Using the PCR and Blood Agar in Diagnosis of Semen Bacterial Contamination of Fertile and Infertile Men

Background: One of the causes of male infertility is Genital tract infections (GTI). Considering the importance of GTI, widespread recognition of them seems necessary. we aimed to characterize and compare semen microbial populations in fertile and infertile men who referred to an infertility clinic in Yazd, Iran. Methods: Semen samples were collected from two groups of fertile (268) and infertile (210) men. Sperm analysis (concentration, morphology, viability and motility parameters) were performed according to the World Health Organization (WHO) 2010 guidelines laboratory manual. Bacterial isolation was performed in Sheep Blood Agar and Eosin Methylene Blue (EMB) agar plates. For PCR, samples were analyzed with genus specific primers. Results: All semen characteristics were poor in the infertile group compared to those in the fertile men (p-value< 0.05). Enterococcus spp. (18.7%, 17.1%; p= 0.814), E. coli (7.9%, 11.4%; p= 0.486), Staphylococcus aureus (6.4%, 2.9%; p= 0.398) and Proteus mirabilis (0%, 2.9%; p= 0.002) were the most common agents, respectively. Also, the results obtained from PCR were confirmed using culture-base method. Conclusions: Proteus mirabilis contamination was identified in the infertile group. While no significant association was observed between male infertility and semen microbial populations, p. mirabilis may be the leading cause of reproduction impairment in men.


Introduction
Male infertility is a worldwide health problem and its etiology is not well defined (1). Male infertility can be responsible for 20-30% of infertility cases that many parameters including anatomical defects, prostatitis, hormonal defects, varicocele, physiological and psychological disorders and immune defects can cause this problem (2)(3)(4)(5)(6). Genital tract infection (GTI) has been suggested as one of the harmful causes for male fertility (7,8).
It is believed that contaminations may exert negative effects on spermatogenesis process or decrease semen quality and sperm function. Scientific reports suggests that the mechanism of contamination process on male reproductive tract and fertility potential are related to sperm agglutination, sperm immobilization, sperm DNA fragmentation[by increasing in reactive oxygen species (ROS) level],induction of chronic inflammations which can lead to reductions in sperm parameters such as count, motility and morphology (9)(10)(11). However, to date the exact mechanism of microorganism actions and its effect on male fertility is not clear.
Many studies showed some microorganisms including bacteria, viruses, and fungi have been isolated from seminal fluid of infertile men (12)(13)(14).

Semen Bacterial Contamination in Males Infertility
Rep. Biochem. Mol. Biol, Vol. 10 Escherichia coli were seen in semen sample of infertile men as its prevalence was higher than those detected in fertile men. Also E. coli was observed in reproductive tract disorders such as prostatitis and epididymitis (15)(16)(17). Staphylococcus aureus is another microorganism that play an important role in male reproductive tract infection (18). Many studies showed S. aureus is one of the most common bacterium isolated from the seminal fluid of infertile men (12, 18,19).Nabi et al in 2013evaluated bacterial contamination in seminal fluid of male partners in recurrent abortion cases. They reported, S. aureus and E. coli were the two main bacteria in the seminal fluid of male partners of women with unexplained recurrent abortion (20). Chlamydia trachomatis is another bacterium found in prostatitis, epididymitis, and urethritis cases. A systematic review in 2016 by Ahmadi et al, pointed to Chlamydia trachomatis as a very important and prevalent pathogen in infertile men as its prevalence was significantly higher than those in fertile men (21).
Baud et al (2019) compared the bacterial composition in 26 normal and 68 abnormal spermiogram parameters using PCR technique. They reported that overall sperm bacterial content didn't play a major role in male infertility (22). The novelty of our study compared to previous studies was to evaluate bacterial contamination semen with blood agar and PCR methods in the high population of fertile and infertile men. There are very limited studies regarding the microbial contaminations in seminal fluid of infertile men in Iran. Since geographic parameters, genetic history and hygiene conditions are contributing factors for prevalence of microorganisms in a population, we aimed to characterize and compare seminal microbial status in the fertile and infertile males referring to Yazd infertility research center and find out which of the two methods blood agar and PCR assay is more suitable semen bacterial contamination.

Study population
In this case-control study, a total of 478 volunteer men who referred to Yazd infertility research center from September 2015 to July 2016 were divided into two groups of Fertile (n; 268) and Infertile (n; 210) males. Diagnosis of fertility or infertility were done by an urologist according to the World Health Organization (WHO) 2010 guidelines (23). Inclusion criteria included; Fertile and infertile men identified by the Andrology Laboratory. Exclusion criteria included infectious diseases sufferers and antibiotics usage of at least two weeks prior to semen collection, exposure to chemotherapeutics or radiation, testis trauma, smokers and variciselectomy. The ethical committee based at the Yazd infertility research center approved the study protocol and use of patients' information. Also, all participants read and signed an informed consent.

Sperm collection and analysis
Semen specimens were collected in sterile container, the abstinence period for all participants was 2-5 days. Semen analysis was carried out according to the world health organization (WHO) laboratory manual for the examination and processing of human semen (Organization, 2010). Following masturbation induced ejaculation and were liquefied at 37 °C for 30 minutes. Sperm analysis including count and motility parameters (e.g., progressive, nonprogressive and immotile) were assessed by Mackler chamber. Papanicolaou and Eosin-Nigrosin staining tests were done for sperm morphology and sperm viability evaluation, respectively (24).

