TITLE: 
Comparative evaluation of subgingival plaque microflora in pregnant and non-pregnant women: A clinical and microbiologic study



ABSTRACT.CONTEXT:
Gingival changes in pregnancy have been attributed to changes in the subgingival biofilm related to hormonal variations.

ABSTRACT.AIMS:
To evaluate the subgingival plaque microflora in pregnant and nonpregnant women to determine if pregnancy induces any alterations in the subgingival plaque and to associate these changes with changes in periodontal status.

ABSTRACT.SETTINGS AND DESIGN:
Thirty pregnant and 10 nonpregnant women within the age group of 20-35 years having a probing pocket depth (PPD) of 3-4 mm were included in the study. The pregnant women were equally divided into 3 groups of 10, each belonging to I, II, and III trimester.

ABSTRACT.MATERIALS AND METHODS:
Plaque index, gingival index, PPD, and microbiologic evaluation for specific bacterial counts for Prevotella intermedia, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Parvimonas micra, and Fusobacterium nucleatum were carried out for all subjects.

ABSTRACT.STATISTICAL ANALYSIS:
Mann–Whitney U test.

ABSTRACT.RESULTS:
Increase in gingival inflammation was observed in II and III trimester as compared with I trimester and control. Plaque scores did not show any significant difference between pregnant and nonpregnant women. Specific bacterial examination revealed an increase in proportion of P. intermedia in pregnant women of both II and III trimester as compared with I trimester and nonpregnant women.

ABSTRACT.CONCLUSIONS:
A definite increase in proportions of P. intermedia occurs in subgingival plaque microflora in pregnancy that may be responsible for the exaggerated gingival response.

BODY.INTRODUCTION:
Gingival changes in pregnancy were described as early as 1898 even before much knowledge about hormonal changes was available.[1] Loe reported a 100% incidence of gingivitis in a group of pregnant women.[2] Many studies have suggested that gingival changes seen during pregnancy are a result of increased levels of progesterone and its effect on microvasculature.[3] A number of investigators have demonstrated that the hormonal influence on the immune system contributes significantly to the etiopathogenesis of the condition.[4] There is increase in gingivitis independent of a change in clinically detectable plaque mass, lead up to the suggestion that altered steroid levels increase the inflammatory response to given plaque scores. One of the widely accepted hypotheses is the change in subgingival biofilm related to hormonal variations. Many investigators have suggested that pregnancy gingivitis is associated with an increase in the numbers of Prevotella intermedia. The present study was undertaken to monitor the gingival crevice microbial flora in pregnant and non-pregnant women to determine if gestation induces alteration in subgingival microflora and the clinical changes associated with it.

BODY.MATERIALS AND METHODS:
Patients in the inpatient and outpatient department of Lady Goshen Hospital and the patients visiting the Periodontics department of A.B. Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India, were taken for the study. Informed consent was obtained from all the participants and the study was approved by the ethical committee of the institution. Thirty pregnant and 10 non-pregnant women within the age group of 20-35 years having a probing pocket depth (PPD) of 3-4 mm were included in the study. The 30 pregnant women were equally divided into 3 groups of 10, each belonging to I, II, and III trimesters. Non-pregnant women (control group) were asked to record the dates of their menstrual period so as to standardize the time of examination. The groups were designated as group I (control group), group II (I trimester), group III (II trimester), and group IV (III trimester). Patients were selected for this study after they satisfied the following criteria:

No history of any systemic disease
No history of dental treatment during the past 6 months, including oral prophylaxis
No history of antibiotic drug therapy or intake during the past 6 weeks
No history of oral contraceptive use (for control group).


Patients with very poor oral hygiene and heavy calculus formers as well as smokers were excluded from the study.
BODY.CLINICAL EXAMINATION:
For each patient, full mouth plaque index (PI)[5] and gingival index (GI)[6] were recorded at 4 sites per tooth (mesial, distal, buccal, and lingual). PPD was recorded using a William's periodontal probe.

BODY.MICROBIOLOGIC ANALYSIS:
BODY.BACTERIAL SAMPLING:
Supragingival plaque was removed from the sites using sterile curettes in order to avoid contamination. Subgingival plaque samples were then collected from the 3-5 mm pockets in each participant using sterile paper points placed in the sulcus for 30 s. They were transferred into the Robertson's cooked meat medium (RCM) for culturing and a smear was taken on glass slide for gram staining.

BODY.MICROBIOLOGIC PROCEDURES:
The paper points were transported to microbiologic laboratory in preheated RCM soon after collection. The isolation of anaerobes was done in the laboratory.

