1 Introduction

2 Materials and methods

2.1 Reagents and instruments

Wright-Giemsa stain solution was prepared in this Lab. UU medium was provided by the Institute of Dermatology, Chinese Academy of Medical Sciences. 1Earle's solution (Lot E7883), 10Earle's solution (Lot E7510), and Percoll solution (Lot P7828) were bought from the Sigma Company. A 45% Percoll solution: add 10Earle's solution 0.8 mL to  Percoll solution 9.2 mL, and 5 mL of the above solution mixed with 1Earle's solution 5 mL. TUNEL Kit was bought from the Borringerman Company (Germany). Microscope (Model BH-2) was bought from the Olympus Company (Japan).

2.2 Subjects

2.2.1 UU infection group  

Thirty-five males, Han race, average age 29 years old , urinary UU cultures were positive in twice and verified by electron microscope. They were most probably infertile, as their spouses had been shown to be gynecologically normal but never conceived.

2.2.2 Control group 

Thirty males, Han race, average age 33 years old, urinary UU cultures were negative in twice. They have fathered children or had their spouses pregnant.

2.3 Preparation of semen sample and cell  

The semen sample was collected in a sterile container after 48 hours of sexual abstinence, 2 mL of which was added onto the bottom of the tube and covered with 2 mL 45% Percoll solution. After centrifugation at 300g for 20 min, the cells were distributed mainly in the boundary region where the semen and Percoll solution meet. The cells were then diluted with normal saline solution, and centrifuged at 200g for 10 min. The supertanant was removed and the sediment was dispersed in normal saline as a cellular suspension. The suspension 5 L was spread on a slide, dried in air at room temperature and fixed in 95% alcohol for 30 min.

2.4 Observation of cellular morphology 

The cell slide was stained with Wright-Giemsa stain and was observed under microscope.

2.5 Examination of cellular apoptosis with TdT-mediated TUNEL technique   

Briefly, dUTP labeled by fluorescein is transferred to the OH of 3-end by Terminal deoxyribonucleotide transferase, and thus apoptosis of the spermatogenic cells can be identified with fluorescing.

2.6 Statistical analysis

4 Discussion

In 1973, Gnarpe first proposed that UU infection could be associated with human infertility^[4]. After that, many investigators have studied the correlation between UU infection and male infertility. It is well documented that infection is one of the important pathogenic causes of male infertility. In the literature it has been shown that UU infection may lead to the following changes^[5]:

(1)  UU attaching to the surface of sperm may increase the hydrokinetic resistance, thus slowing down the sperm movement. Meanwhile, UU may interfere in the sperm-ovum identification and fusion if UU attaches itself to the rear of the acrosome or the equatorial area of sperm. 
(2) UU infection causes sperm tail curling, head shrinkage, neck swelling, sperm agglutination, and an increase in the percentage of abnormal form sperm. 
(3) Essential metabolites of UU are H₂O₂ and NH₃ which are detrimental to sperm. The phosphatase A and C in the UU membrane can decompose the lipid of sperm membrane and destroy its integrity.
(4)   UU sticking to the spermatogenic cells may cause the latter to fall off from the seminiferous tubule and to change the structure of sperm.

On the basis of these observations and our finding that the apoptosis rate of UU infection group is significantly higher than that of the control group, the authors believe that the UU infection may cause pathologic cellular changes, that can possibly activate cellular apoptotic process as follows:

(1) After stimulation by UU, the testicular spermatogenic cell and macrophage will secrete TNF-^[6], which is an important cytokin that may cause apoptosis^[7].
(2) UU Infection increases the number of phagocytes in the reproductive system. After engulfing foreign matter, they produce oxydase of reductive coenzyme II, which transfers single electron. This leads to the liberation of oxygen free radicals that react with poly-unsaturation fatty acid and cholesterol to produce peroxide, thus resulting in apoptosis^[8]. Besides, H₂O₂, the metabolite of UU, is itself an active oxidant, can produce more OH under Fe²⁺. Small amounts of OH can bring about apoptosis of several kinds of cells, including spermatogenic cell^[9].

References

[1] Xu C, Sun GF, Xu S, Wang YF. Establishment of an animal model infected by Ureaplasma urealyticumits effects on the morphology of the testis. Reprod Contracep  1995; 15: 205-8.
[2] Huang YF, Shang XJ, Xu JP, Jing YF, Chen JD. Study on the location of Ureaplasma urealyticum inside androgones. Chin J Androl  1995; 9: 197-200.
[3] Huang YF, Xu JP, Shang XJ, Ge YF. Morphological observation of apoptotic spermatogenic cells. Acta Anat Sin 1996; 27: 58-60.
[4] Gnarpe H, Friberg J. T-mycoplasma as a possible cause for reproductive failure. Nature  1973; 242: 120-1.
[5] Xu C, Xu S, Wang YF, Huang PZ. Pathogenic mechanisms of male infertility caused by Ureaplasma urealyticum infection. I. Morphological investigation on sperm. Chin J Androl  1992; 6: 66-9.
[6] Li WY, Chen GJ, Cheng LP, Zhu YF, Xu C, Wang YF. The effect of Ureaplasma urealyticum on the production of TNF- by mouse macrophage. Reprod Contracep  1996; 16: 55-8.
[7] Forrest VJ, Kang YH, Mclain DE, Robinson DH, Ramakrishnan N. Oxidative stress-induced apoptosis prevented by Trolox. Free Radic Biol Med  1994; 16: 675-84.
[8] Sandstrom PA, Buttke TM. Autocrine production of extracellular catalase prevents apoptosis of the human CEM T-cell line in serum-free medium. Proc Natl Acad Sci USA  1993; 90: 4708-12.
[9] Halliwell B, Gutteridge JM. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol  1990; 186: 1-85.