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Microfluidic isolation of sperm for micro-testicular sperm extraction (MTESE)
varicocele are at a greater risk for sperm alterations than men with varicocele who do not smoke, indicating an important environmental component to male infertility. Supported by: CNPq (160857/2012-0) (Brazil). P-167 Tuesday, October 21, 2014 METHYLATION ANALYSIS OF TESTIS AND BRAIN SPECIFIC GENES ON THE X CHROMOSOME IN KLINEFELTER SYNDROME (KS) PATIENTS PROVIDES INSIGHT INTO PARTIAL ANDROGEN RESISTANCE AND BEHAVIORAL ISSUES IN MEN WITH KS. A. Mielnik,a A. Mehta,b L. J. Dow,a M. Funaro,a P. N. Schlegel,a D. A. Paduch.a aWeill Cornell Medical College, New York, NY; bEmory University School of Medicine, Atlanta, GA. OBJECTIVE: This study examined the inactivation status of genes spanning the X chromosome in Klinefelter Syndrome (KS) patients. We hypothesized that gene inactivation further from the X chromosome inactivation center will be less stringently controlled, inducing the wide phenotypic spectrum seen in KS. Our analysis focused on the inactivation status of 2 genes important to Androgen Receptor (AR) signaling, AR and Filamin A (FLNA), to assess the reasons for partial androgen resistance and underandrogenization in KS. DESIGN: We executed a cohort analysis of gene inactivation from methylation on the X chromosome, comparing KS to non-KS patients. MATERIALS AND METHODS: We chose 8 genes spanning the entire X chromosome for methylation analysis, selecting genes expressed in the testis and brain: VCX, FAM9A, DDX53, AR, XIST, ESX1, GLUD2, FLNA. Methylated and unmethylated primer pairs for each gene were designed using Methyl Primer Express and MethPrimer. Methylation specific PCR was performed on bisulfite treated DNA extracted from five 47, XXY males, five 46,XY males and control females, followed by gel electrophoresis analysis as our group previously described. Expression levels of AR and FLNA mRNA and proteins were assessed using qRT-PCR and Western Blot, respectively. The ratio of methylated to unmethylated products was measured. RESULTS: XIST followed a similar inactivation pattern in KS patients and females, with one copy of XIST methylated (inactive), the other unmethylated (active). AR was hypomethylated in KS patients compared to men and women, with a ratio of 1.7 unmethylated to methylated AR in KS versus the female ratio of 1.2. Overexpression of AR in KS patients was confirmed by qRT-PCR and Western Blot analysis. FLNA, shown to impair trafficking of activated AR to the nucleus when underexpressed, was hypermethylated in KS compared to normal men and women, indicating an FLNA deficit. DDX53, involved in the pathogenesis of autistic symptoms, was completely inactivated in men, while active in women and KS patients. CONCLUSION: Our analysis of the methylation status of 8 genes on the X chromosome found clear differences in the inactivation patterns in KS patients. Hypermethylation of FLNA may explain the partial androgen resistance in KS, providing possible new treatment avenues for KS. Activation of DDX53 in KS suggests an underlying cause of developmental issues in KS, but this hypothesis requires further study. Supported by: Robert S Dow Foundation. Lindsay Dow was Supported by Eric M. Smith scholarship in Andrology and Business. P-168 Tuesday, October 21, 2014 MICROFLUIDIC ISOLATION OF SPERM FOR MICRO-TESTICULAR SPERM EXTRACTION (MTESE). K. Murphy,a J. Son,b J. Hotaling,a B. Gale,b D. Carrell.a aAndrology, University of Utah, Salt Lake City, UT; bMechanical Engineering, University of Utah, Salt Lake City, UT. OBJECTIVE: The purpose of this study is to develop a system for separating limited numbers of non-motile human sperm from somatic testicular cells. DESIGN: A Sperm separation device was designed based on concepts of inertial microfluidic forces in order to separate cell types by size and shape. The separation efficiency of the design was evaluated by comparing the contents of the device outputs to the device inputs. MATERIALS AND METHODS: Spiral channel prototypes were fabricated using plastic laminates and laser cutting to contain four outlet channels corresponding to different flow lanes within the spiral. Device characterization was performed by flowing solutions of fluorescent microsphere particles and quantifying particle separation into specific outlet channels. Device prototypes were further tested for cell separation efficiency by flowing immobilized
