Abstract
OBJECTIVE: This study was designed to evaluate the effect of magnetic-activated cell sorting (MACS) using Annexin V-conjugated microbeads in sperm preparation for patients with unexpalined infertility and repeated intrauterine inseminaiton (IUI) failure. DESIGN: This study collected semen samples from male partners of couples with unexplained infertility and repeated IUI failure. The rearch design was a self-matched case control study. MATERIALS AND METHODS: This study was approved by institutional review board of Chung Shan Medical University Hospital. Semen samples (n=41) with normal semen analysis (sperm count≥20X10, motility>50% and morphology>14%) were collected. After double density gradient centrifugation (DGC), the sperm cell suspension was divided into two separate fractions with or without subsequent MACS. And then, we checked the induced acrosome reaction and the apoptosis of sperm by AnnexinV/PI, TUNEL assay (terminal dUTP nick-end labeling) and mitochondrial membrane potential using JC-1. Wilcoxon signed rank test was utilized to calculate the difference between samples. RESULTS: Following MACS, the induced acrosome reaction was significantly improved (45.9±5.3 vs. 32.8±4.3, respectively, p<0.001). The proportion of sperm DNA fragmentation(9.2±0.7% v.s. 12.5±1.0%), mitochondrial membrane potential disruption (18.7±1.9% v.s. 27.2±3.0%) and the externalization of phosphatidylserine (5.9±1.3% v.s 8.2±2.0%) subsequent to MACS was significantly reduced compared to the DGC. CONCLUSIONS: Magnetic-activated cell sorting using annexin-V microbeads effectively separated apoptotic and non-apoptotic spermatozoa based on the externalization of phosphatidylserine (PS) to the outer membrane leaflet. The acrosome reaction percentage was also significantly elevated. MACS in combination with DGC could be used as a sperm preparation technique to enhance the sperm quality and function for unexplained infertility.
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