Russell Hayden

Testosterone and Varicocele

Varicocele is defined as an excessive dilation of the pampiniform plexus. The association between varicocele and infertility has been well-established as evidenced by negative effects on spermatogenesis. Accumulating evidence now suggests that varicocele presents a pantesticular insult, with resultant impairment of Leydig cell function. The presence of a varicocele has been linked to lower serum testosterone levels and varicocelectomy may reverse some of the adverse effects on androgen production. In this review, the evidence linking varicoceles to impaired steroidogenesis and which cohorts of men may benefit most from varicocele repair are discussed.

Surgical management of nonobstructive azoospermia

Nonobstructive azoospermia (NOA) is characterized by the complete absence of sperm in the ejaculate due to testicular failure. The evaluation and management of patients with NOA offer a challenge to the reproductive urologist. In the era of in vitro fertilization with intracytoplasmic sperm injection, surgical sperm extraction techniques can afford men with NOA biologic paternity. To provide a comprehensive review of surgical sperm retrieval approaches in the patient with NOA emphasizing complications, success rates and outcome optimization, a Medline search was conducted querying surgical approaches used to manage NOA. Four sperm extraction techniques are described including: testicular sperm aspiration, testicular sperm extraction, fine needle aspiration mapping and microdissection testicular sperm extraction. In addition, the roles for pre-extraction varicocelectomy and sperm cryopreservation are discussed. The management of NOA continues to evolve as newer tools become available. Several modalities of sperm acquisition exist. An understanding of their complications and success rates is fundamental to the treatment of NOA.

Manipulation of a locally expressed renin-angiotensin system in the testis: implications for steroidogenesis.

THE renin-angiotensin system (RAS) is best that ACE2, a recently discovered homologue of understood for its effects on blood pressure and fluid balance. Contemporary investigations, however, have begun to identify an expanded role for locally expressed RAS pathways that provide autocrine and paracrine functions. Multiple tissues have now been shown to harbor components of a renin-angiotensin mechanism, including the testis and the epididymis. The RAS in the male reproductive tract has been implicated in sperm maturation, maintenance of seminal plasma electrolytes and steroidogenesis. This issue of The Journal includes an article that aims to better elucidate this poorly understood system by exploring how various RAS blockers may impact testicular steroidogenesis. The study by Alves-Pereira et al (page 1878) provides an analysis of testicular RAS and its relationship to androgen synthesis. Their work expands on previous investigations that have associated alterations of the testis specific RAS to testicular dysfunction, hypertension and dyslipidemia. An early study 3 decades ago by Pandey et al demonstrated the presence of renin, angiotensin converting enzyme (ACE)-1, and angiotensin I and II in purified rat Leydig cells. Although their precise roles remain elusive, strong associations have been established with the endocrine function of the testis. Initial efforts showed that rat Leydig cells begin to stain positively for renin during puberty, an effect mitigated by hypophysectomy. The presumed relationship of gonadotropins and Leydig RAS was further clarified by Khanum and Dufau. Their study provided evidence for a modulatory role of angiotensin II on testosterone synthesis such that increasing concentrations of angiotensin II rendered the Leydig cells unresponsive to luteinizing hormone (LH). Angiotensin II represents the principal enzymatic product of ACE1. Although ACE1 is highly expressed in the germinal epithelium, relatively little ACE1 is expressed in the testicular interstitium, a peculiarity of the Leydig RAS mechanism. Subsequent investigations, however, found

Selective use of percutaneous testis biopsy to optimize IVF-ICSI outcomes: a case series

BackgroundSperm quality may degrade during transit through the male reproductive tract in some individuals. In this setting surgically retrieved testicular sperm may outperform ejaculated samples for use with in vitro fertilization (IVF) and intracytoplasmic sperm injection (IVF-ICSI). We sought to describe one center’s experience with the use of fresh testicular sperm after prior failed IVF-ICSI with ejaculated samples.ResultsA retrospective review was conducted evaluating IVF-ICSI cycles performed at a tertiary IVF unit between 2009 and 2014. Couples who were managed with percutaneous testis biopsy to obtain sperm, despite availability of ejaculated sperm, were included. Four couples who underwent a total of 6 percutaneous testis biopsy/IVF-ICSI cycles were identified. Collectively, the couples had undergone 9 prior IVF-ICSI cycles using fresh ejaculated sperm without successful pregnancy. From the six cycles that used fresh testicular sperm four live births resulted (1 twin gestation, 3 singletons). Only 1 of the 4 couples remained childless.ConclusionsFor patients who have had prior failed IVF-ICSI attempts, this small case series demonstrates a possible therapeutic benefit when freshly procured testicular sperm are used in lieu of ejaculated samples.

