Oral microflora and pregnancy: a systematic review and meta analysis

Oral microflora and pregnancy: a systematic review and meta analysis

                OPEN              Oral microflora and pregnancy:
                                  a systematic review
                                  and meta‑analysis
                                  Hoonji Jang1, Alexa Patoine1, Tong Tong Wu2, Daniel A. Castillo3 & Jin Xiao1,4*

                                  Understanding changes in oral flora during pregnancy, its association to maternal health, and its
                                  implications to birth outcomes is essential. We searched PubMed, Embase, Web of Science, and
                                  Cochrane Library in May 2020 (updated search in April and June 2021), and conducted a systematic
                                  review and meta-analyses to assess the followings: (1) oral microflora changes throughout pregnancy,
                                  (2) association between oral microorganisms during pregnancy and maternal oral/systemic conditions,
                                  and (3) implications of oral microorganisms during pregnancy on birth outcomes. From 3983 records,
                                  78 studies were included for qualitative assessment, and 13 studies were included in meta-analysis.
                                  The oral microflora remains relatively stable during pregnancy; however, pregnancy was associated
                                  with distinct composition/abundance of oral microorganisms when compared to postpartum/non-
                                  pregnant status. Oral microflora during pregnancy appears to be influenced by oral and systemic
                                  conditions (e.g. gestational diabetes mellitus, pre-eclampsia, etc.). Prenatal dental care reduced the
                                  carriage of oral pathogens (e.g. Streptococcus mutans). The Porphyromonas gingivalis in subgingival
                                  plaque was more abundant in women with preterm birth. Given the results from meta-analyses were
                                  inconclusive since limited studies reported outcomes on the same measuring scale, more future
                                  studies are needed to elucidate the association between pregnancy oral microbiota and maternal oral/
                                  systemic health and birth outcomes.

                                   Pregnancy is a unique physiological state, accompanied by temporary changes in women’s physical structure,
                                   hormone levels, metabolism and immune s­ ystems1,2. The changes during pregnancy are vital to maintaining the
                                  stable status of mother and fetus, however, some physiological, hormonal and dietary changes associated with
                                  pregnancy, in turn, alter the risk for oral diseases, such as periodontal disease and dental c­ aries3. The delicate and
                                   complex changes during pregnancy also affect the microbial composition of various body sites of the expectant
                                   ­mothers4, including the oral ­cavity2. The oral cavity is colonized with a complex and diverse microbiome of over
                                   700 commensals that have been identified in the Human Oral Microbiome Database (HOMD)5 and recently
                                   expanded HOMD (eHOMD), including bacterial and fungal ­species6. Given a balanced microbial flora helps to
                                   maintain stable oral and general health, alterations in the oral microbial community during pregnancy might
                                   impact maternal oral ­health7,8, birth ­outcomes9, and the infant’s oral ­health10. Therefore, understanding changes of
                                  oral flora during pregnancy, its association to maternal health, and its implications to birth outcomes is essential.
                                       First, despite the speculated associations between oral flora and oral diseases during pregnancy, two critical
                                  questions that remain to be answered are (1) what changes in the oral microbiota occur during pregnancy; (2)
                                  whether the changes are associated with increased risk for oral diseases during pregnancy. Studies that evalu-
                                  ated the stability of the oral microbiome during pregnancy revealed that the composition and diversity of oral
                                  microbiome components remained stable without significant ­change11,12. However, on the contrary, some studies
                                   reported that pregnant women experienced a significant increase in Streptococcus mutans, a well-known culprit
                                   for dental ­caries13,14. In addition, researchers also reported an increased level of periodontal pathogens, e.g.,
                                   Aggregatibacter actinomycetemcomitans, Porphyromona gingivalis and Prevotella intermedia, among pregnant
                                  ­women15–17. Nevertheless, comprehensive evaluations of available evidence are needed to provide conclusive
                                       Second, a clear understanding of the association between oral microorganisms and adverse birth outcomes
                                   conveys significant health implications. A systematic review from Daalderop et al., reported an association
                                   between periodontal disease and various adverse pregnancy ­outcomes18. Women who have periodontal diseases

                                   Eastman Institute for Oral Health, University of Rochester Medical Center, Rochester, NY, USA. 2Department of
                                  Biostatistics and Computational Biology, University of Rochester Medical Center, Rochester, USA. 3Miner Library,
                                  University of Rochester Medical Center, Rochester, NY, USA. 4Perinatal Oral Health, Eastman Institute for Oral
                                  Health, University of Rochester, 625 Elmwood Ave, Rochester 14620, USA. *email: jin_xiao@urmc.rochester.edu

Scientific Reports |   (2021) 11:16870                 | https://doi.org/10.1038/s41598-021-96495-1                                                     1

Oral microflora and pregnancy: a systematic review and meta analysis

                                            during pregnancy are at higher risk for delivering preterm and low birth-weight i­nfants19–21. In terms of oral
                                            microorganisms, researchers reported a higher level of P. gingivalis among women with preterm d        ­ eliveries22,23. A
                                            higher risk of preterm delivery was also observed among pregnant women with detection of periodontal anaer-
                                            obes in subgingival p ­ laque24. In contrast, Costa et al. reported that the risk of preterm birth is not correlated to
                                            an increased amount of periodontopathogenic b      ­ acteria25. Therefore, a thorough review of all available evidence
                                            on the topic of prenatal oral microorganisms and adverse birth outcomes is critical.
                                                Furthermore, maternal oral health is closely associated with children’s oral health, including maternal related-
                                            ness and vertical transmission of oral pathogens from mothers to ­infants26. Thus, in theory, reducing maternal
                                            oral pathogens during pregnancy is paramount, since it could potentially reduce or delay the colonization of
                                            oral pathogens in the infant’s oral cavity. Interestingly, although some s­ tudies27,28 demonstrated that expectant
                                            mothers who received atraumatic dental restorative treatment during pregnancy resulted in significant reductions
                                            of S. mutans carriage, and pregnant women who received periodontal treatment (scaling and root planning)
                                            had a lowered periodontal pathogen level, a study from Jaramillo et al., failed to indicate decreased periodontal
                                            bacteria in pregnant women following periodontal t­ reatment29.
                                                Therefore, this study aims to comprehensively review the literature on oral microorganisms and pregnancy.
                                            We are focusing on analyzing the evidence on the following subcategories: (1) oral microbial community changes
                                            throughout pregnancy, including changes of key oral pathogens, the abundance, and diversity of the oral fungal
                                            and bacterial community; (2) association between oral microorganisms during pregnancy and maternal oral/
                                            systemic diseases; (3) implications of oral microorganisms during pregnancy on adverse birth outcomes.

                                            This systematic review followed the PRISMA ­guidelines30, the protocol was registered for in the PROSPERO
                                            (CRD42021246545) (https://​www.​crd.​york.​ac.​uk/​prosp​ero/).

