|Year : 2012 | Volume
| Issue : 2 | Page : 108-113
Estimation of tumor necrosis factor-α in chronic periodontitis and its co-relation with preterm gestation: A Clinico biochemical study
N Ravindra Reddy1, DS Madhu Babu2, Vinathi Reddy1, N Sarath1, C Venkat Subba Reddy1, A Kishore Kumar1
1 Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh, India
2 Department of Periodontics, Goverment of Dental College, RIMS, Kadapa, Andhra Pradesh, India
|Date of Web Publication||17-Jan-2013|
N Ravindra Reddy
Department of Periodontics, CKS Teja Institute of Dental Sciences, Tirupati, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: The aim of this study was to estimate the levels of tumor necrosis factor-α (TNF-α) in serum of healthy, term gestation women and periodontitis affected pre-term gestation women and to find out the association between TNF-α levels, gestational age and different periodontal variables in serum. Materials and Methods: Clinical parameters, including pocket depth, Russel's periodontal indexes were recorded from 36 subjects divided into two groups based on presence of pocket beyond 5 mm and gestational age above or below 36 weeks. Blood samples from all 36 subjects were obtained from the fetal side of the umbilical cord of the term and pre-term gestation infants at delivery. Serum was separated from the collected blood samples and TNF-α from the resultant serum was quantified using enzyme linked immunosorbent assay essay. Results: The mean amounts of TNF-α was significantly higher in Group-II when compared to Group-I, which was statistically significant ( P < 0.001). Positive correlation was found between TNF-α concentration and Gestational age ( P < 0.001), Russel's periodontal index ( P < 0.001), Pocket depth ( P < 0.001) which were statistically significant. A positive correlation was found between pocket depth, Russel's periodontal index and gestational age ( R = −0.69, P < 0.001), ( R = −0.71, P < 0.001) i.e., when pocket depth (beyond 4 mm or above) and Russel's periodontal index increase, estimated gestational age shows a steady decline. Conclusion: TNF-α concentrations correlated positively with the extent of periodontal destruction, Russel's periodontal index score and gestational age, With increase in amount of periodontal destruction, there is a substantial increase in clinical parameters and TNF-α level in serum, which is inversely related to premature gestation.
Keywords: Chronic periodontitis, pre-term gestational age, tumor necrosis factor-α in serum
|How to cite this article:|
Reddy N R, Madhu Babu D S, Reddy V, Sarath N, Reddy C V, Kumar A K. Estimation of tumor necrosis factor-α in chronic periodontitis and its co-relation with preterm gestation: A Clinico biochemical study. J Orofac Sci 2012;4:108-13
|How to cite this URL:|
Reddy N R, Madhu Babu D S, Reddy V, Sarath N, Reddy C V, Kumar A K. Estimation of tumor necrosis factor-α in chronic periodontitis and its co-relation with preterm gestation: A Clinico biochemical study. J Orofac Sci [serial online] 2012 [cited 2019 Jun 18];4:108-13. Available from: http://www.jofs.in/text.asp?2012/4/2/108/106201
| Introduction|| |
"Periodontitis is defined as chronic inflammatory disorder of the periodontal supportive tissues of teeth". It has been postulated that periodontal infection may cause bacteremia, including the circulation of endotoxins that might trigger host immune response exaggerating the effects of low grade inflammation in other organs.  Dental epidemiological studies suggest that periodontitis may also contribute to adverse pregnancy outcomes,  diabetes,  myocardial Infarction and other conditions. The concept that periodontal disease might influence systemic health is not new. Miller originally published his "focal infection theory" in 1891 suggesting that "microorganisms or their waste products obtain entrance to parts of the body adjacent to or remote from the mouth.  However, recent progress in identification and characterization of periodontal pathogens, as well as elucidation of potential systemic mechanisms of action of bacterial products and inflammatory cytokines, have opened the way for a more realistic assessment of the systemic importance of periodontal disease. Epidemiological, microbiological and immunological studies have lent credence to the concept that periodontal disease may be a separate risk factor for cardiovascular disease, cerebrovascular disease and respiratory disease, as well as pre-term delivery of low birth-weight infants. ,
Tumor necrosis factor-α (TNF-α) is one among those cytokines, which are major acute phase reactants elicited in various chronic inflammatory diseases and elevation of serum TNF-α is an evidence of active tissue damage process. ,, TNF-α is also a potent pro-inflammatory cytokine and the individual cytokine production is discussed as an important factor involved in etiology of periodontal disease and could have an influence on the therapeutical outcome. TNF-α released at the sites of inflammation  influences the immune response via the activation of cells, such as endothelial and gingival fibroblasts, the induction of cytokine production, the up regulation of adhesion molecules, as well as the stimulation of matrix metalloproteinases. Studies on the human myometrium have shown that cytokine release is stimulated by lipopolysaccharide (LPS). Furthermore, estimation of gingival crevicular fluid (GCF) prostaglandin E 2 (PGE 2 ) levels can be used as an indirect estimate of the amniotic fluid levels of PGE 2 . Thus, an elevated PGE 2 level in gingival fluid has been reported to identify periodontitis activity and has also been associated with low birth weight and pre-term labor  which is defined as delivery before 37 weeks of gestation and birth weight less than 2500 g.
