In our study, we find that a decrease in Lactobacillus and an increase in Gardnerella, Atopobium, and Prevotella had a strong association with IVF failure [14].

These links were significantly stronger after the genera Gardnerella, Atopobium, and Prevotella became overgrown, as patients with a high incidence of Gardnerella, Atopobium, and Prevotella either failed to conceive or underwent abortion after embryo transfer. Therefore, it is important to describe the importance of the vaginal microbiota in IVF and demonstrate its potential as a biomarker for predicting IVF outcomes [15].

This study was conducted to search for an additional etiology for the absence of pregnancy after excluding all possible genetic, hormonal, overt infections, and other organic causes of infertility. All of these subjects appeared to be physically and mentally fit for conception [16, 17]. Fetal development from fertilization is not a sterile process. We found an abundance of bacteria, and normal and abnormal flora in our patient population. Culture yield is inferior in samples from the reproductive tract, so next-generation sequencing, a culture-independent technique, can identify and quantify bacterial DNA signature many times over.

The presence of non-Lactobacillus species such as Bacillus, Staphylococcus, Streptococcus, Acinetobacter, Enterococci, Prevotella, Sneathia, and Gardenella in our patient population at a certain titer individually or collectively exceeding a threshold value may indicate the positive impact of Lactobacillus species on pregnancy in an otherwise normal internal milieu, thereby reducing the live baby rate through various mechanisms [18].

Biofilm properties such as Enterococci, Bacillus species, Gardenella vaginalis, Stenotrophomonas maltophilia, Prevotella amnis, and Sneathia present in our samples help them to remain dormant in the reproductive tract, escape antibiotics such as metronidazole, and trigger biofilm dispersion in certain triggers such as stress and inflammation to generate local milieu [19]. A bacterial biofilm is a structured community of bacteria attached to a biological tissue or inert surface. A biofilm can be entrapped in a mucus substance: an extracellular polymeric (EPS) self-produced matrix [20]. This inflammation can affect gametogenesis, fertilization and implantation, and further embryonic development. Traditionally, follicular fluid, endometrial fluid, and semen samples are considered relatively sterile in conventional culture techniques, but metagenomics analysis has revealed the presence of distinct bacterial populations. These can contribute to infertility and failed fertility treatments. The vaginal microbiota of the woman of reproductive age consists largely of at least five different communities [21]. Lactic acid producing Lactobacillus species dominates four of these common state types (CSTs). While the fifth is generally anaerobic and strictly anaerobic, it is sometimes associated with vaginal symptoms. CST-IV does not contain large numbers of lactobacilli but consists of a polymicrobial mix of strict and facultative anaerobes including Gardnerella, Atopobium, Mobiluncus, Prevotella, and other Clostridiales taxa [22,23,24,25]. More than 90% of the vaginal microbiota consists of Lactobacillus species and further up in the endometrium and follicular fluid it makes up the predominant microbiota in people with a higher live birth rate. When this balance is disrupted, it can lead to infertility, as shown in this study. Semen samples and follicular fluid showed the presence of Bacillus species in abundance in our patient pool. Interestingly, microorganisms such as Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli are isolated in the cervix area of infertile human females, which inhibit motility and agglutinate human sperm due to their elastase-positive activity [26]. Bacillus species, Propionibacterium species, Streptococcus species, Actinomyces species, Staphylococcus species, and some of the Bifidobacterium species of bacteria that have been linked to poor IVF outcomes. Our results show that bacterial species in follicular fluids, as well as follicular fluids themselves, can have both positive and negative effects on IVF outcomes [27]. The normal flora of the reproductive tract includes a number of lactobacilli species that promote a healthy and supportive environment for the embryo in the pre- and peri-conceptual periods. This can promote a supportive environment for implantation through the development of lactic acid hydrogen peroxide (H2O2), bacteriocins, antibiotics, harmful hydroxyl radicals, and probiotics [28, 29]. In our study, Lactobacillus was predominant in vaginal microbes, but other bacterial species dominated the follicular fluid, endometrium, and semen samples, revealing their role in infertility through direct and indirect mechanisms. In the presence of bacterial infections, much of the focus in recent years on intrauterine inflammation has been on the cytokine network. This signaling system is believed to play an important role from conception to implantation [30]. Embryo cytokine receptor expression allows cytokines and growth factors secreted into maternal fallopian tubes and uterine epithelial cells to influence the proper development and adaptation of the embryo to its microenvironment [30].

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and colony-stimulating factor (CSF-1) growth factors are leukemia inhibitory factor (LIF), heparin-binding EGF-like growth factor (HB-EGF), insulin-like growth factor (IGF-1, IGF-2), and the cytokines IL-4, IL-10, and IL-11 which are essential for normal blastocyst production while other growth factors are critical for normal blastocyst development such as B. The male reproductive tumor necrosis factor tract. This bacterial infection can affect sperm activity in the male reproductive system and in the adrenal glands [31].

Staphylococcus aureus infections severely affecting the development of semen and sperm activity. This affects sperm volume and sperm concentration as well as sperm motility, morphology and viability [32]. As a result, there may be a causal relationship between staph infection and male infertility. 20.6% of S. aureus is registered, according to a previous study, S. aureus infection was found in semen samples from men with fertility problems. More directly, S. aureus infection has been found to be closely associated with low semen quality and reduced sperm motility [33]. This may play a role in what is known as idiopathic infertility. Although this bacterial flora is relatively harmless, on the one hand it increasingly exceeds certain threshold values and on the other hand it reduces the lactobacilli population and can lead to infertility. We need larger studies to provide more detailed information on this and the possible use of probiotics or antibiotics in the infertility management toolkit.

In conclusion, this is the first NGS-based study of the entire reproductive microbiota to show the presence of diverse bacterial populations in our population and possible implications for fertility potential and ART treatment outcome. Certain bacteria can affect fertility outcome more than others, making them an avenue for further research.

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