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In this study, we arbitrarily classified the morphological changes of sperm under hypo-osmotic condition and compared the results with four different sperm separation techniques. The morphology of classification ranged from a good swelling (SG 3 > SG 2 > SG 1), to non-swelling but reactive (SG 0-R), and completely non-swelling (SG 0). Thirty fresh semen from patients were divided into 4 groups and each processed by washing, swim up, 2-layer column and real time micro-separation system. The prevalent patterns of the swollen sperm in sequence after treatment was SG 0 > SG 1 > SG 3 > SG2 > SG 0-R in fresh semen, and the proportion of SG 0 was almost half of the total % HOS test results. However, SG 3 became the most common swollen form with a significant increase in number with any method of sperm preparation after treatment. The real time technique yielded the highest % rate of SG 3 type of swollen spermatozoa and was the richest in concentration (53+/-3.9%, P < 0.05) as compared with that of swim up (26+/-4.6%), 2-layer (20+/-4.4%), sperm washing (23+/-3.9%), and fresh semen (17+/- 2.8%), respectively. By contrast, 2-layer collected more number of SG0-R (5.3+/-1.2%, P < 0.05) pattern of spermatozoa when compared to real time and swim up. A positive correlation (r = 0.81, P < 0.002) was shown between the % total HOS spermatozoa and total motility of fresh semen but not with morphology. Collectively, an abnormally functional sperm may exist in almost half of a total sperm count from men. It would appear that a better potential fertilization capacity may reside in the more swollen sperm from HOS test than the less swollen sperm and that may be more related with the motility rather than the morphology. The HOS classification and grading system appears valuable in further evaluating sperm quality.

This paper discusses real-time sperm separation technology and the use of Wang tubes for isolating high-quality and fertile sperm. Several methods have been developed and used to prepare a better sperm specimen, including the conventional swim-up technique, Percoll discontinuous density gradient, Sephadex gel filtration, centrifugation, migration-sedimentation, albumin column, and Sperm-prep. However, none of these methods has the ability to isolate from the original ejaculum a large number of highly motile, morphologically normal, and normal-chromatin sperm, while retaining in an adequate volume of physiological medium, free of less-motile and nonmotile sperm, and free of microorganisms, cell debris, seminal plasma, leukocytes, and foreign bodies. These methods should be classified as incomplete techniques of sperm separation and are of only limited effectiveness in infertility treatment. They may also cause irreparable damage to the mother or fetus. Real-time sperm separation technology is based on the observations that nonpathological spermatozoa do not transfer organisms, that the motility pattern and swim-up capacity of pathological sperm are limited or disturbed, and that the movement of sperm differs from the passive motion of nonciliated microorganisms and the random active motion of ciliated microorganisms in terms of velocity and direction. Real-time sperm separation technology encompasses the Wang tube system, which has two main functions: to isolate organisms-free, normal-chromatin, morphologically normal, and highly motile spermatozoa that are suitable for use in infertility treatment, and to prevent sex-linked inherited diseases. The results of these evaluations indicate that real-time sperm separation technology is the most effective and precise method of isolating and decontaminating sperm to date.

The Wang tube was modified to improve in vitro semen manipulation, by adding another two racks to the anterior (posterior) wall of the low component of the Wang tube and supplying another four convex lenses symmetrical to those on the bottom wall. The former provides a slanted position leading to a complete real-time sperm separation image and benefiting low-quality sperm preparation when the modified Wang tube is turned on its side. The latter converses the microscope's light better and gives a clearer objective image.

Various sperm preparation techniques, swim-up and Percoll gradient, and the newly developed Wang's tube system, were evaluated for their ability to recover normal-chromatin sperm. Twenty human semen samples, collected by masturbation, were studied simultaneously with the three methods. Analysis by Acridine orange fluorescence test was performed on all samples. Pretreated semen contains 58 +/- 22% green sperm (fertile/normal). Treatment with Wang's tube system resulted in 99 +/- 1.0% green sperm; Percoll gradient, 78 +/- 11%; and swim-up technique, 72 +/- 15%. It would appear that Wang's tube system yields a high-quality sperm preparation with enough concentration, very active forward progression, and greatly improved sperm morphology, while containing normal-chromatin, double-stranded DNA.