BODY.SPECIFIC BACTERIAL EXAMINATION:
Fusobacterium nucleatum, Aggregatibacter actinomy-cetemcomitans, Parvimonas micra, P. intermedia, and Porphyromonas gingivalis were the selected periodontal pathogens examined.


BODY.ISOLATION OF ANAEROBES:
The transport media with paper points was kept in a Vortex mixer for 5-10 min for homogenization of plaque sample. The collected plaque samples were directly inoculated into the following selective and nonselective media:

Brain heart infusion blood agar supplemented with cysteine, yeast extract, Vit K, and hemin
Laked blood agar for P. gingivalis and P. intermedia
Neomycin blood agar (selective media for all anaerobes)
Tryptic soy agar supplemented with bacitracin, vancomycin, horse serum, and yeast extract (TSBV), which is the selective media for A. actinomycetemcomitans.


The plates were incubated anaerobically in an anaerobic jar (Himedia Laboratories, Mumbai) and incubated at 37°C for 48-72 h. The tryptic soy agar supplemented with bacitracin and vancomycin for A. actinomycetemcomitans was kept in carbon dioxide incubator at 37°C for 48-72 h with carbon dioxide flow at 5-10%. The isolates were further divided up to species level by gram staining and biochemical characteristics. A semi-quantitative method (Mackie–McCartney) was used to quantitate the bacteria in the plaque.[7]
The standard loop of 4 mm diameter, which can hold 0.01 mm of RCM was used and colony-forming units (CFU) were counted. A count of ++++, roughly correlates with ≥105 CFU. Organisms were identified depending on the biochemical reactions.

BODY.STATISTICAL ANALYSIS:
Mann–Whitney U test was done for comparing the plaque score and gingival score of the various groups as well as for intergroup comparisons of various microorganisms.


BODY.RESULTS:
BODY.CLINICAL FINDINGS:
Table 1 shows the mean ± standard deviation of plaque index (PI) scores, gingival scores (GI), and PPDs in all the 4 groups. The intergroup comparison of PI scores and PPD showed no significant difference (data not shown).
Table 1Mean±standard deviation of plaque scores, gingival scores, and probing pocket depths
Intergroup comparison of gingival index (GI) scores showed that the GI scores were increased in the II trimester and III trimester patients when compared with I trimester patients and non-pregnant patients, which were highly significant There was no significant difference in the GI scores between non-pregnant and I trimester patients as well as between II trimester and III trimester patients [Table 2].
Table 2Intergroup comparison of gingival index score using Mann–Whitney U test
No appreciable gingival enlargement was present in any of the subjects.

BODY.MICROBIOLOGIC FINDINGS:
The frequency of detection of the periodontal pathogens (percentage and absolute number related to group) is given in Table 3. The bacterial colonies were graded as ++++, +++, ++, and + according to the number of CFU found in culture plates. A count of ++++ roughly correlates with ≥105 CFU, +++ denotes ≥104 CFU, ++ denotes ≥103 CFU, and + denotes ≥102 CFU. The intergroup comparison of P. gingivalis, P. intermedia, A. actinomycetumcomitans, P. micra, and F. nucleatum using Mann–Whitney U test is shown in Table 4.
Table 3Frequency of detection of periodontal pathogens (percentage and absolute number related to group)
Table 4Intergroup comparison of Porphyromonas gingivalis, Prevotella intermedia, Aggregatibacter actinomycetumcomitans, Parvimonas micra, and Fusobacterium nucleatum using Mann–Whitney U test
BODY.PREVOTELLA INTERMEDIA:
Comparison of P. intermedia between Group I & III, Group I & IV, and Group II & IV showed statistical significance (P < 0.05). Comparison of Group II and Group III was highly significant (P < 0.01). There was no statistical significance (P > 0.05) when comparison was made between Group I and Group II and between Group III and Group IV. In Group I (Control), P. intermedia was isolated from 2 samples among which one sample had +++ growth and the other one had ++ growth. Rest of the 8 samples did not show the presence of the organism in the culture. In Group II (I trimester), P. intermedia was isolated from one sample which had +++ growth. Remaining 9 samples did not show the presence of the organism in the culture. In Group III (II trimester) P. intermedia was isolated from 8 samples of which, 3 had ++++ growth, 1 sample had +++ growth, and 4 had ++ growth. Remaining 2 samples did not show the presence of the organism in the culture. In Group IV (III trimester) P. intermedia was isolated from 7 samples of which 3 had ++++ growth, 3 had +++ growth, and 1 had + growth. Remaining 3 samples did not show the presence of organism in the culture.

