1 Introduction

Of the three sperm-separation techniques evaluated in this paper, the swim-up and the Percoll discontinuous density gradient methods were well-known traditional methods widely used in andrology laboratories^[1]. On the other hand, the real-time sperm separation technique was a relatively new method invented by Fu-Nan WANG^[2]. It was indicated that without using any reagent, the method could isolate sperm preparation completely free from microorganisms and other impurities, and the pregnancy rates of intrauterine insemination (IUI), in vitro fertilization(IVF) and intracytoplasmic sperm injection (ICSI) could be significantly improved in using sperm preparations obtained by this technique^[2-5]. We have performed a comparative study on the relative effectiveness of these three methods in isolating high-quality sperm preparations.

2 Materials and methods

2.1 Subjects

One hundred and forty infertile males visiting this Hospital for initial investigation, aged 24 to 43 years, with no children 2-13 years after marriage. Their spouses were healthy and gynecologically normal. They were grouped into the following categories:

Twenty cases with apparently normal semen: Their semen samples were allocated at random to the three methods for sperm isolation.

Forty cases with semen samples contaminated with different microorganisms: 10 with Chlamydia trachomaticum (CT), 10 with Ureaplasma urealyticum (UU), 10 with human papilloma virus (HPV) and Neisseria gonorrhea (NG), and 10 with other microorganisms.

Their semen samples were allocated at random to the three methods for sperm isolation.

Eighty cases with oligoasthenoteratospermia: Their semen samples were used for IUI after being isolated by the three methods at random.

2.2 Reagents and instruments

Wang's tube was provided by one of the authors, Ren-Kang LU.

1 and 10 Earle's balanced sodium solution, Percoll solution, penicillin and streptomycin were purchased from the Sigma Company (USA).

Acrodine and eosin were purchased from the Chinese Reagents Co. 

Preovulatory serum: Collect 20 mL of venous blood from preovulatory women. Centrifuge immediately at 300g for 30 min. Transfer the serum to another test tube in a water bath at 56 for 45 min. Keep in refrigerator before use.

The polymerase chain reaction kits were purchased from the Sino-America Biotechnology Co., China.

2.3 Real-time sperm separation technique^[2]

Step 1: Warm the Wang's tube, the silicone stopper, the tissue culture medium and the preovulatory serum in an incubator at 37.

Step 2: Fill the tube with tissue culture medium and add 10% preovulatory serum from point A to D. Incubate at 37 for 10 min.

Step 3: Add liquefied fresh semen or centrifuged semen pellet at point P (the sperm loading area). Keep the tube at a horizontal position.

Step 4: Incubate the tube with the tissue culture medium and the sperm (pellet) at 37 (with 5% CO₂ and 96% humidity) for 1 to 2 h.

Step 5: Estimate the sperm density and motility by observing the collection segment (from point C to D) of the Wang's tube under a microscope at 1020 or 1040 magnification every 30 min.

Step 6: Clean the cutting area around point C with 70% alcohol and then rinse the area with the tissue culture medium. Then break the tube at point C and harvest the post-treated sperm sample from point C to D by pressing the stopper. This is the purified sperm sample.

2.4 Methods

Swim-up and Percoll discontinuous density methods were performed as described elsewhere^[1]. The sperm morphology observation and the chromatin staining technique were done according to Huang^[6]. Routine semen examination was performed following the WHO manual^[1]. The polymerase chain reaction were used to detect CT, UU, HPV and NG. The procedure is indicated in the kit directory.

2.5 Statistical analysis

3 Results

3.1 Comparative efficacy on sperm isolation

As can be seen from Table 1, the quality of sperm isolated by the real-time technique was significantly higher than that by the Percoll gradient or swim-up method (P<0.01). 

Table 1. Routine sperm data. Data expressed in means, if applicable. n=20. ^cP<0.01, compared with Real-time group.  

References

[1] World Health Organization. Laboratory manual for the examination of human semen and sperm--cervical mucus interaction. 3rd ed. Cambridge: Cambridge University Press; 1992.
[2] Wang FN. Real-time sperm separation system: a review of Wang tube and related technologies. Arch Ardol 1995; 34: 13-32.
[3] Wang FN. Clinical application of Wang's Tube for assisted reproductive technology. In: Hafez ESE, editor, Assisted Human Reproductive Technology. Washington DC: Hemisphere Publishinjg Co. Chapter 24, 1991. p 246-53.  
[4] Lu RK, Peng SJ. The real-time separation system. Andrology 1994; 2: 123-5 (in Chinese).  
[5] Wang FN, Cheng CM, Merino G, Hsiung CHC. Wang tube system improves pregnancy in infertility treatment. Mol Androl. 1991; 3: 387-95.
[6] Huang YF. Laboratory diagnostic manual in andrology. 2nd ed, Nanjing: Southeast University Publisher; 1993. p 50-1 (in Chinese). 
[7] Bussolo F, Zanchetta R,Lanzone E, Cusinato R. Microbial flora in semen of asymptomatic infertile men. Andrologia 1984; 16: 269-75.
[8] Leiva JL, Peterson EM, Wetkowski M, de la Maza LM, Stone SC. Microorganisms in semen used for artificial insemination. Obstet Gynecol 1985; 65: 669-72
[9] Swenson CE, Toth A, Toth C, Wolfgruber L. Asymptomatic bacterospermia in infertile men. Andrologia 1980; 12: 7-11.
[10] Forman R, Guillett-Rosso F, Fair A, Volante M, Frydama R, Testart J. Importance of semen preparation in avoidance of reduced in vitro fertilization results attributable to bacteria. Fertil Steril 1987; 47: 527-30.  
[11] Stone SC, Delamaze LM, Peterson EM. Recovery of microorganisms from the pelvic cavity after intracervical or intrauterine artificial insemination. Fertil Steril 1986; 46: 61-5.  
[12] Yan RY. Practical Eugenics. 2nd ed. Beijing: People Health Publishing House; 1998. p 318-9 (in Chinese).