                                            Search methods. Database searches were conducted in May 2020 and updated in April and June 2021
                                            to identify published studies on changes in oral microbiome during pregnancy. A medical reference librarian
                                            (DAC) developed the search strategies and retrieved citations from the following databases: Medline via Pub-
                                            Med, Embase via embase.com, All databases (Web of Science Core Collection, BIOSIS Citation Index, Current
                                            Contents Connect, Data Citation Index, Derwent Innovation Index, KCI-Korean Journal Database, Medline,
                                            Russian Science Citation Index, SciELO Citation Index, and Zoological Record) via Web of Science, Cochrane
                                            Central Register of Controlled Trials via Cochrane Library. A combination of text words and controlled vocabu-
                                            lary terms were used (oral microbiota, oral health, bacterial diversity, pregnancy, periodontal pathogens, preg-
                                            nancy complication). See “ESM Appendix” for detailed search methods used.

                                            Inclusion and exclusion criteria. This systematic review included case–control studies, cross-sectional
                                            studies, retrospective and prospective cohort studies, randomized or non-randomized controlled trials that
                                            examined the changes of oral microorganisms in relation to pregnancy, oral diseases during pregnancy, adverse
                                            birth outcome and the effect of prenatal oral health care on oral microorganisms’ carriage. Two trained inde-
                                            pendent reviewers completed the article selection in accordance with the inclusion/exclusion criteria. Disagree-
                                            ments were resolved by consensus between the two reviewers or by the third reviewer.

                                            Inclusion criteria. Types of participants: women during reproductive age (pregnant and non-pregnant women).
                                                Types of intervention(s)/phenomena of interest: pregnancy.
                                                Types of comparisons:

                                            •   oral microbiota changes throughout pregnancy;
                                            •   oral microbiota profiling between pregnancy and non-pregnancy phases;
                                            •   oral microbiota changes following prenatal oral health care;
                                            •   association between oral microorganisms during pregnancy and adverse birth outcome;
                                            •   impact of systematic or oral health conditions on oral microbiota in pregnancy.

                                              Types of outcomes: detection and carriage of oral microorganisms, oral microbiota diversity and composition.
                                              Types of studies: case–control study; cross-sectional study; retrospective and prospective cohort study; ran-
                                            domized and non-randomized controlled trials.
                                              Types of statistical data: detection and carriage [colony forming unit (CFU)] of individual microorganisms;
                                            Confidence Intervals (CI); p values.

                                            Exclusion criteria. In vitro studies; animal studies; papers with abstract only; literature reviews; letters to the
                                            editor; editorials; patient handouts; case report or case series, and patents.

                                            Data extraction. Descriptive data, including clinical and methodological factors such as country of origin,
                                            study design, clinical sample source, measurement interval, age of subjects, outcome measures, and results from
                                            statistical analysis were obtained.

                                            Qualitative assessment and quantitative analysis. The quality of the selected articles was assessed
                                            depending on the types of studies. For randomized controlled trials, two methodological validities were used. (1)
                                            Cochrane Collaboration’s tool for assessing risk of bias in randomized t­ rials31. Articles were scaled for the follow-

          Scientific Reports |   (2021) 11:16870 |                https://doi.org/10.1038/s41598-021-96495-1                                                        2


                                                     Total identified records (n=3983)

                                                      PubMed (n=911)
                                                      Embase (n=1434)
                                                      Web of Science (n=1546)
                                                      Cochrane (n=91)
                                                      Manually added (n=1)

                                                                                                    Duplicate records removed (n=1821)

                                                        Records screened
                                                                                                    Met exclusion criteria (n=2050)
                                                                                                    Abstract not accessible (n=2)
                                                     Full-text articles assessed

                                                             for eligibility
                                                               (n=110)                              Full text not accessible (n=7)
                                                                                                    Study group not clearly defined (n=8)
                                                                                                    Sampling not meeting criteria (n=8)
                                                                                                    No oral microbial related data (n=5)
                                                        Studies included in                         Short communication (n=3)
                                                       qualitative synthesis                        Dataset published in duplicates(n=1)

                                                                                                    Records without the same outcome
                                                                                                    quantifiable unit were excluded (n=65)
                                               Studies included in quantitative
                                                 synthesis (meta-analysis)

                                            Figure 1.  Flow diagram of study identification. The 4-phase preferred reporting items for systematic reviews
                                            and meta-analyses (PRISMA) flow diagram was used to determine the number of studies identified, screened,
                                            eligible, and included in the systematic review and meta-analysis (http://​www.​prisma-​state​ment.​org).

                                            ing bias categories: selection bias, performance bias, detection bias, attrition bias, reporting bias, and other bias.
                                            (2) Adapted Downs and Black scoring that assesses the methodological quality of both randomized and non-
                                            randomized studies of health care i­nterventions32. A total score of 26 represents the highest study quality. For
                                            cohort and cross-sectional studies, a quality assessment tool for observational cohort and cross-sectional studies
                                            was ­used33. Additionally, ­GRADE34,35 was used to assess articles used clinical interventions during pregnancy.
                                                For the articles selected for quantitative analysis, the OpenMeta[Analyst] was used for meta-analysis (http://​
                                            cebm.b             ​ penme​ ta/). The 95% CI and p values were estimated using an unconditional generalized linear
                                                  ​ rown.e​ du/o
                                            mixed effects model with continuous random effects via DerSimonian–Laird method. Heterogeneity among
                                            the studies was evaluated using I2 statistics and tested using mean difference values. Forest plots were created
                                            to summarize the meta-analysis study results of mean difference of viable counts (converted to log value) of

                                            The literature analyses identified a total of 3983 records from database searches (3982) and manual additions (1).
                                            A total of 1821 duplicate references were removed. From the remaining 2162 records, 2050 were excluded after
                                            title and abstract screening. The remaining 110 studies proceeded to a full text review; 32 studies were eliminated
                                            based on the exclusion criteria and 78 articles were chosen for qualitative assessment (Fig. 1).

                                            Study characteristics. The characteristics of ­studies11–17,21–25,27–29,36–98 included in the qualitative review
                                            are summarized in Tables. A total of 78 studies are categorized into the following subgroups: 18 studies on oral
                                            microbial differences between pregnant and non-pregnant women in Table 114–17,36–49; 11 studies on oral micro-
                                            bial differences between pregnant stages in Table 211–13,50–57; 8 studies on oral microbial differences responding
                                            to prenatal dental treatment in Table 327–29,58–62; 16 studies on association between oral microorganisms during

Scientific Reports |             (2021) 11:16870 |               https://doi.org/10.1038/s41598-021-96495-1                                                     3


