicular sperm extraction

Microdissection testicular sperm extraction (microTESE) is considered the gold standard method for surgical sperm retrieval among patients with non-obstructive azoospermia (NOA). In this review, we will discuss the optimal evaluation of NOA patients and strategies to medically optimize NOA patients prior to microTESE. In addition, we will also discuss technical principles and pearls to maximize the chances of successful sperm retrieval, sperm retrieval rates (SRR) based upon testicular histology, predictors of successful sperm retrieval, gonadal recovery following microTESE, and potential complications.

MP07-09 EXPLORING RNA EXPRESSION PROFILES OF KLINEFELTER'S SYNDROME IN THE SETTING OF NON-OBSTRUCTIVE AZOOSPERMIA

by tumours did significantly correlate with spermatogenesis (28.3% with sperm vs. 48.4% when no sperm found, p1⁄40.05). CONCLUSIONS: Spermatogeneis is present in the majority of testes affected by germ cell tumours (70%), and it does not appear to be related to any tumour pathology, apart from percentage tumour volume. Sperm extraction at the time of orchidectomy is a sensible approach, as testis specimens, destined for the pathology lab, would otherwise be a waste of functionally viable tissue. Given that spermatogenesis was focal in 38%, sperm retrieval is best performed with a microTESE (onco microTESE) to allow identification of these small foci. In our own experience, we have found sperm in 60% of patients with testicular tumour and azoospermia, at first presentation, using this technique. A change in focus to identify the azoospermic patient, prior to orchidecotmy, is vital to allow such an approach to be adopted.

MP89-19 TIMING OF RETURN OF SPERM TO THE EJACULATE AND LATE FAILURES FOLLOWING VASAL RECONSTRUCTION

as well as resident and fellow surgical assistance were documented. Patient and female partner age, natural pregnancy rates, procedure type, preand post-operative semen parameters, time from vasectomy, and postoperative complications were all evaluated. The effects of a fellow surgical assistance on perioperative and postoperative outcomes were then analyzed. Statistical significance, defined as a p-value <0.05, was determined using Student’s T, chi square or Fischer exact test. RESULTS: We identified 183 patients who underwent subinguinal varicocele ligation or vasectomy reversal by a single surgeon at our institution. All patients undergoing surgery for fertility concerns were included for analysis. Vasectomy reversals requiring one or more vasoepididymostomy were excluded from analysis, due to insufficient numbers for meaningful comparison, as were any patients undergoing combined cases in addition to microsurgical surgery. Compared to when faculty was operating without a fellow (n1⁄472), fellow surgical assistance (n1⁄481) results in a statistically significant prolonged operative time for both unilateral varicocele ligation (1:22 vs 1:01, p<0.01), bilateral varicocele ligation (2:13 vs 1:43, p<0.01), and bilateral vasovasostomy (2:49 vs 2:19, p<0.01). Comparing fellow assisted cases to those without a fellow, there were no significant differences in all other outcome measurements or patient demographics. Overall, 69.0% of patients undergoing varicocelectomy experienced a significant improvement in their Total Motile Sperm Count post-operatively, and 100% of patients achieved patency after vasectomy reversal. CONCLUSIONS: Fellowship training was associated with a ~30% increase in operative time for unilateral and bilateral varicocele ligation, and a ~20% increase in operative time for bilateral vasovasostomy, but does not adversely impact outcomes of microsurgical cases.

PD08-06 RNA SEQUENCING. EXPLORING HISTOLOGIC PHENOTYPES OF NON-OBSTRUCTIVE AZOOSPERMIA

Sertoli cells (vimentin/clusterin). MiRNA expression profiles were determined for each segment of STs and normalized to let-7a. GenEx software was used to identify differentially expressed miRNAs using adjusted p<0.0007 and minimum of 2-fold difference. RESULTS: Quantitative PCR showed a statistically significant decrease in the relative expression of GFRa1 between dilated and collapsed STs (p<0.001) indicating an abnormal number of spermatogonial stem cells (SSC) or spermatogonia. A set of 12 miRNA were identified to be differentially expressed and linked to known signaling pathways in Sertoli cells and SSCs CONCLUSIONS: Our data supports the hypothesis that unique miRNA profiles support normal SSC division that correlate into islands of spermatogenesis, especially in men with NOA. This data in conjunction with previous observations that SSCs are likely present in patients with SCO offers new targets for further research and possible therapeutic intervention

PD08-07 LOSS OF GERM CELLS DOES NOT AFFECT LEVELS OF MIRNA202-5P EXPRESSION IN AN LRAT KNOCKOUT MODEL INDICATING THAT LOSS OF MIR202-5P IN SCO IS THE PRIMARY DEFECT IN MEN WITH AZOOSPERMIA

Sertoli cells (vimentin/clusterin). MiRNA expression profiles were determined for each segment of STs and normalized to let-7a. GenEx software was used to identify differentially expressed miRNAs using adjusted p<0.0007 and minimum of 2-fold difference. RESULTS: Quantitative PCR showed a statistically significant decrease in the relative expression of GFRa1 between dilated and collapsed STs (p<0.001) indicating an abnormal number of spermatogonial stem cells (SSC) or spermatogonia. A set of 12 miRNA were identified to be differentially expressed and linked to known signaling pathways in Sertoli cells and SSCs CONCLUSIONS: Our data supports the hypothesis that unique miRNA profiles support normal SSC division that correlate into islands of spermatogenesis, especially in men with NOA. This data in conjunction with previous observations that SSCs are likely present in patients with SCO offers new targets for further research and possible therapeutic intervention

PD68-04 IMPACT OF CFTR MUTATIONS ON PHENOTYPE AND REPRODUCTIVE OUTCOMES IN MEN WITH CONGENITAL ABSENCE OF THE VAS DEFERENS

