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Improvement of sperm motility in surgically retrieved testicular sperm (TESE) samples with in vitro culture
electrophoresis in detecting amplification products. AMH in seminal plasma was determined by ELISA. RESULTS: The frequency of FSHR gene variants among fertile men was 46.7% Asn/Asn (N680S), 33.3% Asn/Ser, and 20% Ser/Ser, whereas among OAT men were 34.6%, 38.5% and 26.9% respectively with nonsignificant differences. Seminal AMH was significantly higher in fertile than infertile OAT men. There was significant increase in seminal AMH with Asn/Asn variant of FSHR gene than those with Asn/Ser or Ser/Ser. CONCLUSION: FSH gene variants showed no difference in distribution between fertile or infertile OAT men. However, when correlated with seminal AMH values, there was an increase in Asn/Asn in men with high seminal AMH. Supported by: Mansoura Faculty of Medicine. O-286 Wednesday, October 22, 2014 12:15 PM SPERM SURFACE GLYCAN MODIFICATION BY CHEMICAL REJ. K. Montclare,a PORTER CONJUGATION. J. A. Frezzo,a J. P. Alukal,b V. Kapila.c aChemical and Biomolecular Engineering, New York University Polytechnic School of Engineering, Brooklyn, NY; bObstetrics and Gynecology and Urology, New York University Langone Medical Center, New York, NY; cMechanical Engineering, New York University Polytechnic School of Engineering, Brooklyn, NY. OBJECTIVE: The sperm surface glycocalyx has long been known to serve an instrumental role in sperm capacitation and zona pellucida penetration; however, utilization of this aspect of sperm for assisted reproduction or diagnostic purposes has been lacking. We describe novel orthogonal chemistry methodologies to characterize the network of sperm sialic acids; this is with the purpose of developing a quantitative measurement of sperm fertility that can be used as a novel sperm separation tool. DESIGN: In this research project, we investigated the terminal sialic acids of surface-exposed glycoproteins, which are well-established as biomarkers for fertility, and are also amenable to periodate oxidation, a chemical modification method that enables reporter labeling. MATERIALS AND METHODS: IRB approval was obtained; donated, de-identified sperm that resulted in a positive live-birth pregnancy were isolated by density gradient and subjected to sodium periodate oxidative treatments (0.25 mM, 0.5 mM, 0.75 mM and 1.0 mM) followed by fluorescent tagging with 100 mM amino-oxy AlexaFluor 488, all while retaining the viability of the cells. Fluorescence of labeled cells was measured via excitation at 488 nanometers after repeated washes of unconjugated label. RESULTS: Isolated sperm cells subjected to periodate oxidation and labeling demonstrates insignificant cytotoxicity across three concentrations of the sodium periodate (p<0.001). While fluorescence labeling did occur with the negative control group, there were substantial increases of 67% and 62% in labeling with two separate periodate concentrations emphasizing the utility of this method. CONCLUSION: Effective isolation of fertile sperm for assisted reproduction technologies is limited by sperm characterization methods which sacrifice the viability of sperm for further usage. Herein we describe an orthogonal chemistry method for live sperm cell labeling that retains pertinent viability characteristics while yielding quantitative measures useful in fertility diagnostics or assisted reproduction. Supported by: This work was Supported by ARO W911NF-10-1-0228 and W911NF-11-1-0449 (J.K.M.), AFOSR FA-9550-08-1-0266 (J.K.M.) and in part by the NSF MRSEC Program under Award Number DMR-0820341, NSF GK-12 fellow grant DGE-0741714 (J.A.F.) and NYU CTSA grant UL1TR000038 from the National Center for Advancing Translational Sciences (NCATS), NIH (J.K.M.). O-287 Wednesday, October 22, 2014 12:30 PM IMPROVEMENT OF SPERM MOTILITY IN SURGICALLY RETRIEVED TESTICULAR SPERM (TESE) SAMPLES WITH IN VITRO CULTURE. C. Lawrence,a J. Byun,a V. Chow,b K. Poon,b J. Havelock,a J. Roberts,a K. Seethram.a aPacific Centre for Reproductive Medicine, Burnaby, BC, Canada; bDept. of Urologic Sciences, University of British Columbia, Vancouver, BC, Canada. OBJECTIVE: For IVF-ICSI cases requiring surgical sperm retrieval, it is essential to determine a reliable incubation time for the sample before use in ICSI or for cryopreservation in order to maximize the amount of motile sperm and to best co-ordinate sperm retrieval with oocyte collection. The objective of this study is to verify the effect of overnight incubation of TESE sperm on sperm motility.