BODY.PORPHYROMONAS GINGIVALIS:
In Group I (Control) P. gingivalis was isolated from 2 samples among which one had +++ growth and one had ++ growth. Rest of the 8 samples did not show the presence of the organism in the culture. In Group II P. gingivalis was isolated from 3 samples among which 2 had +++ growth and 1 had, + growth. Rest of the 7 samples did not show the presence of the organism in the culture. In Group III P. gingivalis was isolated from 2 samples, among which 2 samples had +++ growth. Rest of the 8 samples did not show the presence of the organism in the culture. In Group IV, P. gingivalis was isolated from 2 samples, among which 2 samples had ++++ growth. Rest of the 8 samples did not show the presence of the organism in the culture.

BODY.AGGREGATIBACTER ACTINOMYCETUMCOMITANS:
The organism was isolated from 3 samples in each group. In Group I, 1 sample had +++ growth and 2 samples had + growth. In Group II, 2 samples had +++ growth and 1 sample had + growth. In Group III, 2 samples had ++ growth and 1 sample had +++ growth. In Group IV, 2 samples had ++++ growth and 1 sample had ++ growth. Remaining of the 7 samples in each group did not show the presence of organism in the culture.

BODY.PARVIMONAS MICRA:
In Group I, Parvimonas was isolated from 7 samples, among which 2 samples had ++++ growth, 1 sample had +++ growth, and 4 samples had ++ growth. Rest of the 3 samples did not show the presence of the organism in the culture. In Group II, Parvimonas was isolated from 5 samples, among which 4 samples had +++ growth and 1 sample had ++++ growth. Rest of the 5 samples did not show the presence of organism in the culture. In Group III, Parvimonas was isolated from 6 samples, among which 1 sample had +++ growth and 1 sample had ++++ growth. Rest of the 4 samples did not show the presence of the organism in the culture. In Group IV, Parvimonas was isolated from 6 samples, among which 2 samples had ++++ growth, 3 samples had +++ growth, and 1 sample had + growth. Rest of the 4 samples did not show the presence of organism in the culture.

BODY.FUSOBACTERIUM NUCLEATUM:
In Group I, F. nucleatum was isolated from 2 samples, among which 1 sample had ++++ growth and the other sample had +++ growth. Remaining of the 8 samples did not show the presence of the organism in the culture. In Group II, F. nucleatum was isolated from 1 sample only which had +++ growth. Remaining of the 9 samples did not show the presence of the organism in the culture.
In Group III, F. nucleatum was isolated from 2 samples, among which both samples had +++ growth. Remaining of the 8 samples did not show the presence of the organism in the culture. In Group IV, F. nucleatum was isolated from 3 samples, among which 2 samples had + growth and the other 1 had ++++ growth. Remaining of the 7 samples did not show the presence of the organism in the culture.



