sterile fluidics lines as opposed to other groups using flushing of the lines between sorting. No study to date evaluated the actual risk of genetical mismatch between the assumed parents of the child and the real genetic parents in reproductive medicine. Aims: To confirm that the use of single-use sterile fluidics lines as a part of our sorting protocol eliminates the risk of sperm carry-over. METHODS: Sperm from either C57BL/6-Tg(UBC-GFP)30Sca/J mice expressing green fluorescent protein (GFP) (GFP-sperm) or non- fluorescent C57BL/6J wild type mice (NF-sperm) were obtained from the epididymis and vas. Presence of GFP in sperm was confirmed with epifluorescence microscopy. After GFP-sperm sorting was finished, the fluidics was flushed for 10 minutes using a bleach solution WASH group. In the SINGLE-USE group, single-use sterile fluidics lines and the sorting chip were used for each run. The presence of sperm carry-over was defined as detection of one or more of GFP-sperm events when non-fluorescent sperm was sorted. Both GFP and SYTO-17 channels were recorded at the same time and for each run. Experiments were performed in triplicate. FlowJo, FCS Express 6 Flow, and R were used independently for analysis. RESULTS: Epifluorescence microscopy showed the expres- sion of GFP protein in GFP-sperm and lack of fluorescence in wild type sperm. There was the peak of fluorescence in FL1 channel from GFP- protein detected in the GFP-sperm group, but no signal was detected in FL1 channel when sperm from NF-sperm was sorted. The gates used to sort GFP-positive sperm were highly reproducible and accurate with 15,221/16,465/16,410 sperm with a mean number of GFP-positive sperm 16,032 (CV[4.4%) identified in each run. There was one carry-over sperm event detected in WASH group. Multiparemetric analysis of shape and size of cells confirmed presence of carry-over sperm when flush only method was used. Use of different analytical software did not change the finding. There was no carry-over signal detected when single-use fluidics line and sorting chip were changed for each run (SINGLE-USE group). CONCLUSIONS: Sorting of human sperm with single-use fluidics lines for each patient eliminates the risk of technical carry- over of sperm between samples and as such, should be the only method used in the future clinical trials. Source of Funding: NIH grant 1R03HD094046 e 01 to DAP, Robert Dow, Irena and Howard Laks, and A-Time. MP38-16 THE USE OF CHROMSELECT SORTED SPERM IMPROVES THE RATE OF FERTILIZATION AND THE NUMBER OF LIVE DELIVERIES Darius Paduch, North Bergen, NJ; Russel Hayden*, New York, NY INTRODUCTION AND OBJECTIVE: The selection of sperm for in-vitro fertilization (IVF) is based on the visual and not functional assessment of sperm quality. We have previously shown that ChromaSelect (ChrSel): fluorescence-activated cell sorting (FACS) allows for selection of alive, motile sperm with intact cell membrane; the "best sperm" functionally. Our previous in vitro studies showed no evidence of sperm damage from ChrSel. Aims: to answer if the use of ChrSel sorted sperm improves rates of fertilization and live deliveries, and leads to the birth of healthy pups. METHODS: Controlled animal trial. More than a hundred B6D2F1 female mice, aged 7 to 15 weeks, and male mice at least 12 weeks old. IVF: 7.5 IU of PMSG with 7.5 IU of hCG 48 hours later was used for stimulation. Oocytes were procured 15 to 19 hrs later. Fresh sperm was retrieved from the vas deference for each experiment. IVF was conducted by us in FertiUp/CARD media (Cosmo Bio). Em- bryos were cultured in EmbryoMax KSOM Advanced (Millipore-Sigma) system. Fresh sperm was divided into an unsorted control group (CON) and sorted ChromaSelect sperm sorted group (BEST) based on multi- parametric SYTO-17 and YO-PRO-1 gating using FX500 (SONY). Fertilization rate, blastocyst formation rate, and quality were assessed for 96 hours twice daily. Embryos were transferred into the oviducts by injection into eight recipient females one day after fertilization. A vaginal plug identified eligible recipient females following cohabitation with