MP44-14 SORTING OF SPERM WITH CHROMASELECT DOES NOT SKEW THE RATIO OF X TO Y CHROMOSOME BEARING SPERM

Abstract

You have accessJournal of UrologyInfertility: Therapy I (MP44)1 Apr 2020MP44-14 SORTING OF SPERM WITH CHROMASELECT DOES NOT SKEW THE RATIO OF X TO Y CHROMOSOME BEARING SPERM Darius Paduch*, Anna Mielnik, and Russ Hayden Darius Paduch*Darius Paduch* More articles by this author , Anna MielnikAnna Mielnik More articles by this author , and Russ HaydenRuss Hayden More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000899.014AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: ChromaSelect is a highly sensitive and specific method of identification and isolation of intact and live human sperm from ejaculate, epididymal or testis specimens with a low concentration (up to 100 sperm /ml). As Y ch is much smaller than X, it is theoretically possible that Y or X bearing sperm will show different fluorescence intensity and thus could result in a skewed selection of one of the sexes in sorted sperm.The aim of our study was to evaluate the ratio of Chr X and Y bearing sperm in intact, live sperm and dead/apoptotic sperm fraction after ChromaSelect identification and sorting. METHODS: Ejaculated human sperm were resuspended to a concentration of 10x106/ml in MHM-C medium, stained using ChromaSelect protocol, and then sorted on SONY-FX500 fluorescent activated cell sorting (FACS). Three groups of samples were prepared: CONTROL – unsorted but stained sperm, LIVE- after sorting intact sperm, and DEAD: after sorting apoptotic and dead sperm based on specific gates. Sperm from each group were placed on microscope slides and number of sperm bearing X, Y, and Chr 8 was counted in at least 500 sperm from each group using FISH with Vysis CEP X (DXZ1) Spectrum Green, CEP Y (DYZ3) Spectrum Orange, and Vysis CEP 8 (D8Z2) Spectrum Aqua DNA. Sperm specific decondensation protocol for FISH was developed by us. The automatic FISH protocol for peripheral WBCs was not adequate for the hybridization of DNA probes in sperm. Only sperm with one signal from Chr 8 were used in the analysis. Four channels epifluorescent microscope was used to acquire images that were then processed using the Bioimaging station. Z-stage deconvolution was used to assure a single signal from each probe was counted. DAPI was used for counterstaining. Experiments were performed in triplicates. JIM 14 was used for statistical analysis. RESULTS: The rate of aneuploidy in any of the specimens was less than 0.5% and was negligible. Single signal for Chr 8 and either X or Y was observed in each counted sperm. The mean ratio of X:Y sperm for CONTROL was 0.99 (49.7% of X positive sperm), for LIVE was 0.99 (49.72 % of sperm had Chr X) and for DEAD was 1.03 (50.68% sperm had Chr X). The total number of sperm counted was 829 in CONTROL, 720 in LIVE, and 916 in DEAD groups. There was no statistically significant difference in the number of X or Y chromosomes bearing sperm between any of the groups. (Chi-square) CONCLUSIONS: Sorting of human sperm using the ChromaSelect method does not skew the ratio of X or Y carrying sperm hence it will not result in any change in the sex ratio in children born using sorted sperm. Source of Funding: NIH grant 1R03HD094046 – 01 to DAP, Irena and Howard Laks and Robert Dow Foundation © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e661-e661 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Darius Paduch* More articles by this author Anna Mielnik More articles by this author Russ Hayden More articles by this author Expand All Advertisement PDF downloadLoading ...