A novel approach for the selection of human sperm using annexin V-binding and flow cytometry

Objective: To develop a method whereby sperm with phosphatidylserine externalization can be separated from those without this feature. Because annexin V binds phosphatidylserine, this study is using this feature to select functional spermatozoa. In addition, the relationship between annexin V binding in human spermatozoa and normal sperm morphology according to strict criteria was to be assessed, Design: Prospective study. Setting: Department of Obstetrics and Gynaecology of Stellenbosch University at Tygerberg Academic Hospital, Tygerberg, South Africa. Patient(s): Semen from 14 healthy sperm donors. Exclusion criterion was the presence of less than 20 x 10(6)/mL total motile spermatozoa in the original sample. Main Outcome Measure(s): Annexin V-negative sperm, annexin V-positive sperm, normal sperm morphology. Intervention(s): An aliquot of a semen sample after double density gradient centrifugation was incubated with annexin V fluorescein isothiocyanate conjugate (FITC). Cell fluorescence signals were determined using a FACScalibur flow cytometer equipped with a FACSSort fluidic sorting module. The sorting procedure delivered two sperm subpopulations: annexin V-negative and annexin V-positive. Morphology slides were made and the sperm morphology was assessed according to strict criteria. Result(s): There was a significant enrichment of annexin V-negative sperm as well as morphologically normal sperm in the annexin V-negative subgroup after separation with flow cytometry. The percentage of morphologically normal sperm increased from 8.3% in the control to 11.9% in the annexin V-negative fraction, whereas the percentage of annexin V-positive sperm decreased to 5.7%. Conclusion(s): The annexin V-negative sperm subpopulation had morphologically superior quality sperm compared to annexin V-positive sperm. It is important to select morphologically normal sperm during intracytoplasmic sperm injection (ICSI) as it may contribute to increased implantation and pregnancy rates (PR). (Fertil Sterill (R) 2009;91:1285-92. (C) 2009 by American Society for Reproductive Medicine.)