FERTILITY & STERILITYÒ
mouse sperm, mouse red blood cells, and mixtures of sperm and red blood cells (RBCs). The sperm enrichment in output channels compared to input was calculated, and statistical analyses were performed using student t-tests. RESULTS: We found that microsphere particles with a diameter of nine micrometers could be selectively focused towards the innermost outlet channel of the spiral device at a flow rate of 0.5 ml/minute. Similarly, when mouse RBCs were flowed through the device, they were more concentrated within the innermost outlet channel. Immobilized mouse sperm cells, however, were selectively concentrated within the outermost outlet channel, likely as a result of their unique morphology. In experiments where sperm cells were mixed with RBCs, the sperm:RBC ratio was enriched by more than three fold in the outermost outlet channel, suggesting that our spiral microfluidic design is capable of separating sperm cells from non-sperm cells. CONCLUSION: Our results show that inertial microfluidic forces can be applied to viable cells, and used to enrich sperm cells from a mixed spermsomatic cell population. This proof-of-principle study will serve as the groundwork for further microfluidic device optimization in order to achieve isolation of sperm cells from testicular somatic cell types. Because our design aims to isolate sperm cells without the use of cell lysis or enzymes, it will be amenable to use in a clinical in vitro fertilization (IVF) setting following micro-testicular sperm extraction (mTESE) surgery. P-169 Tuesday, October 21, 2014 VAS DEFERENS ANASTOMOSIS AND EPIDIDIMOVASOSTOMY MALE FERTILITY SURGICAL TREATMENT SIMULATION MODEL USING HUMAN AND BOVINE PLACENTA. A. B. Reis, M. M. R. Oliveira. Surgery, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. OBJECTIVE: Different types of surgical simulations are described, and the most used in urology are cadavers, animals (pigs), and synthetic models. Each one has its advantages and disadvantages. Description of vas deferens and epididimo vasostomy microsurgery training model that is ex vivo, biological, cheap, and has high fidelity is the aim of this study. DESIGN: Urology microsurgery procedures are mostly done for male fertility treatment. Vas deferens anastomosis and epididimovasostomy are high technically demanding procedures that require continuing surgical practice to maintain good quality work. A description of an ex vivo, cheap, high fidelity model using human and bovine placenta was done. MATERIALS AND METHODS: The research project was approved by the ethical committee of the Federal University of Minas Gerais, Belo Horizonte, Brazil. Twelve placentas, six human and six bovine were collected from the pathology department of the University hospital, and from Arro farm, respectively. They were kept at the microsurgical laboratory of that university where the work was developed. All placentas were cleaned in normal sailing solution and had their main vessels canulated with urinary catheters, and the intravascular blood clots removed. RESULTS: All human and bovine placentas had biological properties that allowed the simulation of the proposed surgical exercises. The vas deferens has the exact diameter, vessel wall size, and lumen of medium size bifurcation of the bovine placenta artery. The human placenta vessels have a plethora of different caliber arteries and veins that resembles the vessels needed to be anastomosed to the epididimus in real surgery. CONCLUSION: Urology microsurgery simulation has been used with proved benefits. A description of an ex vivo, high fidelity, low cost, easily available model for microsurgery training in male infertility is done. Further studies with validation methods will be needed to prove its efficacy.
SPERM BIOLOGY P-170 Tuesday, October 21, 2014 SHOULD ICSI BE THE TREATMENT OF CHOICE IF MALE PARTNERS PRESENT WITH INCREASED SPERM DNA DAMAGE? L. Simon, K. I. Aston, J. A. Dorais, J. M. Hotaling, M. Link, E. B. Johnstone, A. K. Moore, A. O. Hammoud, C. M. Peterson, D. T. Carrell. Andrology and IVF, University of Utah School of Medicine, Salt Lake City, UT. OBJECTIVE: The purpose of this study is to examine the two treatments of in-vitro conception, viz. conventional IVF and ICSI, and assess the hypothesis that ICSI should be performed in cases with increased sperm DNA damage.
