THE BACTERIUM Fusobacterium sp. MAY INTERFERE WITH CONCEPTION

This study was aimed to investigate microbiological and molecular markers of periodontal problems and its association with conception in 101 Iraqi women, 50 primary infertile while 27 secondary infertile and 24 pregnant as control group. Major periodontal pathogen in the subgingival plaque and cervical vaginal swab specimens was detected, and molecular marker (16SrRNA) gene used for detection of Fusobacterium in our study. Fusobacterium sp. isolates from oral samples were examined biochemically and confirmed by molecular detection, they were also subjected to antibiotic sensitivity test and were shown to be resistant to β-lactamase family of antibiotics and were mostly resistant to Ciprofloxacin. Four Fusobacterium sp samples were identified in both Oral samples and vaginalcervical specimens of the same four women from the primary infertile group. Fusobacterium sp. was significantly (P<0.01) more frequently detected in the Oral samples and vaginal-cervical specimens among primary infertile group (38.7%) than pregnant (9.1%) as so among secondary (30.0%) than pregnant (9.1%). Analysis identified a model of a significant predictive factor of presence of Fusobacterium sp.: primary infertile [Odd ratio (OR) = 6.3 at 95% CI = 1.33-25.1] and secondary infertile OR = 4.2 at 95% CI = 1.84-12.22. this can show that infertalty state can be more risked than pregnant women to have the Fusobacterium sp and be effected by it.


INTRODUCTION
Fusobacterium, is a Gram negative bacterium, non-spore former, anaerobic species of the Fusobacteriaceae family that consists of nine genera including Fusobacterium and Leptotrichia. There are currently 14 species defined, Within the Fusobacterium genus some of which (including F. nucleatum) are identified as pathogens of human and animals that is gaining importance as a pathogen with a high number of associated diseases. (5). The Fusobacterium is a non-motile, rod-shaped, or bacilli bacterium. It shows a significant role in the progress of biofilm dental plaque as a bridge bacterium interaction with early and late colonizing bacteria in the oral cavity (3), as well as its role as a part of oral plaque, through its adhesive abilities which provide benefit as a link between early and late colonizers of this biofilm (27). It is one of the species found in the oral cavity, in both unhealthy and healthy humans. It is found in different kinds of periodontal diseases including the simple reversible kind of gingivitis and the progressive irreversible forms of periodontitis: chronic periodontitis and localized aggressive periodontitis (33). There are strong correlation between several of the cultivable bacteria for example Prevotella intermedia, and Fusobacterium nucleatum and periodontal disease has been mentioned (49). Fusobacterium sp. may cause with other oral species, e.g. Tannerella forsythia, Porphyromonas gingivalis and Streptococci, interaction in virulence is revealed which may result in increased bone loss, abscess, or death. (44). Previous studies have reported that Fusobacterium sp. was involved in wide variety of systemic diseases including gastrointestinal (GI) disorders, atherosclerosis (21), rheumatoid arthritis and respiratory tract infections (47). There has been an increasing interest in causes of infertility such as ovulation disorders (16), Fallopian tube pathology (35), ovarian insufficiency, endometriosis (46), and uterine or cervical abnormalities, as well as infection and inflammation that play a role and effect fertility in women by affecting the ovary, uterus, the embryo (26), and its relationship with periodontitis that share common risk factors such as age (25), obesity (12) and tobacco smoking (48), but the relation still needs more clarification and data of the effect of periodontitis on the success of becoming pregnant are restricted. However, research has shown that women with decreased clinical periodontal health and symptoms of gingival inflammation are more affected with in vitro fertilization problems (40,4). One research has already shed light on the effect of microbiological markers of periodontal infection on conception (38).This study has been applied with a cohort of young women who failed to get successful conception and aimed to find a connection between the oral microorganisms and conception by using microbiological and molecular methods.

Subjects:
Clinical specimens were collected from (101) of Iraqi women, who were grouped according to their conception status (primary infertility, secondary infertility and pregnant as control group), Subjects were enrolled from the High Institute for infertility Diagnosis and Assisted Reproductive Technologies/Al-Nahrain University, Baghdad-Iraq, from October 2018 to March 2019. Women ages ranged between  years, mean (29.47±2.5). Clinical diagnosis of Infertility and periodontitis was confirmed by consultation of clinical specialists. Samples collection Oral samples: Sub gingival plaque samples were collected by a sterile paper point (Albion/UK) that was inserted into two deep pockets of the periodontal area for 30 econds and removed. Sampling sites were protected from saliva influx with sterile cotton pellets. The paper points were collected in a sterile Eppendorf tube for each with 1 ml of sterile Thioglycolate medium (Hi-media/India), and transported in a cooled box to the microbiology laboratory for further experiments (31).

