The present study aimed to investigate the association between periodontal conditions and abnormal glucose tolerance (GT) in pregnant women with threatened premature labor (TPL). Fifteen normal pregnant women, 14 women with TPL and abnormal GT (TPL/abnormal GT), and 16 women with TPL and normal GT (TPL/normal GT) were enrolled. Periodontal parameters, including probing pocket depth and clinical attachment level, were examined. Detection of Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), Prevotella intermedia (P. intermedia), Aggregatibacter actinomycetemcomitans, Treponema denticola, and Tannerella forsythia (T. forsythia), in saliva, subgingival plaque, and chorionic tissues from women was performed by using polymerase chain reaction. There were no significant differences in periodontal parameters and detection rates of periodontal pathogens from oral samples between women with TPL/normal GT and TPL/abnormal GT. F. nucleatum, P. intermedia, and T. forsythia were detected in chorionic tissues of women with TPL/abnormal GT, whereas F. nucleatum and P. gingivalis were detected in chorionic tissues of those with TPL/normal GT. No periodontal pathogens were detected in the chorionic tissues of normal pregnant women. In conclusion, our data do not indicate that periodontal disease is a risk factor for abnormal GT in TPL. However, the presence of F. nucleatum, P. intermedia, and T. forsythia in chorionic tissues in women with TPL may be involved in abnormal GT.
Periodontal disease is a chronic inflammatory disease caused by multiple periodontopathic bacteria including Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), Prevotella intermedia (P. intermedia), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Treponema denticola (T. denticola), and Tannerella forsythia (T. forsythia), resulting in breakdown of the tooth-supporting apparatus and loss of teeth. Maternal periodontitis is modestly, but independently, associated with adverse pregnant outcomes1,2). Many studies have reported an association between periodontal disease and preterm low birth weight since the first report by Offenbacher et al. in 19963). We have previously shown that the periodontal conditions of Japanese pregnant women with a diagnosis of threatened premature labor (TPL) are worse than those of normal pregnant women4). Additionally, P. gingivalis and F. nucleatum are found in chorionic tissues of pregnant women who are hospitalized because of a high risk of pregnancy, such as TPL, placenta previa, and multiple pregnancies, but not in chorionic tissues of normal pregnant women5,6). Pregnant women with TPL are generally treated with tocolytic agents to prevent premature uterine contractions. Ritodrine, one of the tocolytic agents for treatment of TPL, is a β2-adrenergic stimulant and inhibits myometrial contractions7,8). Ritodrine can affect glucose homeostasis9). Therefore, ritodrine may cause hyperglycemia in some women with TPL10). Maternal hyperglycemia during pregnancy is associated with increased rates of hypertension, preeclampsia, cesarean delivery, diabetes later in life in the mother, macrosomia, neonatal hypoglycemia, and shoulder dystocia in fetuses11,12).
Gestational diabetes mellitus (GDM), which shows abnormal glucose tolerance during pregnancy, is defined as a type of diabetes mellitus (DM) that is first diagnosed during pregnancy13). Risk factors for development of GDM include obesity, previous GDM, and a family history of DM14). Recently, some reports have indicated that GDM is related to the maternal inflammatory environment and abnormality in the placenta15-17). The association between periodontal disease and GDM has been investigated for the past 10years. Periodontal conditions of women with GDM are significantly worse than those of women without GDM18,19). Moreover, some reports have demonstrated that the detection rate of periodontal pathogens in oral samples and levels of inflammatory mediators in gingival crevicular fluid of women with GDM are significantly higher than those of normal pregnant women20,21). However, other reports have concluded that there is no significant association between periodontal disease and GDM22,23). Dasanayake et al. reported that there was no significant difference in clinical periodontal status between women with GDM and those without GDM, even though levels of T. forsythia in the vagina and serum C-reactive protein (CRP) levels in women with GDM were significantly higher than those in women without GDM24). Therefore, the correlation between periodontal disease and GDM is controversial. Consequently, additional studies to prove the association between periodontal disease and abnormal glucose tolerance during pregnancy are required.