                              Country, study      Groups (no. of                      Measurement           Microorganisms      detection                              Quality
           Author (year)      design              subjects)        Sample source      interval              evaluated           methods         Study findings         assessment
                                                                                                                                                The subgingival flora
                                                                                                                                                evolved to a compo-
                                                                                                                                                sition that has more
                                                                                                                                                anaerobes as preg-
                                                                                                                                                nancy progressed
                                                                                                                                                The anaerobe/aer-
                                                                                                                                                obe ratio increased
                                                                                                                                                significantly at an
                                                                                                                                                early stage of preg-
                                                                                      Pregnant group                                            nancy and remained
                                                                                      T1: < 13 weeks GA     A. naeslundii, A.                   high until the third
                                                                                      Follow-ups:           odontolyticus,                      trimester
                                                                                      monthly after until   A. viscosus, B.                     Only B. melanino-
                                                  Pregnant (20)
           Kornman and        USA, prospective                                        delivery              asaccharolyticus,                   genicus ss. inter-
                                                  Non-pregnant     Subgingival plaque                                           Culturing                              Fair
           Loesche (1980)36   cohort                                                  Non-pregnant          P. intermedi),                      midius (currently
                                                                                      group                 B. ochraceus, F.                    P. intermedia) sig-
                                                                                      Monthly visit for     nucleatum, S.                       nificantly increased
                                                                                      4 consecutive         sanguis                             during pregnancy
                                                                                      months                                                    compared between
                                                                                                                                                In the 2nd trimester,
                                                                                                                                                the anaerobe/aer-
                                                                                                                                                obe ratio and the
                                                                                                                                                proportions of B.
                                                                                                                                                melaninogenicus ss.
                                                                                                                                                intermedius different
                                                                                                                                                significantly from the
                                                                                                                                                non-pregnant group
                                                                                                                                                Significant differ-
                                                                                                                                                ences in proportions
                                                                                                                                                of Actinomyces were
                                                                                                                                                found between
                                                                                                            P. intermedia,                      pregnant and non-
                                                  Pregnant (19)
                                                                                      Pregnant group        Black-pigmented                     pregnant group and
           Muramatsu and      Japan, cross-       Non-pregnant     Supragingival
                                                                                      One time point        anaerobic rods,     Culturing       between 2nd tri-       Fair
           Takaesu (1994)37   sectional           (12)             plaque, saliva
                                                                                      during pregnancy      Actinomyces                         mester pregnant and
                                                  Postpartum (8)
                                                                                                            streptococcus                       postpartum group
                                                                                                                                                No statistically
                                                                                                                                                significant changes
                                                                                                                                                in proportions of P.
                                                                                                                                                Positive correlations
                                                                                                                                                between bacteria
                                                                                                                                                carriage and estra-
                                                                                                                                                diol concentrations
                                                                                                                                                C. rectus (r = 0.443,
                                                                                                            C. rectus, P. gin-                  p = 0.006)
                                                  Pregnant (22)                       Pregnant group        givalis, A. actino-                 P. gingivalis
           Yokoyama et al.    Japan, cross-                        Unstimulated
                                                  Non-pregnant                        27.4 ± 5.1 weeks      mycetemcomitans, Real-time PCR      (r = 0.468, p = 0.028) Fair
           (2008)38           sectional                            whole saliva
                                                  (15)                                GA                    F. nucleatum, P.                    F. nucleatum
                                                                                                            intermedia                          (r = 0.452, p = 0.035)
                                                                                                                                                Positive correlations
                                                                                                                                                between C. rectus
                                                                                                                                                levels and sites of
                                                                                                                                                4 mm-pocket depth
                                                                                                                                                (r = 0.568, p = 0.006)
                                                                                                                                                Carriage of subgin-
                                                                                                                                                gival P. intermedia
                                                                                                                                                doubled in the 2nd
                                                                                                                                                trimester, comparing
                                                                                      Pregnant group                                            to the 1st trimester;
                                                                                      T1: 12–14 weeks                                           continued increas-
                                                                                      GA                                                        ing till after the
                                                                                      T2: 25–27 weeks                                           delivery (p < 0.05);
                                                                                      GA                                                        and decreased to the
                                                                                      T3: 34–38 weeks                                           lowest point after
                                                                                                            P. intermedia, P.
                                                  Pregnant (30)                       GA                                       16s rDNA         lactation
           Gürsoy et al.      Finland, prospec-                    Subgingival                              nigrescens (former
                                                  Non-pregnant                        T4: 4–6 weeks                            sequencing and   Carriage of             Fair
           (2009)16           tive cohort                          plaque, saliva                           Bacteroides inter-
                                                  (24)                                postpartum;                              culturing        salivary P. intermedia
                                                                                      T5: After lactation                                       remained stable dur-
                                                                                      Non-pregnant                                              ing the pregnancy
                                                                                      group                                                     and decreased
                                                                                      T1–T3 (once                                               (p < 0.05) after lacta-
                                                                                      per subsequent                                            tion to the same level
                                                                                      month)                                                    as the non-pregnant
                                                                                                                                                P. nigrescens is likely
                                                                                                                                                associated with preg-
                                                                                                                                                nancy gingivitis

          Scientific Reports |     (2021) 11:16870 |                    https://doi.org/10.1038/s41598-021-96495-1                                                                  4