Cervical-vaginal Swabs:
Gynecological Sterile Speculum (China) was used to collect the samples from women by sterile swabs (swabs were taken from vagina and upper cervix by specialist physician). Swabs were immediately suspended in 1 ml of sterilized phosphate buffered saline PBS (pH=7.4), and then stored at -20℃ until use for further molecular experiments (45).

Isolation and identification of oral bacteria
Paper point specimens were mixed by micropipette and diluted in buffer phosphate saline (10 -2 ). An aliquot of 100 µL was spread on culture plates containing blood agar medium, which was supplemented with Human blood 50.0 ml/L, Hemin 5μg /ml (5 mg /L.), Vitamin K1 (1μg /ml) (1mg /L.) and Bacitracin 10.0 mg/L and Nalidixic Acid 15.0 mg/L antibiotics in1000.0 ml of Distilled Water )as selective agents for isolation of such fastidious, strictly anaerobic oral bacteria , Selective medium plates were incubated in a tightly packed anaerobic atmosphere jar at 37°C for (48-72 hr.). Microscopically examination was conducted on pure bacterial culture; they were stained with Gram stain to distinguish their response to the stain to examine their color, shapes and arrangements. Biochemical characteristics was achieved by biochemical tests to evaluate the following characteristics: production of H2S, Indole test catalase, urease and sugar fermentation (rhamnose, adonitol, salicin, arabinose, inositol, sorbitol, sucrose, mannitol, raffinose, and maltose) (19). Antibiotic Sensitivity test Antibiogram of isolated Fusobacterium sp. strains was detected by Disc diffusion method and were based on the susceptibility patterns for selected antibiotics which represented various classes of antimicrobial agents, as show in Table (1). According to Kirby-Baure method used to carry out antimicrobial susceptibility. The isolates were explicated as susceptible, intermediate or resistant to a particular antimicrobial agent by comparison with standard inhibition zones as mention according to CLSI, 2011 (14). Preparation of inoculum: bacterial inoculum was prepared in normal saline to adjust its turbidity, from fresh culture that was incubated anaerobically for 18-24 hr.,3-5 colonies were taken by sterilized loop then suspended in 5 ml of normal saline to get a culture with 10 5 cell/ml (7). Inoculation and applying antibiotic discs: A sterile cotton swab was used to transfer bacterial culture (swab immersed into inoculum tube) then carefully and evenly spread on Mueller Hinton agar plates and left for 10 min., and then the antimicrobial discs (T ab l e 1 ), were placed on the agar medium by using sterile forceps and were compressed to ensure contact with the surface of agar. The plates that contained the antibiotic discs inverted and incubated at 37 ˚C for 24 hr. under anaerobic conditions. Inhibition zone for every antibiotic was measured by a metric ruler in millimeters (mm) CLSI, 2013 (22). The DNA of pure bacterial colonies was extracted by Genomic DNA mini extraction kit (Geneaid /Korea), according to the manufacture protocol: pure bacterial colonies were collected from the plates with a sterile inoculation loop into 1 ~ 2 ml tube of buffer and pellet was collected by centrifugation for 1min at 16,000 rpm, and the supernatant was discarded. A volume of 180 μl of GT Buffer was added into sample tube and then resuspended by vortex or pipette. Microbial fluid was collected from the Cervical-Vaginal swabs suspended in 1ml of sterile Phosphate buffer saline. Microbial fluid was collected by centrifugation for 5 min at 16,000 rpm, and then the supernatant was discarded. Aliquot of200 μl of GT Buffer was added into the sample tube, and then resuspended by vortex or pipette. Proteinase K solution (20 μl) was added into all sample tubes, then mixed for 10 sec by vortex, after that incubated for at least 10 min at 60℃ (tubes were inverted 3-4 times during incubation). GB Buffer (200 μl) was added into the sample tube, then mixed by vortex for 10 sec, incubated at 70℃ for 10 min (tubes inverted every 3 min for complete lysis). After lysis completed, 200 μl of absolute ethanol was added into the sample lysate and mixed immediately by vortex (any precipitate appears had been broken with pipette). About 620 μl of mixture was transferred into GD column (in a 2 ml Collection Tube) without wetting the rim, the cap closed, and centrifuged at 16,000 rpm for 2 min. The filtrate was discarded and the GD column was placed in a new 2 ml Collection Tube. W1 Buffer ( 400 μl) was added to the spin column without wetting the rim, and centrifuged for 30 sec at 16000 rpm. The flow-through was discarded and reused the collection tube. A volume of 600 μl of Wash Buffer was added (ethanol was added) to the GD Column, and centrifuged for 30 sec at 16000 rpm. The flowthrough was discarded and reused the Collection Tube and centrifuged again for additionally 3 min at 16000 rpm to dry the Column matrix. The spin Column was placed into a new 1.5 ml tube, and 50-100 μl of Pre-heated Elution Buffer directly added onto the membrane. Incubated for 3-5 min at room temperature to allow completely absorbed, and then centrifuged for 30 sec at 16000 rpm to elute the purified DNA. (6). PCR detection: To amplify the 16SrRNA gene, the Fusobacterium sp. primers used in the study shown in (Table 2), PCR was achieved on DNA extracts from subgingival bactrial isolates and cervical-vaginal swab samples. Amplification reaction was done by a Thermal Cycler in a 25 μl reaction volume containing 5 μl of Taq PCR premix, 10 picomoles/μ (1 μl) of the forward primer, 10 picomoles/μ (1 μl) of reverse primer, 3 μl of genomic DNA, and 15 μl distilled water. The 16S rRNA PCR was performed for 3 min at 94C and 34 cycles, with each cycle consisting of denaturation at 94 o C for 30 sec, annealing at 49 o C for 45 sec, extension at 72 o C for 1 min, and final extension for 7 min. (32). The amplified products were analyzed by gel electrophoresis on a 1.5% w/v agarose gel in 1x TBE buffer for fifty minutes (80 volt) and visualized by staining with red safe stain by UV transillumination.