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FERTILITY & STERILITYÒ
mouse sperm, mouse red blood cells, and mixtures of sperm and red blood cells (RBCs). The sperm enrichment in output channels compared to input was calculated, and statistical analyses were performed using student t-tests. RESULTS: We found that microsphere particles with a diameter of nine micrometers could be selectively focused towards the innermost outlet channel of the spiral device at a flow rate of 0.5 ml/minute. Similarly, when mouse RBCs were flowed through the device, they were more concentrated within the innermost outlet channel. Immobilized mouse sperm cells, however, were selectively concentrated within the outermost outlet channel, likely as a result of their unique morphology. In experiments where sperm cells were mixed with RBCs, the sperm:RBC ratio was enriched by more than three fold in the outermost outlet channel, suggesting that our spiral microfluidic design is capable of separating sperm cells from non-sperm cells. CONCLUSION: Our results show that inertial microfluidic forces can be applied to viable cells, and used to enrich sperm cells from a mixed spermsomatic cell population. This proof-of-principle study will serve as the groundwork for further microfluidic device optimization in order to achieve isolation of sperm cells from testicular somatic cell types. Because our design aims to isolate sperm cells without the use of cell lysis or enzymes, it will be amenable to use in a clinical in vitro fertilization (IVF) setting following micro-testicular sperm extraction (mTESE) surgery. P-169 Tuesday, October 21, 2014 VAS DEFERENS ANASTOMOSIS AND EPIDIDIMOVASOSTOMY MALE FERTILITY SURGICAL TREATMENT SIMULATION MODEL USING HUMAN AND BOVINE PLACENTA. A. B. Reis, M. M. R. Oliveira. Surgery, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. OBJECTIVE: Different types of surgical simulations are described, and the most used in urology are cadavers, animals (pigs), and synthetic models. Each one has its advantages and disadvantages. Description of vas deferens and epididimo vasostomy microsurgery training model that is ex vivo, biological, cheap, and has high fidelity is the aim of this study. DESIGN: Urology microsurgery procedures are mostly done for male fertility treatment. Vas deferens anastomosis and epididimovasostomy are high technically demanding procedures that require continuing surgical practice to maintain good quality work. A description of an ex vivo, cheap, high fidelity model using human and bovine placenta was done. MATERIALS AND METHODS: The research project was approved by the ethical committee of the Federal University of Minas Gerais, Belo Horizonte, Brazil. Twelve placentas, six human and six bovine were collected from the pathology department of the University hospital, and from Arro farm, respectively. They were kept at the microsurgical laboratory of that university where the work was developed. All placentas were cleaned in normal sailing solution and had their main vessels canulated with urinary catheters, and the intravascular blood clots removed. RESULTS: All human and bovine placentas had biological properties that allowed the simulation of the proposed surgical exercises. The vas deferens has the exact diameter, vessel wall size, and lumen of medium size bifurcation of the bovine placenta artery. The human placenta vessels have a plethora of different caliber arteries and veins that resembles the vessels needed to be anastomosed to the epididimus in real surgery. CONCLUSION: Urology microsurgery simulation has been used with proved benefits. A description of an ex vivo, high fidelity, low cost, easily available model for microsurgery training in male infertility is done. Further studies with validation methods will be needed to prove its efficacy.
SPERM BIOLOGY P-170 Tuesday, October 21, 2014 SHOULD ICSI BE THE TREATMENT OF CHOICE IF MALE PARTNERS PRESENT WITH INCREASED SPERM DNA DAMAGE? L. Simon, K. I. Aston, J. A. Dorais, J. M. Hotaling, M. Link, E. B. Johnstone, A. K. Moore, A. O. Hammoud, C. M. Peterson, D. T. Carrell. Andrology and IVF, University of Utah School of Medicine, Salt Lake City, UT. OBJECTIVE: The purpose of this study is to examine the two treatments of in-vitro conception, viz. conventional IVF and ICSI, and assess the hypothesis that ICSI should be performed in cases with increased sperm DNA damage.
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