MP60-14 DYSREGULATION OF RNA SEQUESTRATION BY YBX2 IS A NOVEL MECHANISM OF MATURATION ARREST AMONG MEN WITH NON-OBSTRUCTIVE AZOOSPERMIA

in the ejaculate was considered suggestive for significant bacteriospermia. Semen analysis values were assessed based on 2010 World Health Organization reference criteria. EAU guidelines for semen culture (leukocytospermia >10 WBCs/mL, according to WHO classification) were used to predict positive semen culture in our cohort and thus validated. Moreover, we tested the predictive performance and accuracy of several clinical parameters and compared them to EAU guidelines RESULTS: Overall, a positive semen culture was found in 56 (10%) men. The most commonly identified pathogens belonged to the Enterobacteriaceae family (15% of all positive examinations). Of all, 141 (26%) patients had leukocytospermia >106 WBCs/mL and would have therefore deserved semen culture testing according to EAU guidelines; of them, 12 (9%) actually displayed a positive semen culture. Conversely, 44 (79%) out of 56 patients with positive semen culture would have been missed. Overall predictive accuracy, sensibility, and specificity of EAU guidelines were 48%, 21%, and 74% respectively. No further clinical parameter was significantly associated with positive sperm culture and could be therefore used as a predictor, except for increased serum neutrophil-to-lymphocyte (NRL) ratio (predictive accuracy 60%, p1⁄40.03 vs. EAU guidelines). CONCLUSIONS: The vast majority (79%) of asymptomatic infertile men with a positive sperm culture may miss a proper diagnostic assessment when relying on EAU guidelines. Of clinical importance, not a single parameter, except for NLR, can assist medical decision making. Therefore, a semen culture should be offered to every infertile man.

MP60-06 UNCOVERING BIOLOGY OF KLINFELTER SYNDROME (47,XXY) INFERTILITY USING NOVEL 10X GENOMICS SINGLE CELL SEQUENCING

been proposed as potential biomarkers of obstructive azoospermia. Our goal was to evaluate sperm specific proteins and microscopic sperm presence in vasal fluid at time of vasectomy reversal. METHODS: Patients undergoing vasectomy reversal were enrolled between 2015-2016. Samples were grouped into groups based on microscopic presence of sperm. Proteomic profiles were generated using liquid chromatography/ tandem mass spectrometry, and MS/MS protein spectral counts compared between individuals and treatment groups controlling for less than 5% false discovery rates (FDRs). Differential Expression was assessed using Bioconductor’s edgeR package. Proteins were then matched with the human swissprot database. Samples were analyzed with regards to content of 8 known sperm specific proteins. RESULTS: We analyzed 53 vasal samples from 27 patients. A total of 1,692 unique proteins were detected. The presence of sperm specific proteins varied regardless of the microscopic presence of sperm (Table 1). Cysteine-rich secretory protein was abundant in all samples, while Transketolase-like protein 1 and Sperm-associated antigen 11B were absent or very low counts in all samples. Thus, using the 5 remaining sperm specific proteins we were able to identify 273 unique proteins as potential sperm biomarkers; however, no potential biomarkers correlated with microscopic sperm findings. Most common GO terms included viral process, signal transduction, and innate immune response, as well as protein folding and spermatogenesis. CONCLUSIONS: Cysteine-rich secretory protein was abundant in samples regardless of microscopic sperm presence, rendering this a potential biomarker. We identified many processes contributing to the variability of the proteomic profile of vasal fluid at time of vasectomy reversal to include spermatogenesis, degradation, immune and inflammatory responses. Further evaluation is needed to determine if a potential protein biomarker exists that could predict vasectomy reversal fertility outcomes that could alleviate the need for intra-operative light microscopy, aid in procedural planning and provide a counseling tool for patients.