Statistical analysis
The Statistical Analysis System-SAS (2012) program was used to detect the effect of difference factors in study parameters. Least significant difference -LSD test was used to significant compare between means. Chisquare test was used to significant compare between percentage (0.05 and 0.01 probability). Estimation of Odd ratio and CI was also used in this study.

RESULTS AND DISCUSSION Study population characters:
The results presented in this study are based on a total of 101 Iraqi women, whom were divided into three main groups depending on the state of infertility or pregnancy: 1-Primary infertile: which included 50 women, ages , they failed to conceive for unknown causes, years of infertility was ranged between (2-13) years. 31 (62%) of them with periodontal problems 2-Secondary infertile: which included 27 women, ages  years, which experienced previous pregnancy and started failing to conceive for unknown causes, years of infertility after last pregnancy (2-12) years. 15 (55.56%) of them periodontal problems 3-Pregnant: which included 24 women, ages  with normal pregnancy 11(45.8%) who had periodontal problems. As shown in (Table 3). The percentage of primary infertile group of women was (50%), which was significantly higher compared to both secondary infertile (27%) and pregnant (24%) groups, (Fig.1a) this was showed in other studies with similar percentage, that showed the percentage of primary and secondary infertility among women was 62.97% and 37.03%, respectively (9). The primary infertile group of women showed the highest percentage (62%) with periodontal problems compared to secondary (55.5%) and pregnant (45.8%) groups at (p<0.05), as shown in (Fig. 1b), this was agreed with studies of unknown causes of conception, this distribution was also compared with other studies that showed varied result, when chronic periodontitis was positively associated with increased time of conception (TTC) and the authors are recommending that women in child bearing age should be encouraged to have regular preventive dental checkups in order to maintain good oral and periodontal health (36) Figure 1. a) The percentage of women according to study groups (Primary, Secondary and Pregnant). b) The percentage of periodontal problems in study groups of (Primary, Secondary and Pregnant). Periodontal diseases was known to be case of inflammation in the oral cavity which involves the teeth and surrounding gum (8). Studies have linked periodontitis with different diseases such as pre-eclampsia with low birth weight (41), diabetes - (42), Rheumatoid -Arthritis(31),increased cardiovascular diseases like Myocardial Infarction, -Atherosclerosis (43) and Alzheimer's Disease (37). Other studies also shed light on conception and abortions that exhibited the relationship between periodontitis and several pregnancy outcomes such as preterm birth, still birth and others (13). Bacterial identification of periodontitis Periodontitis may be caused by different agents, mostly known cause is bacterial agents, in which a wide range of bacterial species are related to periodontitis infections such as Porphyromonas gingivalis (30), Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans (1). Which are mostly anaerobic fastidious microorganisms that need special growth conditions to be isolated. Microbial examination of isolates from this study revealed different kinds of bacteria that varied in microscopic examination and showed different shapes and sizes of colonies, colors, on blood agar enrichment medium as well as for the microscopic examination that exhibited different bacterial shapes, arrangements and Gram positive as well as Gram negative bacteria. These isolates were sub-cultured for purification and a total of 21 (36.8%) different bacterial isolates were recovered of both Gram

Fusobacterium sp. Isolates
The Fusobacterium sp. bacterium was isolated from 4 oral samples of the primary infertile group. Microbial examination showed Fusobacterium sp. as Gram-negative, non-spore forming bacteria, slender or rod shaped bacilli usually with pointed ends. The bacterial colonies appeared on blood agar as small, convex, non-translucent and grey-creamy colonies and generally do not produce hemolytic reaction on blood agar these characteristics were also seen in the same of other studies (34) as shown in (Fig. 2a.b).

