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

ABSTRACT

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).