vasectomized males. JMP 14 was used for statistical analysis. RESULTS: Fertilization rate was 75% (41/55) for IVF with sor- ted sperm (BEST) v 40% (21/53) for unsorted control (CON) p<0.0002. Blastocyst quality was better using the sorted group. A total of 70 em- bryos were transferred into females. Eight pups were born to the four females in the control group (unsorted sperm), and 23 live pups were born to 4 females using sorted sperm OR:2.9 (p < 0.01, Fischer Exact Test). The mother killed one pup in the sorted group. Physical exami- nation of born live pups showed no anatomic defects when either sorted or unsorted sperm was used for IVF. CONCLUSIONS: Ours is the first report confirming that Chro- maSelect sperm sorting with the selection of "best sperm" improves the fertilization rate and rate of live deliveries. There were no teratogenic defects related to the use of ChromaSelect sperm. Further trials in non- human primates will be performed next before clinical trials aimed at improving outcomes of in vitro fertilization by using ChromaSelect sperm sorting in humans. Source of Funding: NIH grant 1R03HD094046 e 01 to DAP, Robert Dow Foundation, A-Time. MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY Antonio Luigi Pastore*, Andrea Fuschi, Yazan Al Salhi, Gennaro Velotti, Lorenzo Capone, Alessia Martoccia, Pietro Paolo Suraci, Chiara Zerbinati, Luigi Iuliano, Antonio Carbone, Latina, Italy INTRODUCTION AND OBJECTIVE: Cholesterol is the main lipid component of sperm cell that is essential for sperm membrane fluidity, capacitation, and acrosomal reaction. Recent data obtained in bovine sperm showed that sperm capacitation is associated with the formation of oxysterols, oxidized products of cholesterol. The main objective of the present study was to identify and quantify, for the first time, the different species of oxysterols in human semen from normo- zoospermic, oligoasthenoteratozoospermic and asthenozoospermic patients. The secondary aim was to investigate the potential role of oxysterols in sperm pathophysiology. METHODS: To investigate the profile of human sperm oxy- sterols in subjects with normal and altered sperm characteristics, we recruited 150 consecutive subjects referring at the Centre of Andrology of Department of Urology for analysis of seminal fluid between March 2017 and June 2018. In order to investigate the possible correlation to oxidative stress and semen oxysterols profile, we included a group of patients with varicocele given the demonstrated relationship between varicocele and oxidative stress. Semen analysis was assessed by light microscope according to World Health Organization guidelines (WHO, 2010). Oxysterols were determined by GCeMS using deuterium- labelled internal standards as described by Dzeletovic et al. RESULTS: Complete oxysterol profile was obtained in 134 human semen of normozoospermic, oligoasthenoteratozoospermic, asthenozoospermic and varicocele patients. Oxysterols analyzed included seven autoxidation- and five enzymatically-generated oxysterols. Among the 12 oxysterols analyzed, 25-hydroxycholesterol (25-HC) resulted the most abundant oxysterol in normozoospermic subjects, and turned out to be the only one that differed significantly (p<0.0001) among the 4 groups. It was higher in the normozoospermic group (21.63Æ18.47 ng/mL, meanÆSD) than oligoasthenoteratozoospermic (2.59Æ2.93, ng/mL, meanÆSD), asthenozoospermic (5.59Æ3.17) and varicocele (13.48Æ11.81, ng/mL, meanÆSD) groups. Furthermore, 25-HC positively correlated with the spermatozoa number (r[0.72, p<0.0001). CONCLUSIONS: In conclusion, to the best of our knowledge, this is the first study providing evidence for the feasibility of detection and quantification of oxysterols in human semen samples. We found in spermatozoa the presence of cholesterol 25-hydroxylase and its preferential accumulation in the neck and the post acrosomal area. Vol. 203, No. 4S, Supplement, Saturday, May 16, 2020 THE JOURNAL OF UROLOGY Ò e575 Copyright © 2020 American Urological Association Education and Research, Inc. Unauthorized reproduction of this article is prohibited.