The present study aimed to investigate the association between periodontal conditions and abnormal glucose tolerance in women with TPL.
The study protocol was approved by the Human Ethical Committee of Kagoshima City Hospital (H19 No8) and the ethical standards of the Kagoshima University Medical and Dental Hospital Human Ethical Committee (No. 20-18).
ParticipantsHospitalized pregnant women who were diagnosed with TPL in the Department of Obstetrics and Gynecology, Kagoshima City Hospital and normal pregnant women in the Women's Hospital Aiiku were selected. Blood glucose levels of women with TPL at fasting and 2 h after eating were measured. Women in whom glucose levels were greater than 120 mg/dL one or more times were diagnosed as having abnormal glucose tolerance by obstetric doctors. Exclusion criteria were a history of DM or GDM, use of corticosteroids and antimicrobials during pregnancy, periodontal therapy during pregnancy, viral infection, and autoimmune disease. Written informed consent was obtained from all of the subjects.
Oral examinationsOral examinations were performed in the women during their second trimester by one periodontist (K.H-N). Periodontal parameters, including the plaque index (PlI)25), gingival index (GI)26), clinical attachment level (CAL), probing pocket depth (PPD), and bleeding on probing (BOP) were examined at four sites (mesial, distal, buccal, and lingual/ palatal) per tooth either at the teeth on the right side of the maxilla and left side of the mandible or at the teeth on the left side of the maxilla and right side of the mandible. The presence of two or more teeth showing PPD of ≥4 mm and CAL of ≥3 mm at the same site was defined as periodontitis, based on modification of the definition previously described by Lopez et al.27). Patients who were not diagnosed with periodontitis and had BOP were diagnosed with gingivitis.
Bacterial examinationsSubgingival plaque samples were obtained by inserting a sterilized paper point for 15 s into pockets of sites with inflammatory signs, including gingival redness and swelling. Saliva samples were collected using Salivettes (Sarstedt, Nümbrecht, Germany) before a periodontal examination. Part of chorionic tissues obtained by obstetric doctors after delivery were immediately fixed in 4% paraformaldehyde and then embedded in paraffin. Total DNA was purified from subgingival plaque, saliva and chorionic tissue samples using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. Polymerase chain reaction (PCR) was carried out using the Taq PCR Core Kit (Qiagen) to detect P. gingivalis, F. nucleatum, P. intermedia, A. actinomycetemcomitans, T. denticola, and T. forsythia, in the DNA samples. The primer sequences, GenBank accession numbers, and predicted sizes of the PCR products used for PCR detection of the bacteria are shown in Table 128,29). PCR amplification was performed by 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. The PCR products were separated by 2% agarose gel electrophoresis, stained with ethidium bromide, and visualized using an ultraviolet light transilluminator (ATTO Corporation, Tokyo, Japan).
Primer sequences used in PCR.
At the time of the oral examination, each participant was interviewed regarding height and weight measured before pregnancy, birth experience, smoking habits, and a history of DM, GDM, preterm birth (PB; birth before 37 weeks of gestation), and low birth weight (LBW; delivery of newborn with birth weight <2,500 g). Blood pressure on the day of the oral examination, medicine taken during the pregnancy, gestational age at delivery, and weight of the newborns were recorded after delivery. The gestational age was determined by measuring the crown rump length of the fetus with ultrasonographic tomography at the first trimester.
Statistical analysisNonparametric analysis of variance (Kruskal-Wallis test) was used to detect statistically significant differences between the three groups. When the Kruskal-Wallis test was significant, the Steel-Dwass post hoc test was performed for multiple comparisons. Differences in the ratios between the three groups were analyzed by Fisher's exact test. When Fisher's exact test was significant, the chi-square test was applied to analyze the differences between two groups. Results were considered statistically significant at P<0.05. Statistical analyses were performed using statistical software (BellCurve for Excel, Social Survey Research Information Co., Ltd., Tokyo, Japan). All results are expressed as mean±SD or the ratio.