                     Country, study    Groups (no. of                          Measurement       Microorganisms       detection                                    Quality
 Author (year)       design            subjects)          Sample source        interval          evaluated            methods              Study findings          assessment
                                                                                                                                           No significant
                                                                                                                                           changes in total
                                                                                                                                           bacterial counts in
                                                                                                                                           the pregnant group
                                                                                                                                           either during or after
                                                                             Pregnant group                                                Significant reduction
                                                                             T1: 12–14 weeks                                               in A. actinomyce-
                                                                             GA                                                            temcomitans after
                                                                             T2: 23–25 weeks     C. rectus, P. gin-                        delivery (p = 0.039)
                                                                             GA                  givalis, A. actino-                       No statistically sig-
 Carrillo-de-                           Pregnant (48)                        T3: 33–36 weeks     mycetemcomitans,                          nificant differences
                     Spain, prospective
 Albornoz et al.                        Non-pregnant      Subgingival plaque GA                  F. nucleatum, P.    Culturing             during pregnancy       Fair
 (2010)39                               (28)                                 T4: 3 months        intermedia, T.                            for any of the
                                                                             postpartum          forsythensis, P.                          pathogens evaluated;
                                                                             Non-pregnant        micra                                     however, significant
                                                                             group                                                         changes from the
                                                                             2 visits 6 months                                             third trimester to
                                                                             apart                                                         postpartum for all
                                                                                                                                           the pathogens
                                                                                                                                           Subjects who were
                                                                                                                                           positive for P.
                                                                                                                                           gingivalis had higher
                                                                                                                                           levels of gingival
                                                                                                                                           The organisms
                                                                                                                                           which were most
                                                                                                                                           commonly detected
                                                                                                                                           in both the groups
                                                                                                                                           were: Vielonella, T.
                                                                                                 Veillonella, T.
                                                                                                                                           forsythia, P. inter-
                                                                                                 forsythia, P.
                                                                                                                                           media, P. gingivalis,
                                                                             Pregnant group      intermedia, P.
                                        Pregnant (15)                        T1: during preg-    gingivalis, Pepto-
 Basavaraju et al.   India, prospective                                                                                                    and F. nucleatum
                                        Non-pregnant      Subgingival plaque nancy               screptococcus, F.    Culturing                                  Poor
 (2012)40            cohort                                                                                                                P. gingivalis was
                                        (15)                                 T2: 3 weeks post-   nucleatum, Pro-
                                                                                                                                           present in 5 patients
                                                                             partum              pionebactierum,
                                                                                                                                           out of 15 in the
                                                                                                                                           pregnant-group as
                                                                                                 Candida spp.
                                                                                                                                           compared to 1 in the
                                                                                                                                           non pregnant group
                                                                                                                                           and the count was
                                                                                                                                           reduced to 3 during
                                                                                                                                           No significant
                                                                                                                                           difference in mean
                                                                                                 A. actinomyce-
                                                                                                                                           total bacterial count
                                                                                                                                           between pregnant
                                                                                                 T. forsythia, C.
                                       Pregnant (20)                                                                  Fluorescence         and non-pregnant
 Machado et al.      Brazil, cross-                                            Pregnant group    rectus, P. gingi-
                                       Non-pregnant       Subgingival plaque                                          in situ hybridiza-   group                   Fair
 (2012)41            sectional                                                 14–24 weeks GA    valus, T. denticola,
                                       (20)                                                                           tion                 No significant dif-
                                                                                                 F. nucleatum, P.
                                                                                                                                           ferences between
                                                                                                 intermedia, P.
                                                                                                                                           groups in the num-
                                                                                                                                           bers of all bactieral
                                                                                                                                           species evaluated
                                                                                                                                           P. intermedia sig-
                                                                                                                                           nificantly increased
                                                                                                                                           in pregnant women
                                                                                                                                           who were in their
                                                                                                                                           second and third tri-
                                                                                                 A. actinomyce-                            mesters as compared
                                       Pregnant (30, 10                                          temcomitans,                              with first trimester
                                                                             Pregnant group
 Emmatty et al.      India, cross-     in each trimester)                                        P. gingivalis, P.                         and non-pregnant
                                                          Subgingival plaque One time point                           Culturing                                   Fair
 (2013)17            sectional         Non-pregnant                                              intermedia, F.                            women
                                                                             during pregnancy
                                       (10)                                                      nucleatum, P.                             Proportions of the
                                                                                                 micra                                     pathogens assessed
                                                                                                                                           did not show any
                                                                                                                                           significant difference
                                                                                                                                           among pregnant
                                                                                                                                           and non-pregnant
                                                                                                                                           The detection of
                                                                             Pregnant group                                                A. actinomycetem-
                                                                             T1: Second          A. actinomyce-                            comitans in pregnant
                                                                             trimester (15–      temcomitans,                              women at 2nd and
 Borgo et al.        Brazil, prospective Pregnant (9)
                                                          Subgingival plaque 26 weeks GA)        P. gingivalis, P.    Real-time PCR        3rd trimester was     Fair
 (2014)15            cohort              Non-pregnant (9)
                                                                             T2: Third trimes-   intermedia, F.                            significant higher
                                                                             ter (30–36 weeks    nucleatum                                 than that in the non-
                                                                             GA)                                                           pregnant women
                                                                                                                                           (p < 0.05)

Scientific Reports |       (2021) 11:16870 |                   https://doi.org/10.1038/s41598-021-96495-1                                                                       5


                                 Country, study     Groups (no. of                     Measurement         Microorganisms       detection                              Quality
           Author (year)         design             subjects)         Sample source    interval            evaluated            methods         Study findings         assessment
                                                                                                                                                A significant dif-
                                                                                                                                                ference in total
                                                                                                                                                cultivable microbial
                                                                                                                                                number between
                                                                                                                                                non-pregnant and
                                                                                                                                                each stage of preg-
                                                                                                                                                More total bacteria
                                                                                                                                                counts at early stage
                                                                                                                                                of pregnancy (T1),
                                                                                                                                                comparing to the
                                                                                                                                                non-pregnant group
                                                                                                                                                (p < 0.05)
                                                                                                                                                Significant higher
                                                                                                           Subgingival                          prevalence of
                                                                                                           A. actinomyce-                       Candida spp. in the
                                                                                       Pregnant group      temcomitans,                         middle (T2) and
                                                                                       T1: 7–16 weeks      P. gingivalis, P.                    late (T3) pregnancy,
                                                    Pregnant (132)                     GA                  intermedia, F.                       comparing to the
           Fujiwara et al.       Japan, prospective                   Subgingival                                               Culturing and
                                                    Non-pregnant                       T2: 17–28 weeks     nucleatum                            non-pregnant group Fair
           (2017)42              cohort                               plaque, saliva                                            real-time PCR
                                                    (51)                               GA                  Saliva                               (p < 0.05)
                                                                                       T3: 29–39 weeks     Above 4 + Strepto-                   The number of peri-
                                                                                       GA                  cocci, Staphylo-                     odontal species was
                                                                                                           cocci, Candida                       significantly lower in
                                                                                                           spp.                                 late pregnancy (T3),
                                                                                                                                                comparing to the
                                                                                                                                                early (T1) and mid-
                                                                                                                                                dle (T2) pregnancy
                                                                                                                                                (p < 0.05)
                                                                                                                                                The prevalence of
                                                                                                                                                P. gingivalis and A.
                                                                                                                                                tans was significantly
                                                                                                                                                higher in the early
                                                                                                                                                (T1) and middle
                                                                                                                                                (T2) stage of preg-
                                                                                                                                                nancy, comparing
                                                                                                                                                to the nonpregnant
                                                                                                                                                women (p < 0.05)
                                                                                                                                                A significant increase
                                                                                       Pregnant group                                           in S. mutans during
                                                                                       T1: 6 weeks GA                                           the 2nd and 3rd
                                                    Pregnant (50)
           Kamate et al.         India, prospective                                    T2: 18 weeks GA                                          trimester and post-
                                                    Non-pregnant      Saliva                               S. mutans            Culturing                              Fair
           (2017)14              cohort                                                T3: 30 weeks GA                                          partum period of
                                                                                       T4: 6 weeks post-                                        pregnancy compared
                                                                                       partum                                                   to the non-pregnant
                                                                                                                                                group (p < 0.01)
                                                                                                                                                No difference in
                                                                                                                                                oral yeast detection
                                                                                                                                                within pregnancy
                                                                                                                                                stages and between
                                                                                                                                                pregnant and non-
                                                                                                                                                pregnant stages
                                                                                                                                                (p < 0.05)
                                                    Pregnant (30)                      Pregnant group                                           More oral yeast were
                                 Portugal, prospec-                   Unstimulated
           Rio et al. (2017)43                      Non-pregnant                       T1: 1st trimester   Yeast                Culturing       found in the 3rd     Fair
                                 tive cohort                          saliva
                                                    (30)                               T2: 3rd trimester                                        trimester than the
                                                                                                                                                1st trimmest, but no
                                                                                                                                                difference comparing
                                                                                                                                                to the non-pregnant
                                                                                                                                                stage (p < 0.05)
                                                                                                                                                Saliva flow rate did
                                                                                                                                                not change in both
                                                                                       Pregnant group
                                                                                                                                                Significant higher
                                                                                       T1: 11–14 weeks
                                                                                                                                                bacterial diversity
                                                                                                                                                of the supragingival
                                                                                       T2: 20–25 weeks
                                                                                                                                                microbiota in third
                                                                                                                                                trimester compared
                                                                                       T3: 33–37 weeks
                                                                                                           Quantity of OUT                      to the non-pregnant
                                 China, prospec-    Pregnant (11)    Supragingival     GA                                       16s rDNA
           Lin et al. (2018)44                                                                             and microbiota                       group                Fair
                                 tive cohort        Non-pregnant (7) plaque, saliva    T4: 6 weeks post-                        sequencing
                                                                                                           diversity                            Neisseriaceae and
                                                                                                                                                and Spirochaetaceae
                                                                                                                                                were significantly
                                                                                       4 visits (same
                                                                                                                                                enriched in pregnant
                                                                                       intervals of the
                                                                                       pregnant group)

