The complex microbiome from native semen to embryo culture environment in human in vitro fertilization procedure

Assisted reproductive technologies (ART) are the cornerstone of contemporary infertility treatment. Despite considerable progress made in ART, the implantation rate of replaced embryos remains low, and has been shown to depend on numerous clinical and laboratory factors. The success and failure in ART have largely been attributed to variables such as the patient’s age, weight, endometrial receptivity, and embryo quality and the transfer technique used. The viability of IVF embryos, in turn, depends on the composition of embryo culture media and physical environmental factors applied in embryo culture. At the same time bacterial contamination of gamete samples used in ART may impair the embryo culture environment, causing damage to or even loss of cultured oocytes and embryos [1].

Semen is not sterile [2] and may contain microorganisms even after processing for ART. Although most of the microorganisms detected in semen samples are non-pathogenic commensals or contaminants, their presence has a great significance on in vitro fertilization (IVF), a treatment in which the natural defense of the female genital tract is largely bypassed [3]. Therefore, different approaches have been proposed to reduce the microbial contamination and load in IVF culture media by improving semen preparation and embryo culture protocols. The majority of IVF laboratories use culture media containing antibiotics to minimize the risks of microbial growth. This has been a common practice since the first successful IVF treatment in 1978, when it was suggested that contamination during IVF procedure could negatively affect treatment outcome [4].

Nevertheless, microorganisms may colonize culture dishes with oocytes and embryos; most likely originating from semen samples, as follicular fluid samples are largely sterile and good laboratory practice of IVF eliminates the possibility that embryo culture media will become contaminated by microbes during the procedure. However, the exact frequency of these microbial contaminations is unknown due to low number of investigations [5]. Moreover, there is very little information available on how to handle embryos derived from culture dishes with obvious bacterial contamination. Therefore, the better understanding of whether seminal-derived bacteria have a negative impact on IVF conception could lead to the adoption of more efficacious interventions that can improve the pregnancy and delivery rate in assisted conception [6].

In the current study we aimed to determine the prevalence and counts of bacteria in native semen samples used for IVF, processed semen samples and IVF culture media, and to associate them with IVF embryo quality and pregnancy rate.

Altogether 50 couples attending IVF procedure participated in the study. For microbiological analyses two molecular approaches were combined, high-throughput sequencing that allows a global systemic view of the microbiome, and qPCR with specific primers that provide an accurate and sensitive method for quantification of individual bacteria in total bacterial count.

This study revealed the qualitative and quantitative bacterial composition of the samples used in IVF. We found that there are considerable bacterial changes in IVF samples with prevalence of classes Bacilli in raw semen and embryo culture media, Clostridia in washed sperm, Bacteroidia in incubated sperm and Alphaproteobacteria in incubated sperm and IVF culture media. The associations between certain clinical data (such as increased counts of neutrophils, sperm motility, embryo quality) and presence of some bacterial phyla and genera (Bacteroidetes, Proteobacteria, Staphylococcus, Corynebacterium spp.) were also found. Although our study is not the first research project describing the presence of some groups of microbes in raw and processed sperm and IVF culture media [2, 20, 21], our study is the first one giving a deep assessment of bacterial composition of IVF culture media based on 16S rRNA gene fragments (454 sequencing platform) to help monitor IVF culture conditions.

The goal of embryo culture in IVF is to keep gametes and embryos in as similar condition to their native environment. For that in IVF laboratory, the maintenance of gametes and embryos requires stringent culture conditions. A high standard of hygiene, cleaning and waste disposal must be followed in order to avoid infection of medical staff and patients, and contamination of the culture dishes and equipment. Every step in the laboratory procedures and manipulations must be carried out with a rigorous discipline of aseptic techniques [22]. Therefore, the sterile culture conditions should be pursued in the conditions, where semen samples and follicular fluid samples – obtained via transvaginal ultrasound-guided aspiration, are believed to contain polymicrobial communities. Indeed, the presence of bacteria in environment and patients’ bodies, like semen and follicular fluid samples, and cervical regions passed in egg retrieval and embryo transfer, have been associated with adverse pregnancy outcomes in IVF [23]. Similarly, a small number of researchers have reported isolating microorganisms from IVF culture media [24, 25]. Semen, technician contamination, for example from oil overlaying human embryo culture, is the most often quoted sources of contamination. The most common species identified are Escherichia coli, Aspergillus, Candida albicans and Gram-negative cocci [23].