Microorganism identification
Homogenized semen samples were cultured on the sheep blood agar and eosin methylene blue agar (EMB) (Condapronadisa, Spain) to isolation of gram positive and negative bacteria, respectively. Differential tests were done for phenotypic identification of the species; Bile Esculin Agar (BEA), growth in 6.5% NaCl, catalase and hemolysis tests. Mannitol salt agar (MSA), DNase agar, coagulase and catalase tests were carried out for identification of Staphylococcus species. Differential tests were used for characterization of Gram-negative bacteria which included: catalase, oxidase, Simmons citrate agar, methyl red-voges-proskauer (MR-VP), lysine decarboxylase, triple sugar iron (TSI), and sulfide-indol-motility (SIM).
PCR reactions were performed for detection of various grown colonies (table 1). Chromosomal DNA was extracted from the pure colonies using the Qiagen extraction kit (QIAamp DNA mini kit, USA). The DNA concentration and purity was assessed using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, UK) and 1% gel electrophoresis, respectively. Templet DNA was kept at -20 °C until more use. The primer sequences, PCR compounds and reactions are listed in Table 1. In general, PCR was performed in a final volume of 25 μl in an Eppendorf Master Cycle Gradient thermocycler (Eppendorf, Hamburg, Germany). PCR products were subjected to a 1% agarose/0.5×TBE gel electrophorese (45 mM-Tris-borate, 1 mM-EDTA) stained with 0.1 μl/ml Gel Red™ (Biotium, USA), then photographed under an UV trans-illuminator (Tanon, China).

Statistical analysis
The statistical package for social science (SPSS) software version 20 (IBM Co., Illinois, USA) was used for statistical analysis of data. Student's t-test and proportional test were performed for numerical variables and categorical variables, respectively. Results were presented as Mean±SD or frequency percentage. A statistically significant difference was considered when p value was less than 0.05.

Results
The mean age for the fertile and infertile groups was 36.3±4.2 and 34.6±3.8 years, respectively. Sperm analysis in both groupwereshown in Table 2. The mean semen volume and sperm count as well as mean frequencies of sperm normal morphology, viability and motility (progressive and non-progressive motility) were significantly lower in infertile individuals compared to fertile (for all, p-value≤ 0.0001).
Bacterial culture showed contamination with various species in 36.5% and 34.3% of fertile and infertile samples, respectively (Table 3). Enterococcus and E. coli were respectively the most common agents identified in both groups. Also, other isolated microorganisms including Klebsiella pneumonia (1.4%), Citrobacterkoseri (0.6%), Streptococcus spp.
Proteus mirabilis (n; 6 of 210, 2.8%) were identified only in infertile patients. There was no significant difference in distribution of different microorganisms between the fertile and infertile groups. PCR using specific primers revealed various microorganisms in 36.6% and 45.7% of the fertile and infertile groups, respectively ( Table 4).
In line with culture-base method, PCR showed that Enterococcus and E. coli were the most common agents in the semen collected from both fertile and infertile men. Molecular characterization showed no significant difference in distribution of different microorganisms between the fertile and infertile groups. According to the culture results, all 6 samples were positive for P. mirabilis in PCR(p-value= 0.002). Candida spp. (0.3%) was isolated only from semen samples of fertile men. The data were presented as mean±SD, * p-value≤ 0.05 was considered as significant between fertile and infertile groups.

Discussion
In the preset study, we isolated different microorganisms from seminal fluid of fertile and infertile men. Our results similar to Baud et al, study indicated that there was no significant difference in the prevalence of identified microorganisms between the fertile and infertile males (1). This data was contrast with other studies that shown the prevalence of microorganism in the semen of the infertile was significantly higher than the fertile group and it may be correlated with sperm quality in infertile men (33). Enterococcus faecalis was commonly isolated from the seminal fluids as it was the most abundant bacterium. However, the prevalence of E. faecalis was not significantly different between fertile and infertile men. Escherichia coli is another common bacterium isolated from semen samples (36,37). There is controversy in scientific reports regarding relation between the presence of E. coli in the semen sample and prevalence of male infertility (38,39). However, there seems to be a positive relation between presence of E. coli and acute epididymitis and prostatitis (40,41).
In a recent study (2018), assessment of microorganisms such as E. coli was evaluated in infertile Nigerian patients. The investigators reported that E. coli was not only a prevalent organism in the seminal fluid of infertile men, but there seems to be a significant relation between its presence and decrease in sperm parameters including sperm count, motility and morphology (14).Although, E. coli was the  (44). In the present study, we found this bacterium to be the third most common microorganism in the semen of both fertile and infertile men. Importantly, the frequency distribution of S. aureus in the infertile group was insignificantly lower than that in the fertile group. This observation casts doubt on the role of S. aureus in male infertility.
We also isolated other microorganisms including Klebsiella pneumonia, Klebsiellaoxytoca, Citrobacterkoseri, Streptococcus spp., Diphtheroids, and yeasts, with very low incidence in the semen of fertile men. Due to lack of supporting evidence, the possibility of implication of these microorganisms in male infertility cannot be discussed at the present time. However, Proteus mirabilis was isolated in six cases of semen culture from infertile men. Similar to our results, Elgozali et al in 2015 also isolated Proteus species from semen of infertile man (45). Mehta et al observed Proteus species in the semen of men with Oligo asthenoterato spermia (46). Therefore, p. mirabilis might be implicated in male infertility, though this hypothesis needs further confirmatory studies.
The results of present study didn't show any significant association between male infertility and microbial populations isolated from semen samples. E. faecalis, E. coli, S. aureus and Klebsiella pneumonia were the most prevalent bacteria in the semen of fertile and infertile men, respectively. However, P. mirabilis was detected in the semen of infertile men which may be responsible for lack of reproductive ability. However, much more studies are essential to confirm this inference.