Figure 2a. Fusobacterium colonies cultured on Blood agar, (b) Micrograph of Fusobacterium isolated from subgingival plaque (by sterile paper point) by using Gram stain (100X). Cells appear as single, in pairs, or in short bacilli chains Biochemical test
Fusobacterium sp. isolates showed negative result for urease test (11), while Fusobacterium gave a positive result for Indole test. Whereas the sugar fermentation test showed that Fusobacterium sp. isolates ferment some sugars by production of acids that convert the indicator chlorophenol red to yellow in one to three days of anaerobic incubation. The sugars (sucrose and maltose) were fermented by Fusobacterium sp. isolates, while six of carbohydrates (Rhamnose, salicin, arabinose, sorbitol, Mannitol and Raffinose) were not fermented by these isolates, these results agreed with that documented by Howe and coworkers (22).

Antimicrobial susceptibility of Fusobacterium. sp isolates
Disk diffusion method was used to detect the antibiotic sensitivity of Fusobacterium.sp against different kinds of antibiotics previously mentioned in (Table 1). Fusobacterium isolates showed resistance against many antibiotics (Ampicillin, Amoxicillin, Penicillin, Imipenem, Cephalothin, Cefixime and Amikacin), with significant differences at (P<0.05). whereas, these isolates showed susceptibility against (Cefotaxime (CTX), Gentamicin (CN), Vancomycin (VA), Clarithromycin (CLR), Tetracycline (TE), Chloramphenicol (C), Ciprofloxacin (CIP) and Meropenem (MEM).) as Shown in (Fig.3) and (  Table 4).  In this study Fusobacterium isolates showed resistance against β-lactams antibiotics. Many studies showed the ability of Fusobacterium sp to produce β-lactams and the first reports of penicillin resistance due to β-lactamase production by Fusobacterium.sp were published in the mid-1980s (50). The frequency of β-lactamase production by Fusobacteria seems to be increasing. In several studies observed surprisingly high frequencies of β -lactamase production by several anaerobic, gram-negative species in oral sites (28,2). Fusobacterium.sp isolates showed resistance against Imipenem that is a semisynthetic thienamycin that has a wide spectrum of antibacterial activity against gram-negative and gram-positive aerobic and anaerobic bacteria including many multiresistant strains while Fusobacterium.sp showed sensitivity against Meropenem is similar to Imipenem but having greater activity against gram-negative bacteria (39), this variation in resistance of Fusobacterium.sp to antibiotics may be related to the presence or absence of plasmids that carry antibiotic resistant genes. Some studies have been reported that high resistance observed in several Fusobacterium strains which suggest the presence of antibiotic-resistance marker such as a plasmid or a chromosomal gene. Bacterial plasmids codifying for antimicrobial resistance have been recurrently observed (10). Sensitivity of Fusobacterium.sp isolates against other antibiotics may result from the differences in their chemical composition, antibiotic family and in concentration which lead to different inhibition zones. The highest inhibition zone was detected for the Ciprofloxacin (31.0 ±0.7). According to Sanford Guide to Antimicrobial Therapy (17), Fusobacterium can show sensitivity against Chloramphenicol, Meropenem and other antibiotics. Detection by molecular techniques DNA extraction from oral and swab samples: The DNA was extracted from oral isolates of the oral samples and swab cervicalvaginal samples using Genomic DNA mini extraction kit. The bacterial isolates were chosen depending on the identification of bacteria by using laboratory procedures like microscopic examination and biochemical tests. The DNA quality and integrity were assessed through observing DNA bands by gel electrophoresis on 0.8% of agarose for 40 minutes. The bands looked sharp, single not dispersed, and no smear which might result DNA degradation .as shown in (Fig.4a,b)  (Fig.5a,b). While reaming oral sample and swabs sample was negative and exhibited no band, which was evidence that it was not correlated to the Fusobacterium isolates. The same gene was used for Fusobacterium isolates detection was targeted by other studies (32).  (1,2,4,5,6,8,9,12,and 14) are positive samples ,whereas lane (3,7,11,and 13) are negative samples. M is a marker, PCR amplified product visualized by U.V. This is the first local study in Iraq using both microbiological and molecular methods to identify the presence of a common periodontal pathogen associated with conception. The chief result was the detection of Fusobacterium sp. in oral (subgingival plaque) and cervical-vaginal specimens, these bacterial species may raise the risk for ineffective conception among Iraqi women. There are relationship between periodontal pathogens and infertility that might share several common risk factors such as age, low socioeconomic standing (as education level) and obesity (38). As well as, there are conventional hazards and confusing