MP60-07 EVALUATING TRANSCRIPTIONAL REGULATION OF DILATED AND COLLAPSED TUBULES AMONG NON-OBSTRUCTIVE AZOOSPERMIC MEN USING 10X SINGLE CELL SEQUENCING PLATFORM

METHODS: Two men with non-mosaic KS and azoospermia underwent microTESE. The SFTs were divided into DSFTs or CSFTs under the operating microscope at the time of surgery. Single cell suspensions were prepared from the DSFTs and CSFTs followed by preparation of libraries for single cell sequencing with 10X Genomics chromium system. Reads were aligned with STAR aligner, normalized with Limma, and analyzed with Seurat v2.0. Single tubules were stained with antibodies against UTF1 and SOX9. RESULTS: 20,207 reads were mapped for each specimen to GRCh38. Markers of SSCs and differentiating spermatogonia (SPG) such as ITGA6, GFRa1, SOHLH2, ENO2, ZBTB16, UCHL1, NEUROG3, OCT4, UTF1, SALL4, CDH1, BCL6B, KIT, ID4 and SOX2/3 were identified in both DSFTs and CSFTs from patients with KS and expressed at similar levels. Thus, indicating that SPGs and SSCs are present in both DSFTs and CSFTs in similar numbers along the SFTs. Presence of SSCs along the SFTs in KS was confirmed with antibody against UTF1 and GFRa1 and normalized to number of Sertoli cells using SOX9. There were statistically significant differences in expression of markers of fibrosis, lymphoand angiogenesis as well as makers involved in Sertoli to SSCs signaling; thus, confirming that rescue of KS is a result of rare events in which SSCs are exposed to optimal spermatogenic niche and are able to lose the additional X ch. CONCLUSIONS: Using single cell sequencing and immunofluorescence we uncovered that existence of optimal spermatogenic niche is critical for biologically occurring rescue of the 47,XXY in some rare areas of SFTs. These findings support our further effort into high resolution 4D analysis along SFTs to pinpoint critical levels of ligands and receptors necessary for KS rescue.

Detection and Management of Obstructive Azoospermia

Introduction: Obstructive azoospermia represents a treatable form of male factor infertility. With greater demand for assisted reproductive technologies the general urologist may be tasked with initiating the infertility evaluation and providing counsel for treatment options. In this review we discuss appropriate laboratory, radiographic and genetic testing for the patient with obstructive azoospermia. We also outline surgical treatment options. Methods: The Medline® database was searched for relevant studies of the evaluation and treatment of men with obstructive azoospermia. Key words included obstructive azoospermia, vasovasostomy, vasoepididymostomy, testicular sperm extraction, sperm aspiration and sperm retrieval. Results: Most published reports were based on small cohorts followed at single institutions. There were sufficient data to characterize the current state of the art and review the standard of practice. Conclusions: The initial evaluation of azoospermia is primarily based on differentiating obstructive from nonobstructive etiologies with a substantial reliance on history, physical examination and screening laboratory studies. Various treatment options exist for obstructive azoospermia, including reproductive tract reconstruction (vasovasostomy or vasoepididymostomy) or numerous surgical sperm extraction approaches.Abbreviations and Acronyms: CFTR: cystic fibrosis transmembrane conductance regulator; EDO: ejaculatory duct obstruction; FSH: follicle‐stimulating hormone; HPT: hypothalamic‐pituitary‐testis; ICSI: intracytoplasmic sperm injection; IVF: in vitro fertilization; OA: obstructive azoospermia; PercBx: percutaneous testicular biopsy; PESA: percutaneous epididymal sperm aspiration; SV: seminal vesicle; TESA: testicular sperm aspiration; TRT: testosterone replacement therapy; TRUS: transrectal ultrasound; VE: vasoepididymostomy; VV: vasovasostomy.