Bacterial infections of the genital tract of infertile couples and in vitro fertilization

Infertility is estimated to affect approximately 9-12% of reproductive aged couples worldwide. The causes of infertility can be attributed to several pathological conditions affecting one or both partners. The introduction of “Assisted Reproduction Technologies” (ART) has allowed remarkable scientific and medical advances in the field of assisted reproduction. ART consists of different strategies to overcome some infertility factors, thereby improving reproduction efficacy. Infections of the urogenital tract may contribute to infertility with different sexually transmitted diseases (STDs) being directly or indirectly associated to infertility. Among the pathogens that have been associated to infertility, there is Enterococcus faecalis. This microorganism, previously considered just as a member of the gut microbiota of both animals and humans, is now acknowledged as an important human pathogen responsible for a variety of infections, including infections of the urogenital tract, but also life-threating infections such as sepsis and endocarditis. The presence of acquired resistance to major antibiotic classes, in addition to natural intrinsic resistance, is a relevant issue for the treatment of enterococcal infections. Aminoglycosides in combination with β-lactams is the frontline drug combination therapy for severe enterococcal infections. Of special concern is the acquisition, by horizontal transfer, of genes coding for aminoglycoside modifying enzymes (AMEs) which confer resistance to high levels of aminoglycosides (HLA) and eliminate the synergistic bactericidal activity with β-lactams. Despite genital tract infections can affect human fertility, there are no consensus guidelines available on their management (i.e., microbial screening, antibiotic treatment, therapy outcome) in infertile couples undergoing ART treatment. In the present thesis, an attempt was made to better understand how infections impact on couple fertility. We have also explored the effect of hormone therapy on vaginal microbiota and reproductive outcomes of females undergoing in vitro fertilization (IVF). In addition, we have characterized the antibiotic susceptibility and population structure of a collection of E. faecalis clinical strains isolated from the genital tract of infertile couples to provide clinicians with relevant data to implement management of urogenital infections in infertile couples. Chapter 2 is a general introduction to infertility-associated infections with a special focus on E. faecalis. In the first section, virulence determinants, disease pathogenesis and clinical manifestations of E. faecalis are described. Then, treatment of enterococcal infections with a broad overview on action and resistance mechanisms of major antibiotic classes is provided. Chapter 3 explores the role of urogenital infections on couple fertility starting from a collection of vaginal/endocervical swabs and semen samples from 285 infertile couples. The impact of different bacterial species on the outcome of IVF was examined. The results showed the negative impact of E. faecalis on sperm quality and the association of different bacterial pathogens with reduced levels of vaginal lactobacilli. Interestingly, the presence of E. faecalis together with Ureaplasma urealyticum/Mycoplasma hominis in genital samples of infertile couples was predictive for a negative outcome of IVF. Chapter 4 describes the phenotypic and genotypic features of 41 “infertility-associated E. faecalis” (IAF) strains described in chapter 3. Antibiotic susceptibility of different drugs was carried out (Vitek, MIC and disk diffusion methods) and assessed using EUCAST guidelines. The majority of IAF isolates were susceptible to clinically relevant antibiotics, except for 8 strains that were resistant to HLA and 1 which was also resistant to fluoroquinolones. In order to characterize the IAF isolates, whole genome sequences were used to get insights into the IAF population structure and analyse the genetic bases of antimicrobial resistance. Multi-locus sequence typing (MLST) showed a high diversity of the IAF population. However, a clonal structure of HLA resistant strains was found, as 6 out of 8 resistant IAF isolates belonged to the same clonal complex (CC)/sequence type (ST) CC16/ST480. The work described in chapter 5 evaluates the effect of treatment with exogenous gonadotropins on the vaginal microbiota of 108 infertile women undergoing controlled ovarian stimulation prior to being subjected to IVF. A significant increase of vaginal diamines was observed following hormonal treatment. Analysis of vaginal swabs revealed that a shift occurred after hormone treatment from a Lactobacillus-based microbiota to a microbial population mostly constituted by streptococci, enterococci, enterobacteria, staphylococci and yeasts. A highly significant association between reduced amounts of vaginal lactobacilli and the presence of above mentioned pathogens was found. Finally, IVF outcome was significantly decreased in the patients whose vaginal samples were positive for high levels of diamines and presence of bacterial pathogens, suggesting that a link may exist between vaginal microbiota dysbiosis due to hormonal treatment and IVF failure. Chapter 6 contains a short paragraph with the main conclusions of this Ph.D. thesis.