BODY.DISCUSSION:
Periodontal tissue destruction is governed by various host factors, such as nutrition, hormones, immune mechanisms, and so on. Several investigators have shown that hormonal imbalance seen during pregnancy can adversely affect the gingival tissues, which usually manifest in the form of an exaggerated inflammation.[8] Increase in gingival inflammation during pregnancy and resolution following parturition made most of the investigators consider the alterations in the progesterone and estrogen levels during pregnancy to be the major etiologic factor. The present study was designed to evaluate the subgingival plaque microbial flora in pregnant and non-pregnant women, to determine whether pregnancy induces alterations in the subgingival microflora and if these changes can be associated with changes in periodontal status. The result of the present study which included 30 pregnant (10 each belonging to I, II, and III trimester) and 10 non-pregnant women showed a highly significant increase in GI score in II and III trimesters as compared with the I trimester and non-pregnant women (P < 0.01). The comparison of PI scores of these individuals did not show any significant difference (P > 0.05). The pocket depth in pregnant and non-pregnant women did not show any significant difference (P > 0.05). These results are in general agreement with previous reports by Kornman and Loeshe,[9] Jensen et al.,[10] and Raber-Durlacher et al.[11] Kornman and Loesche reported a significant increase in gingivitis during pregnancy with peak level during II trimester of pregnancy.[9] In the present study the difference in GI score was marginal between the individuals belonging to the II and III trimester of pregnancy with slight increase during the II trimester. This may be possibly due to the differences in the study design. In the present study, 10 individuals each belonging to the I, II, and III trimesters of pregnancy were evaluated. On the other hand, Kornman and Loesche monitored 20 pregnant women throughout their period of pregnancy.[12] All through their study they did not find any fluctuation in the PI scores. The present study also showed no significant differences in PI scores during the I, II, and III trimesters. Jensen et al. in their study showed a significant increase in the GI score of pregnant women as compared with non-pregnant women.[10] They also showed increased gingival crevicular fluid secretion during pregnancy. They did not find any significant differences in pocket depth between pregnant and non-pregnant women. In the present study also, no significant difference in pocket depth in pregnant and non-pregnant groups could be observed. Raber-Durlacher et al., in their study assessed the clinical, endocrinologic, and microbiologic aspects of experimental gingivitis during pregnancy.[11] They found increased swelling, redness, and bleeding on probing during pregnancy. They suggested that as a result of dental plaque accumulation, gingival inflammation develops superimposed on pregnancy-associated physiologic alterations.
Jonsson et al. determined the relationships between periodontal health, salivary steroids, and Bacteroides intermedius in males, pregnant and non-pregnant women.[13] They reported no significant difference between these groups in clinical and bacteriologic parameters. Microbiologic evaluation of subgingival plaque samples in this study revealed that, proportions of P. intermedia was increased in pregnant women belonging to both II and III trimesters as compared with I trimester and non-pregnant women. P. intermedia were prevalent in II and III trimester of pregnancy. However, proportions of other anaerobes included in the study, such as P. gingivalis, F. nucleatum, A. actinomycetemcomitans, and P. micra did not show any significant difference among pregnant and non-pregnant women. These results can be compared with previous reports by Kornman and Loesche,[9] Jensen et al.,[10] and Raber-Durlacher et al.[11] who also reported an increase in P. intermedia during pregnancy.
Kornman and Loesche studied the subgingival microbial flora during pregnancy.[9] Organisms which they included in the study were Actinomyces naeslundi, Actinomyces odontolyticus, Actinomyces viscosus, B. melaninogenicus, intermedius, Bacteroides sub sp. melaninogenicus, Bacteroides assacharolyticus, Bacteroides ocharaceus, F. nucleatum, and Streptococcus sanguis. Their results showed an increase in the proportions of B. melaninogenicus sub sp. intermedius (currently P. intermedia) during the II trimester. In the present study, levels of P. intermedia did not differ significantly between the individuals who were in their II and III trimesters of pregnancy. But highly significant difference was observed between I and II trimesters. Jensen et al. evaluated the periodontal status of 3 groups of women: pregnant, non-pregnant, and women taking contraceptives, clinically and microbiologically, especially for the percentage of Fusobacterium species and Bacteroides species.[10] Their results showed 55-fold increases in the proportions by Bacteroides species in pregnant women and 16-fold in women taking oral contraceptives as compared with non-pregnant women. They suggested that increased female sex hormones substituting for naphthaquinone requirement of certain Bacteroides species were most likely responsible for their increased proportions during pregnancy. Raber-Durlacher et al.[11] also reported an increase in the mean proportions of P. intermedia in subgingival plaque during pregnancy, unlike the reports by Jonsson et al. who did not find any difference in the proportions of P. intermedia in plaque samples of pregnant and non-pregnant women.[13] The present study showed definite increase in proportions of P. intermedia in II and III trimesters as compared with I trimester and non-pregnant women. Carrillo-de-Albornoz et al. investigated the influence of hormonal variations on the subgingival biofilm during pregnancy.[14] They found that P. gingivalis-positive patients presented an increase in gingival inflammation unrelated to plaque. Correlations were also found between maternal hormone levels and P. gingivalis and P. intermedia.
The increased levels of female sex hormones, that is, progesterone and estrogen present during pregnancy seems to be associated with increased GI score and increased proportions of P. intermedia in II and III trimesters. This is supported by findings that both the hormones substituted for naphthaquinone (menadione), which is an essential nutrient for P. intermedia and it has also been shown that steroid uptake of plaque sample during pregnancy is increased. Since P. intermedia is an important periodontal pathogen, an increase in their proportions in subgingival plaque during II and III trimesters of pregnancy has to be considered as a significant change.
Finally, this study has got certain limitations in terms of sample size and lack of longitudinal evaluation, which would have more clearly depicted the temporal association between pregnancy and gingival changes. Also, biochemical tests were not performed to assess the possible hormonal variations.

BODY.CONCLUSION:
On the basis of clinical and microbiologic findings of the present study, definite increase in the proportions of P. intermedia occurs in the subgingival plaque microflora during pregnancy. The exaggerated gingival response during pregnancy may be associated with increase in the prevalence of P. intermedia in the subgingival plaque. Further studies could be conducted to correlate the concentration of female sex hormones in non-pregnant and pregnant women in all the 3 trimesters with the number of microorganisms and the GI score in pregnancy.

Question: Compared to control, probing pocket depth (PPD) of 3-4 mm - I trimester what was the result of probing pocket depth (PPD) of 3-4 mm - II and III trimester on Plaque scores?
Evidence: Increase in gingival inflammation was observed in II and III trimester as compared with I trimester and control
Answer: no significant difference