Infertility as a consequence of spermagglutinating Staphylococcus aureus colonization in genital tract of female mice.

Various studies have shown Staphylococcus aureus to be one of the most prevalent organism in male and female genital tract but most practitioners dismiss it as mere contamination which is assumed to be of no significance. However, it is now suggested that the presence of this organism should not be ignored, as incubation of spermatozoa with S. aureus results in reduced sperm motility. Although S. aureus has been reported to cause immobilization of spermatozoa, however, its role in infertility has yet to be elucidated. The present study was designed to establish a spermagglutinating strain of S. aureus isolated from the cervix of a woman with unexplained infertility, in mouse and evaluate its effect on fertility outcome. Female Balb/c mice were inoculated intravaginally with different doses of S. aureus (10(4), 10(6) or 10(8)cfu/20 µl) for 10 consecutive days. Microbial colonization monitored every 3(rd) day by vaginal cultures, revealed that strain could efficiently colonize mouse vagina. Mating on day 12, with proven breeder males led to 100% decrease in fertility as compared to control. Even a single dose of 10(6) or 10(8)cfu could lead to vaginal colonization which persisted for 10 days followed by gradual clearing till 21 days, vaginal cultures were negative thereafter. Female mice mated on day 7 (culture positive), were rendered infertile, however, the mice mated on day 22 (culture negative), retained fertility and delivered pups indicating its role in provoking infertility. Further, except infertility, no other clinical manifestation could be seen apparently or histologically. However, when a non-spermagglutinating/immobilizing standard strain of S. aureus MTCC6625 was inoculated intravaginally at 10(8)cfu for 10 days followed by mating on day 12, fertility was observed in all the female mice. This supports the hypothesis that infertility observed in the former groups was as a result of colonization with spermagglutinating strain of S. aureus.

Molecular mimicry between spermatozoa and bacteria.

PURPOSE: Sperm immobilization factor isolated from Staphylococcus aureus immobilizes human spermatozoa as well as motile bacteria, showing that sperm immobilization factor receptor might be shared by bacteria and human spermatozoa. Thus, we sought to identify a common sperm immobilization factor binding receptor on spermatozoa and bacteria. MATERIALS AND METHODS: Sperm immobilization factor was isolated from S. aureus. Sperm immobilization factor binding receptors were isolated from spermatozoa and bacteria. RESULTS: Antisperm antibodies in humans cross-reacted with bacteria antibodies. Thus, molecular similarities between determinants of spermatozoa and pathogenic microorganisms can be inferred. Sperm immobilization factor isolated from S. aureus immobilized spermatozoa (150 µg/ml) and motile bacteria, including Escherichia coli, Pseudomonas aeruginosa and Proteus mirabilis (500 µg/ml). This showed the presence of a common sperm immobilization factor binding conformation on spermatozoa and bacteria. Furthermore, receptors binding sperm immobilization factor were extracted and purified from spermatozoa, E. coli, P. aeruginosa and P. mirabilis. Molecular mimicry between spermatozoa and bacteria was confirmed by observing the blockage of spermatozoa immobilization by sperm immobilization factor in the presence of receptors isolated from spermatozoa, E. coli, P. aeruginosa or P. mirabilis. Also, a higher concentration of sperm immobilization factor (200 µg/ml) caused sperm death. Blocking the death of spermatozoa induced by sperm immobilization factor in the presence of these receptors provided further evidence for a common receptor. CONCLUSIONS: Results provide evidence for molecular similarity between bacteria and spermatozoa.

Receptor dependent immobilization of spermatozoa by sperm immobilization factor isolated from Escherichia coli: proof of evidence.

OBJECTIVE: To examine the receptor-ligand interaction between E. coli and spermatozoa resulting in sperm immobilization. METHODS: Sperm immobilization factor (SIF) was isolated and purified from filtrate of E. coli suspension. Receptor on human spermatozoa for SIF was made to isolated and purified by salt extraction, gel permeation and ion exchange chromatography. Receptor dependent immobilization of spermatozoa by SIF was confirmed by competitive inhibition by addition of the purified receptor and binding to sperm receptor by Fluorescin Isothiocynate (FITC) labelled SIF using fluorescence microscopy. RESULTS: Heat labile, non-dialyzable, sperm immobilization factor of ∼56 kDa was isolated and purified from E. coli. Using SIF as a tool, receptor of 113 kDa could be isolated and purified from human spermatozoa. Addition of purified receptor completely inhibited sperm immobilization and death induced by SIF suggesting receptor-dependent immobilization of spermatozoa. Further incubation of sperm with FITC labelled SIF resulted in staining of whole sperm. CONCLUSION: The study provides evidence of receptor-ligand interaction between E. coli and spermatozoa.

Safety studies of sperm agglutinating factor produced by Staphylococcus aureus as a vaginal contraceptive: in vivo studies.