Present study is undertaken to substantiate an association between periodontitis and pre mature gestation by estimating serum TNF-α levels, which acts a novel inflammatory biomarker.
Aims and objectives
- To assess, if periodontits predicts premature gestation
- To study, umbilical cord blood cytokines and periodontal variables in early-stage pregnancy
- To estimate the levels of TNF-α in serum of healthy, term gestation women and periodontits affected pre-term gestation women
- To find out the association between TNF-α levels and different periodontal variables in serum
- To find out the possibility of using TNF-α as a "Novel biomarker" for periodontal disease progression and its association with early stage pregnancy.
| Materials and Methods|| |
The study population consisted of 36 subjects in the age group of 19-35 years with pregnancy and was distributed into 2 groups. Group-I consisted of 18 subjects without periodontitis and term gestation, Group-II consisted of 18 subjects with periodontitis and pre-term gestation. Verbal and written informed consent was obtained from all subjects prior to their enrolment in the study. This study was reviewed by the board of the ethical committee of the dental college and project was envisaged after the clearance was obtained. Participation of the subjects in this study didn't affect any treatment decisions regarding their medical care.
| Patients Selection Criteria|| |
- Group-I without periodontitis, probing pocket depth < 3 mm. 
- Group-II with periodontitis, probing pocket depth of > 5 mm in each quadrant. 
- Group I gestation age->36 weeks. 
- Group II, gestation age < 36 weeks. 
Pregnant women who were having any systemic complications like diabetes, hypertension etc., were excluded from the study.
Periodontal examination was carried out with mouth mirror and William's graduated periodontal probe and clinical parameters-pocket depth, Russel's periodontal indexes were recorded for all 36 subjects.
36 blood samples from 36 patients were obtained from the fetal side of the umbilical cord of the term and pre-term gestation infants at delivery. 10-20 mL of mixed arterial and venous umbilical cord were collected with a sterile 5 mL syringe and transferred to sterile non heparinized test tube vials.
The umbilical cord blood samples, which were transferred to 5 mL sterile nonheparinized test tube vials were placed in slanting position at room temperature. Once the blood clotted, the serum supernatant was collected into sterile centrifuge vials and centrifuged at 10,000 rpm for 20 min. The resultant straw colored serum was obtained [Figure 1] and stored in 200-250 μl at -70°C until analysis.
Tumor necrosis factor-α estimation
TNF-α was quantified using enzyme linked immunosorbent essay prepared as per the guidelines of the manufacturer (Bendermed systems, company catalog number BMS 223/4 [Figure 2]. Briefly, serum samples were added to micro wells for assay [Figure 3]. TNF-α coating antibody is adsorbed onto micro wells. TNF-α present in the sample and standard binds to antibodies adsorbed to the micro wells. Biotin-conjugated anti-human TNF-α antibody is added, which binds to human TNF-α captured by the first antibody. Following incubation, unbound biotin-conjugated anti-human TNF-α antibody is removed by washing. Streptavidin (horse raddish peroxidase [HRP]) is added, which binds to the biotin-conjugated anti-human TNF-α antibody. Following incubation, unbound streptavidin HRP is removed by washing and substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of TNF-α present in the sample and standard [Figure 4]. The reaction is terminated by addition of phosphoric acid and absorbance is measured at 450 mm. A standard curve was prepared from seven TNF-α standard dilutions and concentration of TNF-α was determined by interpolation from the standard curve.
