The use of GDA-J/F3 monoclonal antibody for the detection of dead spermatozoa (necrosperm) during semen analysis.

The distinction between immotile necrosperm (dead spermatozoa) and those with immotility due to other causes is of the utmost clinical importance. The supravital dyes currently used for the identification of necrosperm are not highly reliable or accurate. In this study, GDA-J/F3 monoclonal antibody (MoAb) which reacts with the fibrous sheath (FS) was used as a specific probe for the detection of necrosperm using indirect immunofluorescence (IIF). Previously, several lines of evidence indicated the reaction of the antibody with necrosperm. This was confirmed in the current study where GDA-J/F3 MoAb failed to react with viable swim-up separated spermatozoa; such cells were only stained following sperm demembranation with 1% Triton X-100. Furthermore, by using immunogold electron microscopy of a normozoospermic sperm sample, all the spermatozoa which reacted with GDA-J/F3 MoAb showed damaged cytoplasmic membranes. Following these initial studies, sperm samples were obtained from 42 men attending infertility clinics and assessed by conventional semen analysis and GDA-J/F3 MoAb screening using IIF. The results showed a wide variation in sperm immotility and GDA-J/F3 reactivity; the ranges were 19-99 and 0-50% respectively. This novel immunological approach provides a simple and specific method of necrosperm enumeration for the investigation of male infertility.

Comparative analysis of motility characteristics of Percoll-selected spermatozoa populations from fresh and cryopreserved semen.

The proportion and quality of motility of spermatozoa in normozoospermic ejaculates were assessed using computer-assisted semen analysis. The ejaculate was split and the motility re-assessed following separation on a Percoll gradient with or without cryomedium and cryopreservation. Cryopreservation caused a significant decrease in the proportion of motile spermatozoa and in their velocity and amplitude of lateral head displacement. The initial decrease in the proportion of motile spermatozoa was found to be in part an effect of the cryomedium. The use of Percoll gradient separation did not initially change these effects but after 4 h incubation differences in velocity and amplitude of lateral head displacement between samples were no longer evident. Percoll-selected, cryopreserved spermatozoa had both a stable proportion of motile spermatozoa and a stable velocity for at least 48 h, whereas in fresh spermatozoa populations, similarly separated using Percoll, the proportion of motile spermatozoa had decreased by 24 h and the velocity was lower at 48 h. Percoll preparation is an effective method for the selection of motile spermatozoa from cryopreserved semen which, after a short incubation, have similar motility characteristics to fresh spermatozoa.

The effects on in-vitro fertilization of autoantibodies to spermatozoa in subfertile men.

Thirty-six infertile couples underwent treatment by in-vitro fertilization. In 16 couples (group 1) the male partner was positive for antisperm antibodies measured by direct mixed antiglobulin reaction, direct immunobead test, and serum and/or seminal plasma tray agglutination test. In 20 couples (group 2) the men had no such antibodies. Men with poor sperm motility were excluded. The female partners had no antisperm antibodies, and in the controls (group 2) infertility was due to a known female factor. The fertilization rate in couples without antisperm antibodies (group 2) was 72.7% compared to 50.5% when the men had antibodies. However, the pregnancy rate per embryo transfer was not significantly different in the two groups (46.1% in group 1, 33.3% in group 2). This indicates that antisperm antibodies in the male interfere with sperm--egg fusion and subsequent fertilization but once fertilization has occurred, the pregnancy rate remains the same.

Comparison of mixed antiglobulin reaction and direct immunobead test for detection of sperm-bound antibodies in subfertile males.

OBJECTIVE: To compare two methods of detection of surface bound antibodies on spermatozoa from subfertile males. DESIGN: Prospective comparison of direct mixed antiglobulin reaction (MAR) for immunoglobulin (Ig)G with direct immunobead test (IgG and IgA) applied to spermatozoa from male partners of infertile couples. Circulating unbound antibody measured by tray agglutination test in serum and seminal plasma in a representative proportion. SETTING: Seminology laboratory. PATIENTS: One hundred nine male partners of infertile couples. RESULTS: Highly significant correlation between direct MAR (IgG) and direct immunobed test (IgG) and between both of these tests and serum unbound antibody measured by tray agglutination test. Highly significant correlation between direct immunobead test (IgA) and seminal plasma unbound antibody measured by tray agglutination test, but no correlation with MAR (IgG). CONCLUSIONS: Mixed antiglobulin reaction (IgG) is a cheap, quick, and sensitive screening test, but immunobead test (IgA) provides useful additional information on class of antibody on spermatozoa that may be clinically more important.