          Scientific Reports |        (2021) 11:16870 |                    https://doi.org/10.1038/s41598-021-96495-1                                                               6


                        Country, study    Groups (no. of                        Measurement       Microorganisms        detection                               Quality
 Author (year)          design            subjects)         Sample source       interval          evaluated             methods         Study findings          assessment
                                                                                                                                        Salivary S. mutans
                                                                                                                                        carriage was higher
                                                                                                                                        in pregnant than
                                                                                                                                        women (p < 0.05)
                                                                                                                                        No difference
                                                                                                                                        between pregnant
                                                                                                                                        and non-pregnant
                                                                                                                                        salivary C. albicans
                                                                                                                                        carriage (p > 0.05)
                                                           Whole non-                             C. albicans, C.
                                          Low SES pregnant                                                                              Tonsil (57%) was the
                                                           stimulated saliva,   Pregnant group    glabrata, C. tropi-
                        USA, cross-       (48)                                                                          Culturing and   most prevalent site
 Xiao et al. (2019)45                                      supragingival        3rd trimester     calis, C. krusei,                                            Fair
                        sectional         Low SES Non-                                                                  Colony PCR      for C. albicans detec-
                                                           plaque, mucosal      (> 28 weeks GA)   C. dubliniensis, S.
                                          pregnant (34)                                                                                 tion among pregnant
                                                           swabs                                  mutans
                                                                                                                                        Untreated decayed
                                                                                                                                        teeth is associ-
                                                                                                                                        ated with higher
                                                                                                                                        carriage of salivary
                                                                                                                                        S. mutans and C.
                                                                                                                                        albicans detection in
                                                                                                                                        both pregnant and
                                                                                                                                        non-pregnant groups
                                                                                                                                        (p < 0.05)
                                                                                                  S. aureus, N.
                                                                                                  catarrhalis, K.
                                                                                                                                        E. coli was the most
                                                                                                                                        common species in
                                                                                                  E. coli, P. mel-
                                          Pregnant (26)                                                                                 non-pregnant group
 Aikulola et al.        Nigeria, cross-                                         Pregnant group    aninogenicus, P.
                                          Non-pregnant      Oral swab                                                Culturing          while N. catarrhalis    Poor
 (2020)46               sectional                                               20–28 weeks GA    propionicum, V.
                                          (32)                                                                                          was the most com-
                                                                                                  pervula, S. viri-
                                                                                                                                        mon in the pregnant
                                                                                                  dans, Coagulase
                                                                                                  negative Staphylo-
                                                                                                                                        P. nigrescens had
                                                                                                                                        higher prevalence in
                                                                                                                                        the pregnant group
                                                                                                                                        (p < 0.01)
                                                                                                                                        P. nigrescens
                                                                                                                                        exhibited more
                                                                                                                                        frequently in late
                                                                                                                                        pregnancy than
                                                                                                                                        early and middle
                                                                                                  P. gingivalis, P.
 Huang et al.           China, cross-     Pregnant (84)     Unstimulated        Pregnant group                                          pregnancy (p < 0.05
                                                                                                  intermedia, P.        16s rRNA PCR                          Fair
 (2020)47               sectional         Postpartum (33)   saliva              One time point                                          and p < 0.01)
                                                                                                                                        P. gingivalis in the
                                                                                                                                        postpartum group
                                                                                                                                        exceeds all of the
                                                                                                                                        pregnant stages
                                                                                                                                        (p < 0.01)
                                                                                                                                        P. intermedia did not
                                                                                                                                        show any significant
                                                                                                                                        differences among
                                                                                                                                        Significant differ-
                                                                                                                                        ences in the relative
                                                                                                                                        abundance of oral
                                                                                                                                        microbiome in
                                                                                                                                        pregnant women
                                                                                                                                        A significant
                                                                                                                                        dominance of
                                                                                                                                        Streptococcus and
                                          Pregnant (42)                                           Quantity of OUT
 Sparvoli et al.        Brazil, cross-                                          Pregnant group                          16s rRNA        Gemella in pregnant
                                          Non-pregnant      Oral swab                             and microbiota                                                Fair
 (2020)48               sectional                                               28–36 weeks GA                          sequencing      women (p < 0.01 and
                                          (18)                                                    diversity
                                                                                                                                        0 = 0.03)
                                                                                                                                        Shannon diversity
                                                                                                                                        index were higher
                                                                                                                                        in the non-pregnant
                                                                                                                                        group, while the
                                                                                                                                        Simpson diversity
                                                                                                                                        index was higher in
                                                                                                                                        the pregnant group
                                                                                                                                        S. mutans were
                                                                                                                                        more abundant in
                                                                                                                                        pregnant women
                                          Pregnant (38)
 Wagle et al.           Norway, cross-                                          Pregnant group    S. mutans, Lacto-                     (p = 0.03)
                                          Non-pregnanr      Saliva                                                      Culturing                             Fair
 (2020)49               sectional                                               18–20 weeks GA    bacillus                              Lactobaciilus did not
                                                                                                                                        have the significant
                                                                                                                                        difference between
                                                                                                                                        the groups

                                          Table 1.  Oral microbial differences between pregnant and non-pregnant women.