factors, the finding of this study propose that Fusobacterium sp. can play a role in deferred conception or at least may be a sign of this association. According to PCR analysis result the percentage that gave positive result to Fusobacterium sp of swab samples in study groups with periodontal problems was (64.5%) in primary infertility; (60.0%) secondary infertility compared to (6.4%) in pregnant, with significant difference at (P<0.01) as shown in (Fig. 7). Women with healthy oral state showed very less percentage of Fusobacterium sp, (21%) in primary infertility; (16.6%) secondary infertility, whereas the pregnant had no result, with significant difference at (P<0.01) as shown in (Fig. 6). These results show a high percentage of Fusobacterium sp. in infertile groups compared to pregnant group, Although previous studies state that Fusobacterium sp. is part of the normal flora of the human, the current studies show that it should always be treated as a pathogen, but the increased percentage of presence raise a question if it is really normal flora or is it bacterial transmission from the oral cavity. Prior studies that have noted the importance of link between periodontal microorganisms and adverse pregnancy outcomes emphases on preterm birth and premature rupture of membranes (PPROM) by documented intra-amniotic Fusobacterium sp in patients with preterm birth and in placenta and fetal tissues, comprising amniotic fluid, cord blood, fetal membranes (52) and neonatal gastric aspirates from pregnancies affected with PPROM as well as preterm birth with intact membranes (20). These studies support the result of our study in which the percentage of Fusobacterium sp in pregnant women (18.1%) who had periodontal problems, these pregnant women suffered of abortion and did not complete the pregnancy stages (Fig.6). problems or with healthy oral state Fusobacterium sp. was isolated from 4 women with periodontal disease. They were from primary infertility (Fusobacterium sp presented in the oral samples and swabs samples of the same women). The presence of this bacterium in oral and cervical-vaginal sites can suggest the association between this bacterium and delay of conception in group of Iraqi women .According to the total percentage of Fusobacterium sp. from both swab and oral samples, the percentage of Fusobacterium sp. in women with primary infertility (38.7%) showed significant difference compared to pregnant women (9.1%) at (p > 0.01), while in women with secondary infertility (30.0%) also showing significant differences compared to pregnant women (9.1%) at (p > 0.01). As showed in (Fig.7 a.b).   58-7.85. this can show that infertalty state can be more risked than pregnant women to have the Fusobacterium sp and be effected by it. The strains of Fusobacterium sp identified in amniotic fluid and placenta appear to match those from the maternal or the partner subgingival sites rather than the lower genital tract, the previous studies showed many evidences to support our result in the presence of Fusobacterium sp in the samples of pregnant women who suffered from abortion (19).Recently, many studies suggest that detection of the virulence mechanisms of Fusobacterium sp is a key to understanding its relationship with such an extensive spectrum of adverse pregnancy outcomes, Fusobacterium sp considered as an invasive bacteria, it can bind and invade both of epithelial and endothelial cells (51). In accordance with the present results, previous studies have demonstrated that Fusobacterium sp might translocate from the oral cavity of young girl ̕ s to their uterus when the immune system was weakened through the respiratory infection. In any case of the transmission route, the complications of pelvic inflammation disease (PID), including chronic pelvic pain, ectopic pregnancy, and tubal infertility with intra-abdominal scarring, highlight the potential importance of detecting Fusobacterium sp in females. Even though most of Fusobacterium infections are periodontal in nature, translocated infections can have long term effects for reproductive health (24).In this study detection of Fusobacterium sp by using specific primer in a group of Iraqi women and the results showed the presence of this bacterium in cases with primary and secondary infertility in addition to abortion but there are not presented in pregnant women, there is no data showing the relationship between the presence of Fusobacterium sp. and conception but this result provide further support for the hypothesis that several organisms like Mycoplasma hominis, Bacteroides spp., Gardnerella vaginalis, and Fusobacterium nucleatum usually display low virulence unless they reach the intrauterine environment (15). Fusobacterium sp, in usual is nonpathogenic oral anaerobic bacteria, that has been proposed to spread hematogenously to the placenta and change vascular endothelium permeability, potentially allowing for the colonization of other potentially pathogenic organisms, such as Pseudomonas sp. (23).