The Journal of Clinical Embryology™ Volume 13, Issue 4 43 Introduction The goal of all sperm preparation techniques is to improve sperm quality in ART. The ejaculated spermatozoa have to be separated quickly and efficiently from the seminal plasma so that they can be functional [1]. The two routinely used preparation techniques are swim-up and discontinuous density gradient techniques. Since men with abnormal semen parameters do not yield adequate motile sperm, it needs to be processed prior to its use in ART [2]. Spermatozoa are sensitive to temperature changes, dilution and centrifugation as it may adversely affect sperm quality, especially in infertile patients, by generating harmful reactive oxygen species (ROS) [2-4]. Sperm centrifugation is an essential prerequisite for men with severe oligozoospermia and it is commonly used for ART. Semen samples that appear azoospermic upon initial wet mount microscopy should be centrifuged at a minimum of 1,000 X g for 15 minutes in order to identify presence of sperm [5]. Studies have shown harmful effects of centrifugation on human sperm at speeds greater than 500g and for longer than 5-7 minutes continuously [6]. The process of centrifugation leads to the generation of reactive oxygen species [7]. Free oxygen radicals such as super oxide radical (O 2 - ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (OH) cause damage to the spermatozoa. Both time and speed of centrifugation affect sperm quality [3]. The changes in the temperature during centrifugation generate ROS from the spermatozoa compromising the quality and quantity of normal healthy spermatozoa [8]. The objective of our study was to examine the affect of centrifugation with a temperature regulated centrifuge (Spermfuge) at 34 o C on sperm quality as compared to a standard non-temperature regulated centrifuge. Materials and Methods The study was approved by the Institutional Review Board of the Cleveland Clinic. Semen samples were collected from 12 healthy men with unknown fertility. Semen samples were obtained by masturbation after at least 48 hours of abstinence. Samples were collected into sterile containers. Semen analysis was performed according to WHO (4 th ed.) guidelines [9] and included manual and computer assisted sperm analysis (CASA). Sperm viability and seminal levels of reactive oxygen species were assessed after centrifugation. Motility characteristics assessed by CASA Manual and computer assisted semen analysis (CASA) (IVOS 10.7s; Hamilton Thorne Research, Beverly, MA) were performed. For each measurement, a 5μL sample was loaded on a MicroCell slide (Conception Technologies, San Diego, CA). Four to eight representative fields containing 200 or more spermatozoa were examined. Samples were analyzed for concentration, percent motility, and complex motion characteristics. Sperm motion kinetics measured by CASA included the following: sperm concentration (X10 6 /mL), percentage motility, curvilinear velocity (VCL, µm/sec), straight-line velocity (VSL, µm/sec), average path velocity (VAP, µm/sec), linearity (%), and amplitude of lateral head displacement (ALH, µm). In addition to the computerized results, manual results were calculated for sperm concentration and motility. CASA results were used when the difference between the manual and CASA values was less than 20%. In cases where the difference was greater than 20%, the manual results were reported. Use of Temperature Regulated Centrifuge (Spermfuge) Improves Sperm Quality Ashok Agarwal, PhD, Reecha Sharma, MD, Rakesh K. Sharma, PhD, Vaishali Kale, MD, Aparna Thiyagarajan, MSc, Sajal Gupta, MD, Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio, USA Corresponding author: Ashok Agarwal, Ph.D, HCLD Professor and Director, Center for Reproductive Medicine , 9500 Euclid Avenue, Desk A19.1 Cleveland, Ohio 44195 • E-mail: Agarwaa@ccf.org Website: http://www.ClevelandClinic.Org/ReproductiveResearchCenter