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                               Country, study      Groups (no. of                         Measurement         Microorganisms      detection                              Quality
           Author (year)       design              subjects)          Sample source       interval            evaluated           methods        Study findings          assessment
                                                                                                                                                 A. naeslundii gsp
                                                                                                                                                 2 level decreased
                                                                                                                                                 with increased GA
                                                                                                                                                 (p = 0.05)
                                                                                                                                                 L. casei carriage
                                                                                                                                                 increased with
                                                                                                                                                 increased GA
                                                                                                                                                 (p = 0.04)
                                                                                                                                                 L. casei levels at the
                                                                                                                                                 third trimester were
                                                                                                                                                 positively associated
                                                                                                              S. mutans, S. sobri-
                                                                                                                                                 with birth weight
                                                                                                              nus, S. sanguinus,
                                                                                                                                                 (β = 34.1 g; SE = 16.4;
                                                                                                              L. acidophilus,
                                                                                                                                                 p = 0.04)
           Dasanayake et al.   USA, prospective    First time preg-                       T1: 3rd trimester   L. casei, A.
                                                                    Stimulated saliva                                              Culturing     Total Streptococci      Fair
           (2005)50            cohort              nant women (297)                       T2: Delivery        naeslundii, Total
                                                                                                                                                 and total cultivable
                                                                                                              Streptococci, Total
                                                                                                                                                 organism levels at
                                                                                                              cultivable organ-
                                                                                                                                                 delivery were nega-
                                                                                                                                                 tively associated with
                                                                                                                                                 birth weight
                                                                                                                                                 After multivariate
                                                                                                                                                 analysis with average
                                                                                                                                                 bacterial levels, A.
                                                                                                                                                 naeslundii gsp 2,
                                                                                                                                                 L. casei, pregnancy
                                                                                                                                                 age, and infant
                                                                                                                                                 gender remained sig-
                                                                                                                                                 nificantly associated
                                                                                                                                                 with birth weight
                                                                                                                                                 N. mucosa increased
                                                                                                                                                 throughout the preg-
                                                                                                                                                 nancy (p < 0.001)
                                                                                                                                                 Total bacterial
                                                                                                                                                 No significant dif-
                                                                                                                                                 ferences between T1
                                                                                                                                                 and T2
                                                                                                                                                 Significant reduc-
                                                                                                              37 species includ-                 tion from T1 to
                                                                                                              ing                                T3 (p < 0.05), and
                                                                                          T1: 12 weeks GA
                                                                                                              S. mutans, F.                      further reduction to
                                                                                          T2: 28 weeks GA
           Adriaens et al.     Switzerland, pro-   Healthy pregnant   Subgingival                             nucleatum, P.      DNA–DNA         T4 (p < 0.01)
                                                                                          T3: 36 weeks GA                                                                Fair
           (2009)51            spective cohort     women (20)         plaque                                  intermedia, P.     hybridization   Between T1 and T4,
                                                                                          T4: 4–6 weeks
                                                                                                              gingivalis, A.                     significant differ-
                                                                                                              actinomycetem-                     ences were found
                                                                                                              comitans                           for 8 of 37 species,
                                                                                                                                                 including S. mutans,
                                                                                                                                                 S. aureus, polymor-
                                                                                                                                                 phum, P. micra
                                                                                                                                                 Between measure-
                                                                                                                                                 ment intervals, no
                                                                                                                                                 statistical differences
                                                                                                                                                 identified for the
                                                                                                                                                 levels of four peri-
                                                                                                                                                 odontal pathogens
                                                                                                                                                 Increase of S. mutans
                                                                                                                                                 during the 2nd and
                                                                                                                                                 3rd trimester among
                                                                                                                                                 women 25–35 years
                                                                                          T1: 1st trimester
                                                                                                                                                 Increase of Lacto-
                                                                                          (11–12 weeks GA)
                                                                                                                                                 bacilli in the 2nd
           Molnar-Varlam       Romania, pro-       Healthy pregnant                       T2: 2nd trimester S. mutans, Lacto-
                                                                      Stimulated saliva                                           Culturing      trimester among       Fair
           et al. (2011)13     spective cohort     women (35)                             (20–22 weeks GA) bacillus
                                                                                                                                                 women 20–24 years
                                                                                          T3: 3rd trimester
                                                                                                                                                 old and 30–35 years
                                                                                          (34–35 weeks GA)
                                                                                                                                                 The salivary pH
                                                                                                                                                 increased as the
                                                                                                                                                 pregnancy pro-
                                                                                                                                                 No statistically sig-
                                                                                                                                                 nificant changes in
                                                                                                                                                 counts of S. mutans
                                                                                                                                                 and Lactobacillus
                                                                                                                                                 spp., but a tendency
                                                                                          T1: Between 2nd
                                                                                                                                                 of higher numbers
           Martinez-Pabon      Colombia, pro-      Pregnant women                         and 3rd trimester   S. mutans, Lacto-
                                                                      Stimulated saliva                                           Culturing      during pregnancy         Fair
           et al. (2014)52     spective cohort     (35)                                   T2: 7 months        bacillus spp.
                                                                                                                                                 A statistically signifi-
                                                                                                                                                 cant difference in the
                                                                                                                                                 pH and the buffering
                                                                                                                                                 capacity of saliva;
                                                                                                                                                 both lower during
                                                                                                                                                 pregnancy (p < 0.05)

          Scientific Reports |      (2021) 11:16870 |                     https://doi.org/10.1038/s41598-021-96495-1                                                                  8