|Figure 4: Micro well plate after adding of reagents into appropriate wells|
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Descriptive statistical analysis
The clinical parameters and the total amount and concentration of TNF-α levels in both the study groups were expressed as mean standard deviation. Student's t-test is used to find a significant difference between two means. Kruskal Wallis test was applied to find out significant difference between the study groups. Measures of relationship between continuous variables were determined by Pearson "R" correlation.
| Results|| |
The clinical characteristics and TNF-α levels of both the groups included in this study are grouped in [Table 1]. The mean age of the control group (22.50 years with SD 1.75) is slightly higher than that of the experimental group (22.39 with SD 2.63) but the difference was however, not statistically significant. Thus the groups are not comparable with regard to age. The mean gestational age in weeks of the Group-II is considerably found to be lesser (33.33 with SD 1.78) compared to that of the Group-I (38.72 with SD 1.44) and the difference is statistically significant. The mean concentration of TNF-α in Group-I was 58.19 Pg/mL with SD 61.81 and in Group-II was 460.55 Pg/mL with SD 93.64. The mean concentration of TNF-α levels was significantly higher in Group-II when compared to Group-I, which was statistically significant. The mean sulcus depth in Group-I was 2.16 mm with SD 0.51.The mean pocket depth in Group-II was 6.05 mm with SD 1.69. The mean pocket depth was significantly higher in Group-II when compared with Group-I, which was statistically significant. The Mean Russel's index score in Group-I was 0.53 with SD of 0.19. The Mean Russel's index score in Group-II was 3.37 with SD of 1.43. The Mean Russel's index score was significantly higher in Group-II when compared to Group-I, which was statistically significant. When inter correlation of variables in both the groups were compared [Table 2], Positive correlation was found between TNF-α concentration and Gestational age (R = 0.81, P < 0.001), Russel's periodontal index (R = 0.74, P < 0.001), Pocket depth (R = 0.78, P < 0.001) which were statistically significant. Positive correlation was found between Pocket depth and Gestational age (R = 0.69, P < 0.001), Russel's periodontal index scores (R = 0.94, P < 0.001) which was statistically significantly. Positive correlation was found between Russel's periodontal index and Gestational age (R = 0.71, P < 0.001), which was statistically significant.
|Table 1: clinical characteristics and tumor necrosis factor-β levels of Group-I and II (mean±SD)|
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| Discussion|| |
Periodontitis can be considered as a continuous pathogenic and inflammatory challenge at a systemic level, due to the large epithelium surface that could be ulcerated in the periodontal pockets. This fact allows bacteria and their products to reach other parts of the organism, creating lesions at different levels.  Based on above evidence, the present study was undertaken to determine the influence of periodontitis in pregnant women.
Periodontitis is a gram-negative infection and it may have the potential to influence pregnancy outcome. During the second trimester of pregnancy, the proportion of gram-negative anaerobic bacteria in dental plaque increases with respect to aerobic bacteria. Fusobacterium nucleatum and other subspecies coming from the oral flora have been found in the amniotic fluid of women with pre-term births. The gram-negative bacteria associated with progressive disease can produce a variety of bioactive molecules that may directly affect the host. A microbial component LPS, can activate the macrophages and other cells to synthesize and secrete a wide spectrum of molecules, including cytokines IL-1ß, TNF-α, IL-6 and PGE 2 and matrix metalloproteinase's.If these components travel to the blood stream and cross over the placental barrier, the physiological levels of PGE 2 and TNF-α in the amniotic fluid may increase and induce a pre-term birth. 
In the present study, an increase of PGE 2 and TNF-α is observed as periodontitis increases in severity. Furthermore, it is known that amounts of PGE 2 and TNF-α increase progressively during pregnancy until they reach a threshold where they induce labor, cervical dilation and birth. Consequently, it is not surprising to find that increasing periodontal disease severity in the mother may result in decreasing birth weight and gestational age.