Table 2 shows the clinical and anthropometric parameters in this study. Fifteen women with a normal pregnancy (normal pregnancy group) and 30 women with TPL were analyzed in this study. Women with TPL had intravenous infusion of ritodrine. Fourteen women with TPL were diagnosed with abnormal glucose tolerance (TPL/abnormal GT group). Sixteen women with TPL did not have abnormal glucose tolerance or any clinical signs of other obstetric diseases (TPL/normal GT group).
The body mass index (BMI) of women in the TPL/abnormal GT group was significantly higher than that of women in the TPL/normal GT group (P=0.0325). Blood glucose levels of 11 women in the TPL/abnormal GT group were controlled by injection of insulin, and three women in this group were treated with diet. Neonatal weight in the TPL/normal GT group was significantly lower than that in TPL/abnormal GT group or normal pregnancy group (TPL/normal GT vs TPL/abnormal GT groups, P=0.0316; TPL/normal GT vs normal pregnancy groups, P<0.001). Gestational weeks at delivery in the TPL/abnormal GT and TPL/normal GT groups were significantly lower than those in the normal pregnancy group (TPL/abnormal GT vs normal pregnancy groups, P=0.0047; TPL/normal GT vs normal pregnancy groups; P<0.001). The number of PBs and LBWs were nine and seven in 14 women in the TPL/abnormal GT group, and 14 and 12 in 16 women in the TPL/normal GT group, respectively. There were significant differences in the ratios of PB (P<0.001) and LBW (P<0.001) among the three groups. The ratios of PB and LBW in the TPL/abnormal GT or TPL/normal GT group were significantly higher than those in the normal pregnancy group (PB: TPL/abnormal GT vs normal pregnancy groups, P<0.001; TPL/normal GT vs normal pregnancy groups, P<0.001 and LBW: TPL/abnormal GT vs normal pregnancy groups, P=0.002; TPL/normal GT vs normal pregnancy groups, P<0.001). One pregnant woman in the TPL/abnormal GT group delivered a giant baby (neonatal weight ≥4000 g).
Comparison of clinical and anthropometric parameters
Table 3 shows the oral conditions of the participants in this study. Mean PPD, the percentage of PPD ≥4 mm, CAL, and the percentage of CAL ≥3 mm in the TPL/abnormal GT and TPL/normal GT groups were significantly higher than those in the normal pregnancy group (TPL/abnormal GT vs normal pregnancy groups: PPD, P=0.009; percentage of PPD ≥4 mm, P=0.0131; CAL, P<0.0001; percentage of CAL ≥3 mm, P=0.0011 and TPL/normal GT vs normal pregnancy groups: PPD, P=0.0042; percentage of PPD ≥4 mm, P=0.0316; CAL, P=0.0149; percentage of CAL ≥3 mm, P=0.0051). Mean BOP (P=0.0078), PlI (P=0.0175), and GI (P=0.0102) in the TPL/abnormal GT group were significantly higher than those in the normal pregnancy group. There were no significant differences in periodontal parameters between the TPL/abnormal GT and TPL/normal GT groups. The ratios of subjects with periodontitis in the TPL/abnormal GT and TPL/normal GT groups were 6/14 and 4/16, respectively. None of the normal pregnant women were diagnosed with periodontitis.
Comparison of oral conditions.
Table 4 shows the incidence of bacterial detection in subgingival plaque and saliva. Periodontal pathogens were detected in some oral samples from women in the TPL/abnormal GT group, TPL/normal GT group, and normal pregnancy group. There was a significant difference in the detection ratio of P. intermedia in saliva among the three groups (P=0.013). The detection ratio of P. intermedia in saliva of women in the TPL/abnormal GT (P=0.0006) or TPL/normal GT group (P=0.021) was significantly higher than that of women in the normal pregnancy group.