                   Country, study     Groups (no. of                          Measurement          Microorganisms        detection                                    Quality
 Author (year)     design             subjects)           Sample source       interval             evaluated             methods               Study findings         assessment
                                                                                                                                               The progression of
                                                          Saliva, vaginal,    Weekly from                                                      pregnancy is not
                                     Pregnant women
                                                          stool, oral swab    early pregnancy                                                  associated with a
 DiGiulio et al.                     (49)                                                                                16 s rDNA
                   USA, case–control                      from molar tooth    until delivery and   Not specified;                              dramatic remodeling Fair
 (2015)11                            Full term (34)                                                                      sequencing
                                                          surface & gum       monthly until 12                                                 of the diversity and
                                     Preterm (15)
                                                          lines               postpartum                                                       composition of a
                                                                                                                                               woman’s microbiota
                                                                                                                                               Klebsiella species
                                                                                                                                               was the predomi-
                                                                                                                                               nant isolate from
                                                                                                                                               101 (25.6%) of the
                                      Pregnant women
                                                                                                   Klebsiella spp.,                            The pattern of
                                                                                                   E. coli, S. albus,                          microbial culture
                                      1st trimester (3)                                                                Culturing, API
 Okoje-Adesomoju Nigeria, cross-                                                                   Proteus spp., S.                            whether normal
                                      2nd trimester       Mucosal swab        One time point                           20A identifica-                                Poor
 et al. (2015)53 sectional                                                                         aureus, Streptococ-                         for the oral cavity
                                      (100)                                                                            tion kits
                                                                                                   cus spp., Pseu-                             or not did not vary
                                      3rd trimester
                                                                                                   domonas spp.                                significantly with
                                                                                                                                               parity (p = 0.98),
                                                                                                                                               trimester of preg-
                                                                                                                                               nancy (p = 0.94) or
                                                                                                                                               oral hygiene status
                                                                                                                                               (p = 0.94)
                                                                                                                                               Changes in the
                                                                                                                                               percentage of P.
                                                                                                                                               intermedia, F. nuclea-
                                                                                                                                               tum, P. gingivalis, T.
                                                                                                                                               denticola, C. rectus
                                                                                                                                               and an increase in
                                                                                                                                               A. actinomycetem-
                                                                                                   T. forsythia, C.
                                                                                                                                               comitans was noted,
                                                                             T1: 19 ± 3.3 weeks    rectus, P. gingivalis,
                                                                                                                                               but differences were
                                                                             GA;                   T. denticola, F.
                                                                                                                          Fluorescence         not statistically
 Machado et al.    Brazil, prospective Healthy pregnant   Supragingival &    T2: 48 h postpar-     nucleatum, P.
                                                                                                                          in situ hybridiza-   significant            Fair
 (2016)54          cohort              women (31)         subgingival plaque tum;                  intermedia, P.
                                                                                                                          tion                 - A significant reduc-
                                                                             T3: 8 weeks post-     nigrescens A.
                                                                                                                                               tion was seen for P.
                                                                             partum                actinomycetem-
                                                                                                                                               nigrescens when all
                                                                                                                                               three time points
                                                                                                                                               were compared
                                                                                                                                               (p = 0.01, Friedman
                                                                                                                                               test), with a reduc-
                                                                                                                                               tion from T1 to T3
                                                                                                                                               (p = 0.002), and T2
                                                                                                                                               to T3 (p = 0.037)
                                                                                                                                               Species richness
                                                                                                                                               and diversity of the
                                                                                                                                               subgingival plaque
                                                                                                                                               and saliva samples
                                                                                                                                               were relatively stable
                                                                                                                                               across the pregnancy
                                                                                                                                               The abundance of
                                                                                                                                               Prevotella, Strepto-
                                                                              T1: 1st trimester
                                                                                                                                               coccus and Veillonella
                                                                              (< 12 weeks GA)
                                      Pregnant women                                                                                           in both subgingival
                                                                              T2: 2nd trimester
                                      (30)               Subgingival                                                                           plaque and saliva
 Balan et al.      Singapore, pro-                                            (21–24 weeks GA) 12 Phyla, 65 gen-         16s rDNA
                                      1st trimester (10) plaque, unstimu-                                                                      samples were more      Fair
 (2018)12          spective cohort                                            T3: 3rd trimester era, 131 species         sequencing
                                      2nd trimester (10) lated saliva                                                                          during pregnancy
                                                                              (32–36 weeks GA)
                                      3rd trimester (10)                                                                                       A significant decline
                                                                              T4: 6 weeks post-
                                                                                                                                               in the abundance of
                                                                                                                                               pathogenic species,
                                                                                                                                               e.g., Veillonella
                                                                                                                                               parvula, Prevotella
                                                                                                                                               species and Actinob-
                                                                                                                                               aculum species, was
                                                                                                                                               observed from preg-
                                                                                                                                               nancy to postpartum
                                                                                                                                               Alpha diversity, both
                                                                                                                                               inter-individual and
                                      Pregnant (10)                           Every 3 weeks
 Goltsman et al.   USA, retrospec-                        Saliva, vaginal,                                               16 s rDNA             intra-individual,
                                      Term delivery (6)                       over the course of   1553 taxa                                                         Fair
 (2018)55          tive cohort                            stool, rectal swabs                                            sequencing            remained stable
                                      Preterm (4)                             gestation
                                                                                                                                               across the pregnancy
                                                                                                                                               and postpartum

Scientific Reports |    (2021) 11:16870 |                     https://doi.org/10.1038/s41598-021-96495-1                                                                           9


                              Country, study    Groups (no. of                           Measurement       Microorganisms          detection                           Quality
           Author (year)      design            subjects)           Sample source        interval          evaluated               methods      Study findings         assessment
                                                                                                                                                No significant
                                                                                                                                                differences in total
                                                                                                                                                amount of bacteria
                                                                                                                                                between the groups
                                                                                                                                                T. forsythia showed
                                                                                                                                                significant differ-
                                                                                                                                                ences in quantifica-
                                                Pregnant (52)                                              A. actinomyce-                       tion between 1st
                                                1st trimester (16)                                         temcomitans,                         trimester and 3rd
           de Souza Massoni   Brazil, cross-                       Subgingival
                                                2nd trimester (21)                       One time point    P. gingivalis, T.       qPCR         trimester, and 1st     Fair
           et al. (2019)56    sectional                            plaque
                                                3rd trimester (15)                                         forsythia, S. oralis,                trimester and non-
                                                Non-pregnant (15)                                          Universal                            pregnant (p = 0.048
                                                                                                                                                and p = 0.014)
                                                                                                                                                Amount of T.
                                                                                                                                                forsythia positively
                                                                                                                                                correlated with the
                                                                                                                                                diagnosis of gin-
                                                                                                                                                givitis in pregnant
                                                                                                                                                women (p = 0.031)
                                                                                                                                                No difference in
                                                                                                                                                Chao1 and Shannon
                                                                                                                                                diversity for the
                                                                                                                                                vaginal, oral, or gut
                                                                                                                                                microbiome across
                                                                                                                                                pregnancy for the
                                                                                                                                                group overall
                                                                                                                                                For the oral micro-
                                                                                                                                                biota, having a low
                                                African American    Vaginal, oral        T1: 8–14 weeks
                                                                                                                                                level of education
           Dunlop et al.      USA, retrospec-   Pregnant women      (tongue, hard pal-   GA                                        16S rDNA
                                                                                                           Not specified                        and receipt of antibi- Fair
           (2019)57           tive cohort       (122)               ate, gum line) and   T2: 24–30 weeks                           sequencing
                                                                                                                                                otics between study
                                                Oral samples (97)   rectal swabs         GA
                                                                                                                                                visits were associated
                                                                                                                                                with greater Bray–
                                                                                                                                                Curtis dissimilarity,
                                                                                                                                                with some attenu-
                                                                                                                                                ation of the effect
                                                                                                                                                of education when
                                                                                                                                                additionally control-
                                                                                                                                                ling for prenatal

                                                Table 2.  Oral microbial differences between pregnancy stages.

                                                pregnancy and adverse birth outcome in Table 421–25,63–73; eight studies on impact of periodontal disease on oral
                                                microorganisms during pregnancy in Table 574–81; six studies on impact of gestational diabetes mellitus (GDM)
                                                on oral microorganisms during pregnancy in Table 682–87; 11 studies on impact of systemic health conditions on
                                                oral microorganisms during pregnancy in Table 788–98. Quality and risk of bias for randomized controlled trials
                                                was assessed and are shown in Fig. 2. Quality assessment for cohort and cross-sectional studies are included in
                                                the last column of all tables.
                                                    The quality of the selected articles was assessed using two methodological validities: (1) Cochrane Collabora-
                                                tion’s tool for assessing risk of bias in randomized ­trials31. (2) Adapted Down and Black ­scoring32 that assess the
                                                methodological quality of both randomized and non-randomized studies of health care interventions. A total
                                                score of 26 represents the highest study quality.