We found bacterial load of embryo media in around 8% of the samples by 454 sequencing and more than 70% by real-time PCR method. Previously, Kastrop et al. examined > 14,000 and Ben-Chetrir et al. > 700 IVF cycles by cultivation and found that in both studies 0.7% of IVF cycles had isolated microorganisms [5, 26]. The differences in results may be explained by methods used for bacteria examination. In our study the lower limit of PCR amplicons for 454 sequencing was 0.5 ng/μl but for real-time PCR we used DNA of all 197 isolated samples. Additionally, qPCR specific primers for Enterobacteriaceae group (Gammaproteobacteria) may amplify some other bacterial species such as Moellerella, Morganella, Proteus, Leminorella and etc. [13] that was not found by 454 pyrosequencing. It may equally be explained by the fact that the qPCR used in the current study is more sensitive for specific groups of bacteria that agrees with the study of Al-Mously et al. [27].

The microbiome of semen has been studied mostly in connection with male infertility or prostatitis [28‐30]. We found that the predominant bacterial genera in semen samples were Lactobacillus, Incertae sedis XI, Staphylococcus, Prevotella, Phyllobacterium and Corynebacterium. Previously, the high abundance of Lactobacillus in semen was also published [2, 21, 29]. Most abundant genera presented in semen by Hou et al. study were also identified abundant in our data, such as Lactobacillus, Prevotella, Corynebacterium, Staphylococcus and Veillonella [31]. Semen quality in Lactobacillus-predominant semen samples is higher than in the case of the other community types, as lactobacilli prevent sperm lipid peroxidation, thus preserving sperm motility and viability [28]. Some authors indicated that Gram-positive bacteria such as Lactobacillus and Corynebacterium, might protect against the negative influence of Gram-negative bacteria such as Prevotella, Aggregatibacter and Pseudomonas [21]. Prevotella is a genus of Gram-negative anaerobic bacteria [32], that is a member of both semen and vaginal microbial communities while its increased counts have been described in patients with low-quality semen [21, 33]. The clinical significance of strict anaerobes in sperm samples is a subject of dispute. Anaerobic bacteria are not routinely sought in sperm samples, because they are fastidious to cultivate. In fact, by using molecular methods Kiessling et al. detected and identified many anaerobes in the semen of men undergoing fertility evaluation [34]. Our study demonstrated correlation between presences of gram-negative bacteria (Bacteroidia, Sphingobacteria (class), Proteobacteria (phylum), Alphaproteobacteria (class)) with sperm motility. The gram-negative bacteria contain in their cell walls lipopolysaccharide that associated with more pro-inflammatory and oxidant environment and due to these mechnanism may disrupts sperm motility [35].

There is no data about the presence of Incertae sedis XI in semen samples. Previous publications indicated that Clostridiales Family XI Incertae Sedis bacteria are enriched in the colons of healthy adults and are also found on skin and genitals of women suffering from bacterial vaginosis [36‐38].

Similar to semen, Lactobacillus genus was also dominating in embryo culture media. Next generation sequencing revealed that Lactobacillus sp. are present in endometrial and ovarian follicular microbiome [25, 39]. The authors associated it with embryo development, and difference in the microbiome between the left and right ovaries, which was attributed to differences in haematogenous spread, was also demonstrated [40]. In contrast, the presence of some other species, such as Propionibacterium and Actinomyces among others, has been associated with adverse IVF outcomes. In addition, E. coli and Streptococcus spp. in follicular fluid might inhibit follicle-stimulating hormone (FSH) from binding to its receptor on granulosa cells [41, 42]. To conclude, the follicular fluid bacteria have been associated with both positive and negative IVF outcomes [25, 40]. In our study, the presence of bacteria in IVF culture media did not influence the pregnancy rate. Also, we were unable to determine the origin of the microbiota in embryo culture media.

