MP44-15 EFFICIENT SELECTION METHOD FOR HUMAN ROUND SPERMATID IN TESE NEGATIVE MEN (2020)

You have accessJournal of UrologyInfertility: Therapy I (MP44)1 Apr 2020MP44-15 EFFICIENT SELECTION METHOD FOR HUMAN ROUND SPERMATID IN TESE NEGATIVE MEN Banafsheh Nikmehr*, Cihan Halicigil, Mark J Pettenati, Anthony Atala, and Hooman Sadri-Ardekani Banafsheh Nikmehr*Banafsheh Nikmehr* More articles by this author , Cihan HalicigilCihan Halicigil More articles by this author , Mark J PettenatiMark J Pettenati More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Hooman Sadri-ArdekaniHooman Sadri-Ardekani More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000899.015AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Around 1% of general population of men suffer from azoospermia who divided into two categories: Obstructive (OA) and non-obstructive azoospermia (NOA). The most precise surgical biopsy methods (Micro-TESE) have been succeeded up to 70% to find sperm in testis tissue of NOA patients. The rest of NOA patients without any spermatozoa in their testis biopsies (TESE negative) are mostly faced with two main options: Adaption or donation (either Sperm or Embryo). In past few years, a well-known Japanese group (Tanaka et al 2015 and 2018) introduced a novel method of round spermatid injection (ROSI) and consequently over 90 healthy babies were born. This ROSI method is expected to be used in the United Stated soon. The most challenging issue utilizing ROSI is separating of spermatids (haploid cells) from other testicular cells (diploid cells). Therefore, a practical method to enrich and select human round spermatid is required. METHODS: Frozen testis tissue samples (from brain-dead patients via NDRI) were thawed and subjected for two-step enzymatic digestion with collagenase NB4, Trypsin and DNAse I. Cells were separated at a discontinuous density gradient column, prepared from a Gradient medium (100%) that is routinely used at IVF clinics. Different concentrations of gradient medium were prepared (70%, 45%, 35%, 25% and 15%) with dilution in a basic medium (M10). Cell suspension was layered over the top of the column and centrifugation was done to force the cells to put at different layers. After centrifugation, cells were collected from each layer separately and analyzed by X and Y chromosome Fluorescence in Situ Hybridization (FISH) and cell-cycle Flow Cytometry (using Propidium iodide staining). For FISH analysis, cells with expected morphology and just one sex chromosome (either X or Y) were considered as spermatid. RESULTS: FISH counting data indicated up to 65% spermatid enrichment in 15% gradient layer which was double in compared with initial cell suspension. Cell cycle flow cytometry showed four different peaks for 4N (dividing cells), 2N (diploid cells), N (spermatid) and also sub-N (sperms) cells. Percentage of cells in N peak were also double in 15% layer in compared with initial cells suspension, this confirmed FISH counting data. CONCLUSIONS: we were able to establish an efficient method of human round spermatid separation using clinically available gradient reagents. This can be used for ROSI in Micro-TESE patients or in vitro produced spermatid (using our recently established human 3D testis organoid system). Source of Funding: Wake Forest Institute for Regenerative Medicine internal fund © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e662-e662 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Banafsheh Nikmehr* More articles by this author Cihan Halicigil More articles by this author Mark J Pettenati More articles by this author Anthony Atala More articles by this author Hooman Sadri-Ardekani More articles by this author Expand All Advertisement PDF downloadLoading ...