Sperm agglutinating factor (SAF) isolated from Staphylococcus aureus when applied at concentration 10 µg before mating completely prevented conception in the mouse. The objective of the present study was to evaluate its safety, as safety is an important concern to be addressed before a compound is selected for contraceptive use. Our results showed that SAF has a very high safety profile. Vaginal application of SAF at 10 µg to the mouse for 14 consecutive days caused no systemic toxicity and vaginal irritation as indicated by lack of effect on organ weights and histology. Moreover, no adverse effect was observed on the subsequent reproductive capability, perinatal outcome and growth and development of the offspring. SAF (10 µg) did not irritate the skin or penile mucosa. Oral administration of 2 mg/kg body weight of SAF did not show any toxicity to reproductive and non-reproductive organs. Therefore, SAF with spermicidal activity and lack of toxicity may have the potential to become the active ingredient of a vaginal contraceptive.

Receptor mediated agglutination of human spermatozoa by spermagglutinating factor isolated from Staphylococcus aureus.

PURPOSE: We examined spermagglutinating factor isolated from Staphylococcus aureus for evidence of receptor mediated agglutination of human spermatozoa. MATERIALS AND METHODS: Binding to spermatozoa by spermagglutinating factor isolated from S. aureus with a high degree of specificity indicates receptor-ligand interaction. To examine this interaction we isolated and purified the ligand and the receptor. To assess receptor mediated agglutination of spermatozoa further we blocked spermagglutination induced by spermagglutinating factor in the presence of receptor. RESULTS: Spermagglutinating factor induced spermagglutination was competitively inhibited by adding purified receptor, indicating that sperm agglutinating factor isolated from S. aureus attaches to specific receptors on human spermatozoa. The spermagglutinating factor receptor was a protein with a molecular weight of approximately 57 kDa. Spermagglutinating factor induced spermagglutination and at higher concentrations had a spermicidal effect, which was inhibited by introducing the receptor. As observed on scanning electron microscopy studies, incubating spermatozoa with spermagglutinating factor showed profound morphological alterations. However, spermatozoa with normal morphology were noted when incubated with spermagglutinating factor in the presence of receptor, indicating that morphological alterations may account for spermatozoa agglutination by spermagglutinating factor. CONCLUSIONS: Results suggest that spermagglutinating factor isolated from S. aureus may bind specifically to sperm surface receptor sites before causing spermagglutination.

Mechanism of sperm immobilization by Escherichia coli.

AIM: To explore the influence of Escherichia coli on the motility of human spermatozoa and its possible mechanism. METHODS: Highly motile preparations of spermatozoa from normozoospermic patients were coincubated with Escherichia coli for 4 hours. At 1, 2 and 4 hours of incubation, sperm motility was determined. The factor responsible for sperm immobilization without agglutination was isolated and purified from filtrates. RESULTS: This report confirms the immobilization of spermatozoa by E. coli and demonstrates sperm immobilization factor (SIF) excreted by E. coli. Further this factor was purified by ammonium sulfate precipitation, gel permeation chromatography, and ion-exchange chromatography. Purified SIF (56 kDa) caused instant immobilization without agglutination of human spermatozoa at 800 microg/mL and death at 2.1 mg/mL. Spermatozoa incubated with SIF revealed multiple and profound alterations involving all superficial structures of spermatozoa as observed by scanning electron microscopy. CONCLUSION: In conclusion, these results have shown immobilization of spermatozoa by E. coli and demonstrate a factor (SIF) produced and secreted by E. coli which causes variable structural damage as probable morphological correlates of immobilization.

Isolation of a spermatozoal immobilization factor from Staphylococcus aureus filtrates.

Staphylococcus aureus isolated from the cervix of an infertile woman was found to cause complete immobilization of human spermatozoa in vitro. Only the cell culture and cell-free supernatant showed immobilization activity, indicating that the sperm immobilization factor might be released extracellularly by the organism because no activity was observed with the washed cells. Heat treatment of the supernatant at 60 degrees C for 10 min waived its immobilizing activity, indicating that the active component may be a protein. The bioactive molecule from the supernatant was purified to homogeneity by ammonium sulfate precipitation, gel permeation chromatography, and ion exchange chromatography. Sperm immobilization factor (SIF) was found to be an approximately 20 kDa protein. SIF at a concentration of 10 microg/mL was required to cause 100% immobilization of human spermatozoa after 30 min of incubation at 37 degrees C, whereas a concentration of 150 microg/mL caused immediate immobilization, and a concentration of 200 microg/mL resulted in instant loss of viability of human spermatozoa, observed by eosin-nigrosin staining. Scanning electron microscopy showed that the treatment of human spermatozoa with SIF caused multiple defects in the head, midpiece, neck, and tail region of human spermatozoa.