Since incubation temperature is a determining factor for bacterial growth, incubation of IVF media at 37 °C could influence the bacterial growth and activity. We found that washed and incubated sperm samples had quite heterogenous microbial composition with prevalence of genera Prevotella and Staphylococcus, and class Alphaproteobacteria. Interestingly, Alphaproteobacteria was the most prevailed class of bacteria in processed sperm samples without and with further incubation, including the highest prevalence of genera Phyllobacterium in all treated sperm samples as well as Methylobacterium in incubated sperm and Novosphingobium in washed sperm and embryo culture media. Presence of these genera in IVF culture media was not previously published. At the same time previous studies have indicated that coliform bacteria, including E. coli that belong to Alphaproteobacteria were found in higher concentration in semen and media used during IVF procedure [5, 21, 43]. Kala et al. demonstrated that inoculation of E. coli caused adhesion to the sperm membrane and subsequent destruction leading to reduced motility and viability in washed samples [44]. Presence of S. aureus and E. coli may induce apoptosis in human sperm with two possible putative mechanisms: a direct cytotoxic activity of bacterial toxins and the contact with pili and flagella [28].

The majority of IVF laboratories use culture media containing antibiotics to avoid the risks of microbial growth during IFV procedure. The most commonly used antibiotics are penicillin (β-lactam), streptomycin and gentamicin [45]. In our study, both semen incubation media and embryo culture media were supplemented with gentamicin sulphate. Gentamicin is a broad-spectrum bactericidal agent of aminoglycoside group that is effective against Gram-positive and Gram-negative aerobic bacteria. Gentamicin binds to four nucleotides of 16S rRNA and a single amino acid of protein S12. This leads to interference with the initiation complex and misreading of mRNA so that incorrect amino acids are inserted into the polypeptide leading to non-functional or toxic peptides and the breakup of polysomes into non-functional monosomes. Though the counts of Enterobacteriaceae decreased with treatment (qPCR), the abundance of some species (Methylobacterium, Phyllobacterium) belonging to Alphaproteobacteria classes increased. Some of these bacteria species may be resistant to gentamicin. The resistance of E. coli to both penicillin and streptomycin in culture media has been reported previously [5, 24]. Although, in the study of 70 bacterial strains isolated from contaminated culture media were subsequently found to be sensitive to gentamicin, we may support the view that antimicrobials in the culture media probably provide little inhibition to the potentially large numbers of bacteria, including anaerobic bacteria. Moreover, it has been shown that aminoglycosides have toxic effects on sperm motility [46]. A review analyzing randomized controlled trials investigating the effect of antibiotics on embryo transfer concluded that the administration of amoxycillin and clavulanic acid prior to embryo transfer reduced the upper genital tract microbial contamination but did not influence clinical pregnancy rates [47]. Furthermore, there are no data about randomised controlled trials to support or refute other antibiotic regimens in this setting [47, 48].

Our findings showed that the simple presence of bacteria might alter the sperm quality. In the present study the counts of Staphylococcus sp. were correlated with presence of neutrophils in semen. Previously, Moretti et al. demonstrated the sperm concentration and the percentage of progressive motility were significantly decreased in sperm samples containing S. epidermidis, S. aureus and E. coli [28]. In addition, we found that counts of Alphaproteobacteria and Enterobacteriaceae may influence embryo quality. In accordance with our results, it has been previously indicated that if the embryo culture dishes are contaminated with bacteria the quality of the developing embryos is poor [5].

Our study has some limitations. First, the number of samples was moderate. In addition, qPCR did not cover wide spectrum of bacteria.

In conclusion, our study demonstrated that IVF does not occur in a sterile environment. The prevalence and counts of bacteria in the IVF procedure decrease during the semen treatment. We demonstrated the prevalence of classes Bacilli (Lactobacillus genera) in raw semen and IVF culture media, Clostridia in washed sperm and Bacteroidia in incubated sperm samples. The presence of Staphylococcus sp. and Alphaproteobacteria are associated with clinical indicators such as sperm and embryo quality. Therefore, future research should focus on the methods aiding to reduce the adverse impact of these microorganisms on IVF embryo development and helping to avoid IVF failure.