Usefulness of the acrobead test in evaluating human acrosome function in fresh and cryopreserved sperm

Assays for the acrosome reaction are usually cumbersome and lack reproducibility. Accurate determination of acrosomal status is important in patients diagnosed with male infertility before proceeding with intrauterine insemination or in vitro fertilization. We determined the optimum capacitation time and acrosomal status of fresh semen specimens in normal fertile men with the Acrobead test, and whether the assay could be used to evaluate cryopreserved semen specimens.

Semen samples from 13 normal donors were divided, with half of the fresh ejaculate used for the Acrobead test and half cryopreserved for a minimum of 24 hours in liquid nitrogen before testing. Fresh and frozen specimens were prepared with the swim-up technique. Sperm concentration was adjusted to 4, 2, 1 and 0.5 x 10(6)/ml. in 4 wells of a 96-well tissue culture plate. Ten microl. polyacrylamide beads (1.5 x 10(6)/ml.) coated with anti-CD46 monoclonal antibodies (MH61 beads) were added to each well. The attachment of beads with acrosome reacted spermatozoa was scored after 0, 1, 3, 6 and 24 hours of incubation. Results were graded on a scale of 0 (no bead binding to the sperm) to 4 (complete attachment to the beads). Specimens with scores of at least 2 were considered normal.

A score of at least 2 was noted in 3 of 13 fresh specimens (15.3%) at 1, 9 (69.2%) at 3, 11 (84.6%) at 6 and 13 (100%) after 24 hours. However, a significantly greater number of frozen specimens (8 of 13, or 62%) had a score of 2 or more at 1 hour of incubation and 100% bead attachment to sperm occurred at 3 hours.

Our results indicate that in fresh semen specimens an incubation period of 6 to 24 hours can be used to screen individuals who present with normal sperm characteristics but have slow acrosome reactions. Early acrosome reaction observed in cryopreserved specimens indicates that these spermatozoa may have membrane damage and leakage of acrosome contents as a result of the freeze-thaw process. The Acrobead assay is a simple and objective test that can be used at a clinical andrology laboratory to evaluate the acrosomal status of fresh but not frozen human spermatozoa.