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ASRM Abstracts
DESIGN: This prospective study was performed with samples from twenty obstructive azoospermia patients and collected in a private health care facility. MATERIALS AND METHODS: The TESE samples were finely minced in Gamete Buffer (COOK Sydney IVF) immediately upon collection. Sperm motility was assessed after sample preparation, at 4 hours and at 24 hours of incubation. Samples were incubated at 37 C in 6% CO2, 5% O2 balance N2. All samples were cryopreserved and post-thaw assessment performed. A linear regression model, controlling for baseline motility, was used to assess the motility change between 0 and 4 hours and between 4 and 24 hours of incubation. McNemar’s test was used to assess the data categorically. RESULTS: After 24 hours sixteen of the twenty incubated samples either had a motility increase or maintained the motility measured at 4 hours of incubation. This result is significant for overnight incubation (P<0.05). The linear regression of sperm motility at 0 hours to 4 hours showed a significant increase in motility (P<0.01) and also a significant increase (P<0.05) in motility between 4 hours and 24 hours of incubation. All samples had motile sperm present after cryopreservation and thawing. Fourteen of the cryopreserved study samples have been used for treatment resulting in ten clinical pregnancies. CONCLUSION: Incubation of testicular sperm without the use of motility enhancers results in an increased number of motile sperm available for ICSI or cryopreservation at 4 hours and 24 hours post sample collection. The methods used also ensured that all samples had motility post cryopreservation and thawing. The increased sperm motility with incubation validates our current practice of culturing the samples prior to ICSI and prior to cryopreservation which maximizes the total number of motile sperm available. This model for incubation reassures us of our practice of incubating microTESE samples when oocytes may not be quite ready for collection at the same time as sperm retrieval.
O-288 Wednesday, October 22, 2014 12:45 PM PROGNOSTIC VALUE OF PREIMPLANTATION GENETIC SCREENING (PGS) FOR PATIENTS WITH ALTERED RESULTS IN SPERM FISH (FLUORESCENT IN SITU HYBRIDISATION) ANALYSIS. P. Colls,a E. Garcia-Guixe,b S. Munne,a C. Gimenez,b M. Sandalinas.b aReprogenetics, Livingston, NJ; bReprogenetics Spain, Barcelona, Spain. OBJECTIVE: The aim of this study is to find out whether an altered FISH analysis in sperm (AFAS) (increased aneuploidy rate) has a direct influence on embryo chromosomal abnormalities and if these individuals are good candidates for PGS. DESIGN: A retrospective study was performed in couples with just AFAS as indication which underwent a PGS by means of arrays of CGH (Comparative Genome Hybridization). Results were compared with a control group of couples with oocyte donation and without male factor who underwent PGS for SET (Single Embryo Transfer) or to avoid transfer of aneuploid embryos. MATERIALS AND METHODS: The study group is formed by 36 couples with AFAS that underwent a total of 39 PGS cases (289 biopsied embryos). The control group is formed by 13 patients that underwent 16 PGS cases (100 biopsied embryos). Maternal age mean for each group was 30.4 and 29.1 respectively (p¼0.2108). One blastomere of each embryo was biopsied on Day +3 and processed for WGA (Whole Genome Amplification). The amplification product was analyzed by means of arrays of aCGH according to manufacturer’s protocol (24Sure, BlueGnomeÒ). Statistical analysis was performed using Fisher test (Graph Pad Instat 3). RESULTS: Results were obtained in 93% of the analyzed embryos. In the study group, 44.2% of the analyzed embryos were found to be euploid, 47.6% aneuploid and 8.2% were classified as complex abnormal. In the control group, 57.0% of the analyzed embryos were found to be euploid, 38.7% aneuploid and 4.3% were classified as complex abnormal. There are statistically significant differences between the euploid embryos between the 2 groups (p<0.05). Patients with altered sperm FISH results have 1.3 times more risk of producing aneuploid embryos than the control group. For the study group, pregnancy rate was 39.4% per cycle and 46.4% per transfer (ongoing pregnancy: 33.3% per cycle and 39.3% per transfer). For the control group, pregnancy rate was 37.5% per cycle and 46.2% per transfer (ongoing pregnancy: 31.3% per cycle and 38.5% per transfer). No statistically significant differences were observed in the pregnancy rates between both groups. CONCLUSION: This study shows that individuals with AFAS are at risk of producing chromosomally abnormal embryos and PGS can be recommended in order to increase their IVF success. Sperm FISH analysis is a useful tool in the genetic counselling of infertile couples.