RESULTS: A total of 198 men (mean age 35 8 years) were included. Fertilization and pregnancy rates were 44% and 38%, respectively. On univariable analysis, sperm motility and the lack of acrosome defects were associated with higher fertilization and pregnancy rates while an increasing number of total sperm abnormalities was negatively associated with fertilization, but not pregnancy, rates (Table). On MVA, only sperm motility was associated with fertilization rates (p<0.001) while both sperm motility (p1⁄40.004) and the lack of acrosome abnormalities (p1⁄40.018) were associated with pregnancy rates. Female age (mean age 31 5 years) was not associated with fertilization or pregnancy rates on MVA. CONCLUSIONS: This study is the first to assess the relationship between injected testicular sperm characteristics and reproductive outcomes in ICSI. All men with NOA have abnormal sperm morphology and our experience suggests that no specific sperm abnormality, including injection of non-motile sperm, precludes the chance of pregnancy. Further study of sperm characteristics may influence how aggressively surgeons should seek better sperm quality during microTESE procedures. Use of elongating spermatids (blunt-tailed sperm) did not adversely affect the chance of pregnancy despite concerns for lower fertilization with severely blunt-tailed sperm.

MP84-19 BOTHER ASSOCIATED WITH CLIMACTURIA AFTER RADICAL PROSTATECTOMY: PREVALENCE, DEGREE AND PREDICTORS

INTRODUCTION AND OBJECTIVES: While erectile dysfunction (ED) and incontinence are well-understood complications of radical prostatectomy (RP), climacturia remains under-appreciated despite its prevalence in this population. Clinical experience suggests that many men and partners are bothered by this RP sequela. Our objective was to characterize and define predictors of climacturia-associated bother in men and their partners after RP. METHODS: The study was a retrospective review of all men presenting to a single center for management of sexual dysfunction following RP. Patients were queried on various domains of sexual dysfunction including ED, orgasmic and ejaculatory dysfunction, sexual incontinence, and penile deformity. Patients were specifically asked to quantify the amount and frequency of climacturia as well to report their level of bother and to quantify the degree of partner bother. Predictors of bother in patients with climacturia were determined using multivariable analysis (MVA). RESULTS: 252 patients post-RP were included. Mean patient and partner ages were 60 8 years and 55 9 years, respectively. 81% of patients were white and 82% of patients were in stable relationships with a mean duration of 24 14 years. 59% of patients reported at least one occurrence of climacturia. Significantly more men reported bother related to climacturia than partner bother related to OAI (39% vs 13%, respectively; RR1⁄42.95, 95% CI: 2.29-3.80, p<0.01). Patient and reported partner bother were related (r1⁄40.31, p<0.001). Both patient and partner bother were negatively related to length of relationship (r1⁄4-0.14, p1⁄40.05; r1⁄4-0.18, p1⁄40.02). In MVA predicting patient bother, only perceived partner bother remained significant (OR1⁄44.49, 95% CI: 1.6412.27, p<0.01). In MVA predicting reported partner bother, both patient bother (OR1⁄44.49, 95% CI: 1.64-12.27, p<0.01) and duration of relationship (OR1⁄40.96, 95% CI: 0.92-1.00, p1⁄40.05) were significant predictors. CONCLUSIONS: Climacturia is highly prevalent (59% of men) and bothersome in men and their partners following RP. While patient and partner bother from climacturia are related and more commonly seen in shorter relationships, patients are more bothered by climacturia than are their partners. Physicians treating patients with RP should both counsel patients about climacturia pre-operatively and specifically inquire about climacturia post-operatively.

PD68-03 IMPACT OF INJECTED TESTICULAR SPERM CHARACTERISTICS ON REPRODUCTIVE OUTCOMES IN INTRACYTOPLASMIC SPERM INJECTION

INTRODUCTION AND OBJECTIVES: Testicular sperm extraction (TESE) is successful in identifying a small number of sperm in 50% of men with non-obstructive azoospermia (NOA). Traditionally, sperm are isolated from testicular tissue using a combination of standard light microscopy, tissue digestion and time analyzing the specimen in hope to isolate rare spermatozoa. Here we discuss our results utilizing fluorescence-activated cell sorting (FACS) of testis tissue to increase the efficiency in the isolation of spermatozoa. METHODS: Testicular tissue was obtained from 10 patients: 2 cadaveric specimens with normal spermatogenesis and 8 specimens from wasted testicular tissue from microTESE. The specimens were prepared by sharp cutting followed by mechanical disaggregation with a Medimachine (BD Biosciences, USA) and passed through a 50and 30micron filter. The specimens were then stained with DNA-stains To-Pro3 or SYTO 17(ThermoFischer, USA) and incubated. Sperm from a normal semen sample were stained similarly and used as controls for gating during flow cytometry . Then, cell sorting was completed using a FACSAria II (BD Biosciences, USA) to isolate spermatozoa. Each sorted specimen underwent standard light microscopy to identify spermatozoa. RESULTS: Using this technique, spermatozoa were successfully isolated and recovered in both cadaveric specimens. Of the 8 patients undergoing microTESE , 3 (38%) had spermatozoa recovered using standard tissue processing and 4 (50%) had spermatozoa recovered using FACS. Notably, in our cohort, both patients with maturation arrest had a negative microTESE with standard tissue processing, but had successful isolation of spermatozoa using FACS. CONCLUSIONS: Our initial experience using fluorescenceactivated cell sorting for rare spermatozoa isolation from testicular tissue proves the technical feasibility of this process. As this research continues to be refined and implemented, the clinical application of this technique has the potential to increase the rate of successful TESE to isolate spermatozoa.