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The present study was carried out on semen samples of human fertile and infertile subjects, teratozoospermics (TZs) and idiopathics (IDs), with neat semen and sperm prepared by swim up or Percoll density gradient centrifugation procedures. Sperm morphology analysis revealed that only head and midpiece defects in TZs and IDs were significantly (P < 0.001) higher compared to fertile subjects. Infertile subjects indicated significantly higher (P < 0.001) sperm DNA damage compared to fertile subjects. Fertile subjects with sperm prepared from neat and Percoll density gradient centrifugation exhibited a comet tail DNA percentage of 20% and 15%, respectively. The TZs and IDs infertile subjects had higher levels of comet tail DNA of 33% and 25% and 25% and 19%, respectively. A significant (F = 24.01; P = 0.0059) decrease in mean comet head DNA percentage or sperm DNA integrity was observed in neat samples from fertile and infertile subjects by Repeated Measures ANOVA. In Percoll prepared samples from fertile, TZs, and IDs, there was a significant increase in sperm DNA integrity. Similarily, there was a decrease in abnormal sperm morphology in swim up and Percoll prepared sperm compared to neat samples. The Percoll density gradient centrifugation procedure yields sperm with an increase in sperm DNA integrity relative to swim up. Sperm DNA damage of TZs with both sperm preparation methods was significantly (P < 0.01) higher when compared to fertile and IDs. But the level of DNA damage was higher in IDs compared to fertile subjects. Compared to the other methods tested, the Percoll method yielded sperm with improved DNA integrity. In conjunction with semen analysis, the assessment of nuclear integrity improves the characterization of the semen sample and may be used as a tool for allocating the patients to specific assisted reproductive treatments.

The possibility was evaluated that the production of reactive oxygen species (ROS) by human sperm is stimulated by the repeated cycles of centrifugation and resuspension involved in conventional sperm preparation. ROS generation by human sperm was monitored before and after the washing of sperm from 55 men (43 men with suspected subfertility and 12 normal volunteers). The ROS activity of all 55 specimens before washing was inversely correlated with original sperm motility (r = .278, p < .05). The mean level of ROS activity was significantly higher after washing than before processing (p < .05) for the 26 specimens with normal sperm motility, the 20 specimens with normal sperm morphology, and the 12 specimens with both normal motility and normal morphology. In contrast, the mean ROS level was not significantly changed after washing in the 27 specimens with poor sperm motility, the 16 specimens with abnormal sperm morphology, or the 13 specimens with both abnormal motility and abnormal morphology. It would appear that repeated centrifugation, resuspension, and vortexing cause excessive generation of ROS in the motile sperm population of the washed specimen. Washing procedures involving excessive manipulation of sperm may, in fact, cause the most harm to motile sperm, i.e., those that the method is trying to select. Procedures that minimize multiple centrifugation, resuspension, and vortexing steps should therefore be used for the preparation of semen specimens for assisted-reproduction techniques.

There are many clinicians who believe that standard sperm morphological criteria as determined by the World Health Organization (WHO) may be one of the most reliable indices of male fertility potential. Many previous studies of sperm morphology have lacked meticulous correction of all female infertility factors in forming their conclusions. Other studies may have compared morphological analysis to either the zona-free hamster oocyte penetration test or fertilization rates with in vitro fertilization/embryo transfer (IVF-ET). The former may not be a valid "gold standard" with which to compare, and the latter might not truly reflect the in vivo situation in view of the markedly different type of sperm-oocyte interaction. These data failed to find a strong correlation of abnormal sperm morphology (as determined by WHO criteria) to identify the subnormal male. When motile densities were normal (greater than 10 x 10(6)/ml), 87% of the female partners of men with normal morphology achieved pregnancies within 6 months compared to 82% with subnormal morphology. Even subnormal motile densities and morphology did not predict the subfertile male. These data strongly suggest that the normals established by WHO for morphology are inadequate to distinguish normal from subnormal. Perhaps increasing the percentage of abnormal sperm morphology to be considered subfertile might prove more useful, or perhaps using another type of morphological assessment might be more effective.

Acridine orange fluorescence, semen analysis, and zona-free hamster egg penetration tests were performed on 100 consecutive semen samples. No significant correlation was detected between sperm motility and red fluorescence. However, abnormal sperm morphology correlated significantly with red fluorescence (p less than or equal to 0.001) and fluorescence index (p less than or equal to 0.001). Presence of a high percentage of red fluorescing sperm did not prevent their penetration of zona-free hamster egg, indicating that the presence of abnormal DNA had no effect on the sperm fertility. The significance of acridine orange fluorescence male fertility test requires further elucidation.