**6. Animal model research**

spermatozoa [33] was followed by another report showing, also *in vitro,* that *E. coli* directly reduces sperm ΔΨm and alters plasma membrane stability [34]. From the point of view of sperm function, it has been observed that ΔΨm is positively correlated with sperm motility *in vivo* [35, 36]. However, after contact with some strains of *E. coli*, which decrease sperm motility *in vitro*, they had no effect on ΔΨm [37]. This study also found that some *E. coli* isolated from different patients was unable to decrease sperm motility, remarkably even an O6, which is thought to be a highly uropathogenic strain in urinary tract infections [38]. These facts con‐ trast with the notion that *E. coli* in general alters sperm function. These differences could be attributed to the fact that different strains bear specific but different characteristics from other *E. coli* strains. Evidence confirming this was observed in our work, when sperm were incu‐ bated with a hemolytic strain of *E. coli.* This strain caused a decrease in motility, ΔΨm, vitality and an increase in intracellular ROS in normal spermatozoa. These effects were not observed with other strains non‐hemolysis producers [39]. These differences among strains highlight the importance of knowing what kind of toxins are effectively produced by the *E. coli* strain infecting a patient, because it could indicate the level of sperm damage to be expected. As example, hemolytic *E. coli* strains produce the alpha‐hemolysin (HlyA) toxin [40], a calcium‐ dependent pore‐forming toxin which has intracellular effects, inactivating pathways related to cell survival [41]. This toxin can be highly relevant, particularly if we consider that between 40 and 50% of *E. coli* strains isolated from patients with epididymitis release this toxin [42]. Evidence of *E. coli* effects on human spermatozoa shows that this bacterium impairs sperm qual‐ ity, principally causing decreased motility; nevertheless, there are other consequences for sperm quality, specifically the incubation of sperm with *E. coli* decreases the ability of the male gamete to penetrate the oocyte, the most important step in the function of the spermatozoon [43].

74 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

**5. Soluble products of** *E. coli* **also affect human sperm function**

effects of *E. coli* soluble factors were inhibited [44].

The effects of *E. coli* soluble products on sperm have been studied using supernatants of *E. coli* culture as a source of bacterial metabolic product. It has been reported that although the direct contact with *E. coli* was able to alter sperm motility, the metabolic products of *E. coli* had no effect on decreasing motility in human spermatozoa [24]. However, after this report, it was shown that incubation with the soluble factors of *E. coli* reduced sperm motility and ΔΨm [33]. Added to this, the ability of *E. coli* soluble factors to decrease motility, viability, ΔΨm and increase ROS in spermatozoa can be prevented by lactobacilli. In an *in vitro* experiment, after adding lactobacilli to simulate the normal condition in the female genital tract, the harmful

Among the soluble factors of *E. coli,* a component called spermatozoa immobilization factor (SIF) was first described in 1977. The effect of SIF, as the name implies, is to immobilize spermatozoa, and this effect could be reversed by washing the spermatozoa [45]. Years later, an apparently similar SIF of 56 kDa was isolated and purified from supernatants of a strain of *E. coli.* SIF‐56 decreases sperm motility completely and almost instantaneously. It was also observed that SIF‐56 at very high concentrations can even induce sperm death [46]. Sperm immobilization mediated by SIF‐56 has been shown to depend on 115 kDa‐receptor present in sperm [47]. Another *E. coli*

*In vitro* investigations have the disadvantage of not necessarily representing what would happen *in vivo*. Hence, *in vivo* studies in animal models allow us to get closer to the reproductive reality of a man with accessory glands infected by *E. coli.* That is how the progressive reduction of testic‐ ular size with a consequent decrease in sperm count caused by the necrotic death of the testicular germ cell has been described in rats inoculated with *E. coli* [49]. After three days of injecting rats with HlyA producing *E. coli,* the epididymis had epithelial damage, leukocyte infiltration and edema and the sperm‐fertilizing potential was lost, because despite being motile, the spermato‐ zoa had a premature acrosome reaction [50]. The above‐mentioned greater pathogenicity of the HlyA‐producing *E. coli* strains was reported in the work of Lang [50], where the *E. coli* strains that did not produce HlyA induced only slight damage to the epididymis. As already stated, sperm recognize peptidoglycans and LPS through TLR‐2 and TLR‐4, and this recognition event induces sperm cell death. This was confirmed by using knockout mice for both TLR‐2 and ‐4 and by observing that in these animals LPS or peptidoglycans did not induce sperm death [32].

Further evidence of the contribution of some *E. coli* strains to infertility was observed after inoculating the vaginal tract of rats with SAF‐producing strains. Control rats were inoculated with *E. coli* non‐SAF producers. It was observed that the rats inoculated with SAF‐producing strains were incapable of pregnancy, demonstrating that these toxin‐producing strains affect fertility profoundly [51].