The detection ratios of periodontal pathogens in chorionic tissues are shown in Table 5. F. nucleatum, P. intermedia, and T. forsythia were detected in three chorionic tissues of 14 women in the TPL/abnormal GT group. The ratios of chorionic tissues in which P. gingivalis and F. nucleatum was detected in the TPL/normal GT group were 2/16 and 2/16, respectively. Periodontal pathogens were not detected in any chorionic tissues from normal pregnant women. Periodontal pathogens were detected in chorionic tissue in 6/14 women in the TPL/abnormal GT group and in 3/16 women in the TPL/normal GT group. There were significant differences in the detection ratios of P. intermedia (P=0.026) and T. forsythia (P=0.026), and for the ratio of any periodontal pathogen detected in chorionic tissue (P=0.012) among the three groups. The ratio of chorionic tissues in which any periodontal pathogen was detected in the TPL/abnormal GT group was significantly higher than that in the normal pregnancy group (P=0.004).
Table 6 shows the clinical profiles of nine women in whom periodontal pathogens were detected in chorionic tissues. One woman in the TPL/abnormal GT group in whom P. intermedia, F. nucleatum, and T. forsythia were detected in chorionic tissues delivered a giant baby.
comparison of detection of periodontal pathogens in oral samples.
Comparison of detection of periodontal pathogens in chorionic tissues.
Clinical profiles of the women in whom periodontal pathogens detected in chorionic tissues.
Periodontal conditions in women with GDM are worse than those in women without GDM18,19). Gogeneni et al. reported that GDM was significantly associated with increased levels of P. gingivalis, Filifactor alocis, and T. denticola in saliva20). In the present study, we could not show any significant differences in periodontal conditions and detection rates of periodontal pathogens from oral samples between women in the TPL/normal GT and TPL/abnormal GT groups. In our study, as soon as women with TPL were diagnosed with abnormal GT, their blood glucose levels were controlled by medical treatment. Therefore, hyperglycemia in women in the TPL/abnormal GT group might not have been high enough to affect their periodontal condition. Additionally, the periodontal condition of women with GDM reported by Gogeneni et al.20) (mean BOP: 70.3% and mean PPD: 2.3 mm) was worse than that of women in the TPL/abnormal GT group in our study (mean BOP: 29.7% and mean PPD: 2.1 mm). These findings may explain why we did not find any significant differences in the detection ratios of periodontal pathogens from oral samples between the TPL/normal GT and TPL/abnormal GT groups. In the present study, the periodontal condition of women in the TPL/normal GT or TPL/abnormal GT group was significantly worse than that of women in the normal pregnancy group, which is consistent with our previous reports that worsening periodontal status is associated with TPL4,6). TPL with a poor periodontal condition might have masked an association between periodontal disease and abnormal GT in the present study. It has been reported that higher pre-pregnancy BMI is associated with a higher prevalence of GDM30). In the present study, the mean BMI before pregnancy in women in the TPL/abnormal GT group (25.4±6.7) was significantly higher than that of women in the TPL/normal GT group (19.3±4.5). This finding suggests that the risk of pathogenesis of abnormal glucose tolerance in this study might be associated with a higher BMI before pregnancy, but not periodontal disease. Further studies focusing on the severity of periodontal disease in women with GDM are required.
