Objective To determine the membrane integrity in the head and tail regions of individual spermatozoon, and observe sperm morphology for samples with totally immotile sperm. Methods Ten infertile men with immotile sperm were enrolled into this study （group A）. The membrane integrity in the head and tail regions of individual spermatozoon of immotile sperm was examined by using the combined hypo-osmotic swelling-eosin Y exclusion test （HOS-EY test）. Sperm morphology was observed by light, scanning and transmission electron microscopy. Ten semen samples from normospermic donors were used as the control （group B）. Results The percentage of sperm with intact both head and tail membranes in group A was significantly lower than that in group B （P〈0.01）, whereas the value of sperm with defective head membrane but intact tail membrane in group A was significantly higher than that in group B （P〈0.01）. Abnormal sperm morphology in group A had a high incidence, and immotile sperm with viability and normal morphology could be observed in some cases. Most sperm had multiple ultrastructural defects. Conclussion Some immotile sperm had intact tail membrane but defective head membrane. Immotile sperm with viability and normal morphology could exist in some cases though abnormal sperm were in a great proportion. Carefully evaluating immotile sperm membrane integrity and morphology should benefit the treatment of patients with immotile sperm.
To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.
Objective To investigate effects of cryopreservation on changes of the ultrastructure of human testicular sperm and evaluate the efficacy of cryopreserving testicular tissue as a source of sperm for assisted reproduction. Methods Testicular biopsy tissues were obtained from infertile patients （n=12） with obstructive azoospermia and cryopreserved. Testicular sperm motility was observed after in vitro culture procedure. The ultrastructure of testicular sperm （n=6） was examined by transmission electron microscope. Results After cryopreservation, 10 biopsy tissues frozen revealed motile sperm, and 2 samples showed non-motile sperm. Some testicular sperm in frozen-thawed group had normal morphology in fine structures. Sperm head in frozen-thawed tissue showed a proportion of nuclei with more electron-dense granules of chromatin. In a few frozen-thawed sperm heads, formation of vesicles and degeneration were observed. The frozen-thawed testicular sperm frequently showed swollen or/and ruptured of the plasma membrane and acrosome membranes. Conclusion Cryopreservation of testicular tissue is simple and efficacious for testicular sperm extraction. And the freezing-thawing procedure of testicular tissue causes damage to ultrastructural morphology of human testicular sperm.