A study that evaluated relationship the between levels of PGE 2 and TNF-α in GCF and intra amniotic fluid in pregnant women with periodontal disease concluded that there is positive relationship between PGE 2 levels in the crevicular fluid and those in the intra amniotic fluid. 
Different studies on association between periodontitis and pre-term/low birth weight have used different criteria for the definition of periodontitis. (Offenbacher et al.,  Davenport et al.  ). Therefore, in the present study both probing pocket depth, Russel's periodontal index score, were used for the diagnosis of periodontitis requiring at least two sites with a probing depth ≥5.0 mm. ,,
In the present study, the comparison of Group-II parameters i.e., TNF-α, Pocket depth and Russel's periodontal index score mean values shows significant difference from Group-I.
In the present study, the mean concentration levels of TNF-α were higher in Group-II than Group-I (T = 15.21, P < 0.010) and a positive correlation between TNF-α levels and pocket depth, Correlation co-efficient (R = 0.78, P < 0.001) was statistically significant. This study also shows that steady increase in Russel's periodontal index score and pocket depth of 0.81, 4 mm respectively, indicates higher concentration of TNF-α levels. In the present study, with an increase in pocket depth, the estimated TNF-α concentration levels showed a steady increase. A 4 mm or above pocket depth indicates a higher concentration of TNF-α levels. These results are in agreement with Bogges et al.,  Lin et al.,  Casey et al. 
Bogges et al.  suggested that pre maturity risk may increase when focus is exposed to periodontal bacteria and inflammatory response is generated. As per the results of Lin et al.  it was found that the concentration of TNF-α was increased in maternal serum mice infected with Porphyromonas gingivalis, with a significant increase in dams with Fetal growth restriction (FGR) fetuses compared to levels in the mice having normal fetuses, which was statistically significant. 
In the present study mean Russel's periodontal index scores and mean pocket depth were higher in Group-II (T = 8.31, P < 0.001), (T = 15.21, P < 0.001). A Positive correlation was found between pocket depth, Russel's periodontal index and gestational age (R = 0.69, P < 0.001), (R = 0.71, P < 0.001) i.e., when pocket depth (beyond 4 mm or above) and Russel's periodontal index increase estimated gestational age shows a steady decline. These results are in accordance with Romero et al.,  Radani et al.,  Moss et al. 
Belkys et al.  suggested that periodontal disease in pregnant women could be a clinically significant risk factor for pre-term delivery. It was found that correlation analysis demonstrated a highly significant clinical relationship between more severe periodontal disease and low birth weight (R = 0.49, P < 0.01), a highly significant relationship was also clinically demonstrated between increasing periodontal severity and decreasing gestational age of the new-born babies (R = 0.59, P < 0.01). 
As per the results of Moss et al.  it was found that probing depths ≥4 mm and a concomitant greater frequencies were the most important periodontal parameters in connection with pre-term birth in the group of pregnant women (P = 0.001 or 3.76 univariate analysis). 
It has also been reported that these high levels of PGE 2 and TNF-α in GCF are correlated with the degree of Periodontal disease (PD) severity and can be used to determine the degree of PD activity. 
| Conclusion|| |
The following conclusions were drawn from the study
With the findings of present study, it can be concluded that poor periodontal health of the pregnant woman is a potential independent risk factor for pre-term gestation. The importance of this finding lies in the fact that poor periodontal health is a factor that is easily amenable to prevention. Future additional studies are needed to provide a more robust measure of attributable risk as well as a multicenter, randomized controlled clinical trail to measure specific periodontal pathogens in the fetal environment and the measurement of the resulting inflammatory mediator levels.
- TNF-α is present in serum samples of term and pre-term gestation subjects.
- With an increase in amount of periodontal destruction, there is a substantial increase in clinical parameters and TNF-α level in serum, which is inversely related to premature gestation.
- TNF-α concentrations correlated positively with the extent of periodontal destruction, Russel's periodontal index score and gestational age, from this it can be postulated that TNF-α is actively involved in progression of periodontal disease and also leads to pre-term gestation.
- This study shows the link, that TNF-α is a novel inflammatory mediator of periodontal disease progression, plays an important part in the initiation of labor and cause pre-term gestation. This provides a probable link between these two conditions providing evidence for above hypothesis.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]