Vol. 102, No. 3, Supplement, September 2014
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ASRM Abstracts
DESIGN: This prospective study was performed with samples from twenty obstructive azoospermia patients and collected in a private health care facility. MATERIALS AND METHODS: The TESE samples were finely minced in Gamete Buffer (COOK Sydney IVF) immediately upon collection. Sperm motility was assessed after sample preparation, at 4 hours and at 24 hours of incubation. Samples were incubated at 37 C in 6% CO2, 5% O2 balance N2. All samples were cryopreserved and post-thaw assessment performed. A linear regression model, controlling for baseline motility, was used to assess the motility change between 0 and 4 hours and between 4 and 24 hours of incubation. McNemar’s test was used to assess the data categorically. RESULTS: After 24 hours sixteen of the twenty incubated samples either had a motility increase or maintained the motility measured at 4 hours of incubation. This result is significant for overnight incubation (P<0.05). The linear regression of sperm motility at 0 hours to 4 hours showed a significant increase in motility (P<0.01) and also a significant increase (P<0.05) in motility between 4 hours and 24 hours of incubation. All samples had motile sperm present after cryopreservation and thawing. Fourteen of the cryopreserved study samples have been used for treatment resulting in ten clinical pregnancies. CONCLUSION: Incubation of testicular sperm without the use of motility enhancers results in an increased number of motile sperm available for ICSI or cryopreservation at 4 hours and 24 hours post sample collection. The methods used also ensured that all samples had motility post cryopreservation and thawing. The increased sperm motility with incubation validates our current practice of culturing the samples prior to ICSI and prior to cryopreservation which maximizes the total number of motile sperm available. This model for incubation reassures us of our practice of incubating microTESE samples when oocytes may not be quite ready for collection at the same time as sperm retrieval.
O-288 Wednesday, October 22, 2014 12:45 PM PROGNOSTIC VALUE OF PREIMPLANTATION GENETIC SCREENING (PGS) FOR PATIENTS WITH ALTERED RESULTS IN SPERM FISH (FLUORESCENT IN SITU HYBRIDISATION) ANALYSIS. P. Colls,a E. Garcia-Guixe,b S. Munne,a C. Gimenez,b M. Sandalinas.b aReprogenetics, Livingston, NJ; bReprogenetics Spain, Barcelona, Spain. OBJECTIVE: The aim of this study is to find out whether an altered FISH analysis in sperm (AFAS) (increased aneuploidy rate) has a direct influence on embryo chromosomal abnormalities and if these individuals are good candidates for PGS. DESIGN: A retrospective study was performed in couples with just AFAS as indication which underwent a PGS by means of arrays of CGH (Comparative Genome Hybridization). Results were compared with a control group of couples with oocyte donation and without male factor who underwent PGS for SET (Single Embryo Transfer) or to avoid transfer of aneuploid embryos. MATERIALS AND METHODS: The study group is formed by 36 couples with AFAS that underwent a total of 39 PGS cases (289 biopsied embryos). The control group is formed by 13 patients that underwent 16 PGS cases (100 biopsied embryos). Maternal age mean for each group was 30.4 and 29.1 respectively (p¼0.2108). One blastomere of each embryo was biopsied on Day +3 and processed for WGA (Whole Genome Amplification). The amplification product was analyzed by means of arrays of aCGH according to manufacturer’s protocol (24Sure, BlueGnomeÒ). Statistical analysis was performed using Fisher test (Graph Pad Instat 3). RESULTS: Results were obtained in 93% of the analyzed embryos. In the study group, 44.2% of the analyzed embryos were found to be euploid, 47.6% aneuploid and 8.2% were classified as complex abnormal. In the control group, 57.0% of the analyzed embryos were found to be euploid, 38.7% aneuploid and 4.3% were classified as complex abnormal. There are statistically significant differences between the euploid embryos between the 2 groups (p<0.05). Patients with altered sperm FISH results have 1.3 times more risk of producing aneuploid embryos than the control group. For the study group, pregnancy rate was 39.4% per cycle and 46.4% per transfer (ongoing pregnancy: 33.3% per cycle and 39.3% per transfer). For the control group, pregnancy rate was 37.5% per cycle and 46.2% per transfer (ongoing pregnancy: 31.3% per cycle and 38.5% per transfer). No statistically significant differences were observed in the pregnancy rates between both groups. CONCLUSION: This study shows that individuals with AFAS are at risk of producing chromosomally abnormal embryos and PGS can be recommended in order to increase their IVF success. Sperm FISH analysis is a useful tool in the genetic counselling of infertile couples.
Vol. 102, No. 3, Supplement, September 2014