In this study, F. nucleatum, P. intermedia, and T. forsythia were detected in chorionic tissues of the TPL/abnormal GT group, whereas F. nucleatum and P. gingivalis were detected in chorionic tissues of the TPL/normal GT group. No periodontal pathogens were detected in chorionic tissues of normal pregnant women. Additionally, there were significant differences in the ratios of any periodontal pathogen detected in chorionic tissue among the three groups. The number of periodontal pathogens (F. nucleatum, P. intermedia, and T. forsythia) in chorionic tissue from women in the TPL/abnormal GT group tended to higher than that from women in the TPL/normal GT group. Women with GDM have higher levels of T. forsythia in the vagina compared with women without GDM, although Campylobacter rectus, F. nucleatum, T. forsythia, P. gingivalis, and T. denticola have been detected in the vagina and cervical secretions in pregnant women with/without GDM24). Miranda et.al. showed that there is an association between higher levels of T. forsythia, F. nucleatum, and P. intermedia in the oral cavity and poor glycemic control in patients with DM31). Therefore, F. nucleatum, P. intermedia, and T. forsythia might be involved in abnormal glucose tolerance. Feng et al. suggested that activation of the toll-like receptor (TLR) 4-Myd88-NFkB pathway is related to upregulation of insulin resistance and higher maternal hyperglycemia32). The TLR4/Myd88/NFκB pathway is thought to be one of pathways activated by periodontal pathogens33). Some reports have indicated that GDM is related to the maternal inflammatory environment and abnormality in the placenta15-17). Our results and previous findings suggest the following: (i) some types of periodontal pathogens are more likely to invade and stay in the intrauterine tissue of women with abnormal glucose tolerance; and (ii) inflammatory reactions that are enhanced by periodontal pathogens in chorionic tissue affect abnormal glucose tolerance. However, the number of chorionic tissues that were analyzed was small and there was a small number of women in whom periodontal pathogens were detected in this study. Additionally, only two parts of chorionic tissues could be analyzed. We could not perform quantitative analysis of periodontal pathogens in chorionic tissues and histological analysis of chorionic tissues focusing on inflammatory reactions because of the limitation of sampling of chorionic tissues. To investigate the effect of periodontal pathogens in chorionic tissues on pregnancy, large-scale in vivo and in vitro studies are required.
There are two possible pathways through which periodontal pathogens translocate to chorionic tissues: (i) bacteria in the oral cavity migrate to the chorionic tissues as a result of homogenous spread; and (ii) bacteria in the vagina and cervix spread to the uterus. Han et al. reported that F. nucleatum translocates from the mother's mouth to the placenta and fetus, and causes acute inflammation and stillbirth34). Furthermore, intravenous injection of F. nucleatum and P. gingivalis to pregnant mice are transmitted to the placenta and cause adverse pregnancy outcomes35,36). Bacteremia occurs after chewing and tooth brushing in individuals with gingival inflammation37). Some periodontal pathogens that are detected in atherosclerotic plaques might be derived from the oral cavity through bacteremia38,39). Therefore, the source of periodontal pathogens that were detected in chorionic tissues in this study might have been the oral cavity. However, in a few subjects, periodontal pathogens that were detected in chorionic tissues were not detected in oral samples. Therefore, we could not exclude the possibility that bacteria in chorionic tissues came from the vagina and cervix because we could not analyze vaginal and cervical samples. Further studies focusing on the dissemination of periodontal pathogens in chorionic tissues are required.
In this study, neonatal weight in the TPL/normal GT group was significantly lower than that in the TPL/abnormal GT group or normal pregnancy group. Additionally, abnormal pregnant outcomes of the TPL/abnormal GT group tended to be lower compared with those of the TPL/normal GT group. There are several risk factors for PB and LBW, including age, race, smoking, alcohol, experience of delivery, and a history of PB and LBW40,41). In this study, there were no significant differences in age, smoking, experience of delivery, and a history of PB and LBW among the three groups. However, the ratios of birth experience and a history of PB and LBW in the TPL/abnormal GT group tended to be lower than those in the TPL/normal GT group. These findings may be due to lower abnormal pregnant outcomes in the TPL/abnormal GT group.
Our data do not indicate that periodontal disease is one of the risk factors for abnormal glucose tolerance in TPL women. However, this study shows the following. (i) The species of periodontal pathogens detected in chorionic tissues are different between women with TPL with normal glucose tolerance and those with TPL without glucose tolerance. (ii) The number of periodontal pathogens (F. nucleatum, P. intermedia, and T. forsythia) in chorionic tissue from women with abnormal glucose tolerance tends to be higher than that from women without abnormal glucose tolerance. These results suggest that F. nucleatum, P. intermedia, and T. forsythia s in chorionic tissues in women with TPL may be involved in abnormal GT. To investigate the association between periodontal disease and abnormal glucose tolerance during pregnancy, further clinical, large-scale studies of pregnant women without obstetric diseases other than GDM are required.
This study was partly supported by a Grant-in-Aid (23792483 and 26463139) for Scientific Research from the Japan Society for the Promotion of Science.
The authors have no conflicts of interest directly relevant to the content of this article.