                                                Oral microbial differences between pregnant and non‑pregnant women. Evident changes of
                                                oral microbiota were seen among pregnant women, comparing to those of non-pregnant women. A significantly
                                                higher amount of total cultivable microorganisms were found in pregnant women comparing to the non-preg-
                                                nant at each stage of ­pregnancy42. The plaque bacterial community was more diverse in 3rd trimester pregnant
                                                women compared to non-pregnant ­women44.
                                                    Regarding oral pathogens, the prevalence of A. actinomycetemcomitans was significantly higher in pregnant
                                                women in each stage compared to non-pregnant women (p < 0.05)15,42. Two ­studies14,45 assessed S. mutans car-
                                                riage in saliva, and found that S. mutans carriage increased significantly throughout the pregnancy; particularly,
                                                significant differences were seen between women in their first trimester and non-pregnant women (p < 0.0114 and
                                                p < 0.0545). The detection of P. gingivalis and P. intermedia increased significantly in pregnant women compared
                                                to non-pregnant w  ­ omen17,42. Although no difference was found in terms of C. albicans carriage between pregnant
                                                and non-pregnant ­women45, two studies revealed a higher detection of Candida spp. among women in their late
                                                pregnancy stage, comparing to the non-pregnant g­ roup42,43.

                                                Oral microbial differences throughout pregnancy stages. Interestingly, seven ­studies11,12,51,52,54,55,57
                                                revealed a stable oral microbial community during pregnancy. All four ­studies11,12,55,57 that performed sequenc-
                                                ing analysis revealed that microbiota species richness, diversity and composition were relatively stable across the
                                                pregnancy stages. The level of S. mutans and Lactobacillus spp. were assessed in two s­ tudies13,52. The levels of S.
                                                mutans and Lactobacilli increased in both studies, but without statistical ­signficance52.

          Scientific Reports |      (2021) 11:16870 |                   https://doi.org/10.1038/s41598-021-96495-1                                                                  10


                      Country, study     Groups (no. of                         Measurement          Microorganisms         detection                              Quality
 Author (year)        design             subjects)            Sample source     interval             evaluated              methods         Study findings         assessment
                                                                                                                                            A reduction in
                                                                                                                                            salivary S. mutans
                                                                                                                                            levels in treatment
                                         Treatment group                                                                                    group became sig-
                                         (33) Dietary coun-                                                                                 nificant (p < 0.01)
                                         seling + Dental                                                                                    six months
                                         Prophy + system-                                                                                   after the study
                                         atic fluoride (1 mg                                                                                began (at T3); S.
                                         per day from the                       T1: 3rd month GA                                            mutans reduction
                                         last week of 6th                       T2: 6th month GA                                            remained signifi-
                                         month GA) + daily                      T3: 9th month GA                                            cant (p < 0.001) at
                                         fluoride and CHX                       T4: 6 months                                                the end of the
 Brambillia et al.                                           Unstimulated
                      Italy, RCT​        mouth rinse                            postpartum       S. mutans                  Culturing       study               See Fig. 2
 (1998)                                                      saliva
                                         Control group                          T5-T7: 12, 18,                                              Children of
                                         (32)                                   24 months                                                   mothers in
                                         Dietary coun-                          postpartum,                                                 treatment group
                                         seling + Dental                        respectively                                                had significantly
                                         Prophy + system-                                                                                   lower salivary S.
                                         atic fluoride (1 mg                                                                                mutans levels than
                                         per day from the                                                                                   those of control-
                                         last week of 6th                                                                                   group mothers
                                         month GA)                                                                                          at 18 months old
                                                                                                                                            (p < 0.05) and
                                                                                                                                            24 months old
                                                                                                                                            (p < 0.01)
                                         Treatment group
                                         (74)                                                        P. gingivalis, P.
                                                                                                                                            Mothers who
                                         Prenatal Peri-                                              intermedia, P.
                                                                                                                                            had pre‐term
                                         odontal interven-                                           nigrescens, B. for-
                                                                                                                                            low birth weight
                                         tion (Hygiene                          Treatment group      sythus, A. actino-
                                                                                                                            DNA-DNA         had significantly
                                         instruction + full                     T1: During preg-     mycetemcomitans,
 Mitchell-Lewis       USA, prospective                        Subgingival                                                   hybridization   higher levels of
                                         mouth debride-                         nancy                F. nucleatum,                                                 Fair
 et al. (2001)59      cohort                                  plaque                                                        checkerboard    B. forsythus and
                                         ment)                                  Control group        T. denticola, P.
                                                                                                                            method          C. rectus, and
                                         Control group                          T1: After delivery   micros, C. rectus,
                                                                                                                                            elevated counts
                                         (90)                                                        E. corrodens,
                                                                                                                                            for the other spe-
                                         Postpartum                                                  E. nodatum, S.
                                                                                                                                            cies examined
                                         periodontal inter-                                          intermedius
                                                                                                                                            No significant
                                                                                                                                            changes from
                                         Treatment group                                                                                    baseline to
                                         (40)                                                                                               postpartum in the
                                         SRP + polish-                                                                                      levels of any single
                                         ing + OHI + sonic                                        Red cluster                               bacterial species
                                         power toothbrush                                         P. gingivalis, T.                         or cluster among
                                         during 2nd                                               forsythensis, T.                          control mothers
                                         trimester                                                denticola                 DNA-DNA         P. intermedia
                                                           Gingival cervical
 Offenbacher et al.                      Control group                          T1: < 22 weeks GA Orange cluster            hybridization   and P. nigrescens
                      USA, RCT​                            fluid, subgingival                                                                                    See Fig. 2
 (2006)60                                (34)                                   T2: Postpartum    F. nucleatum, P.          checkerboard    reduction
                                         (Supragingi-                                             intermedia, P.            method          detected in the
                                         val debride-                                             nigrescens, C.                            treatment group
                                         ment + manual                                            rectus, A. actino-                        (p < 0.05)
                                         toothbrush during                                        mycetemcomitans                           A composite
                                         pregnancy) + (SRP                                                                                  score of orange-
                                         6 weeks postpar-                                                                                   cluster organisms
                                         tum)                                                                                               decreased in
                                                                                                                                            treatment group
                                                                                                                                            (p = 0.03)
                                                                                                                                            Women in
                                                                                                                                            treatment group
                                                                                                     P. gingivalis,                         had significantly
                                         Treatment group
                                                                                                     T. denticola, T.                       greater reductions
                                         (413): SRP before                      T1: 13–16 weeks
                                                                                                     forsythia, P. inter-                   (p < 0.01) in
 Novak et al.                            21 weeks GA          Subgingival       GA
                      USA, RCT​                                                                      media, C. rectus,      Realtime PCR    counts of P. gingi- See Fig. 2
 (2008)61                                Control group        plaque            T2: 29–32 weeks
                                                                                                     F. nucleatum, A.                       valis, T. denticola,
                                         (410): SRP after                       GA
                                                                                                     actinomycetem-                         T. forsythia, P.
                                                                                                     comitans                               intermedia, and
                                                                                                                                            C. rectus than
                                                                                                                                            untreated women
                                         Treatment group                                                                                    A statistically sig-
                                         (30)                                                                                               nificant decrease
                                         Oral Environ-                          T1: Before treat-                                           (p < 0.0001) in S.
                                         ment Stabilization                     ment (70% in 2nd                                            mutans counts
 Volpato et al.       Brazil, prospec-
                                         (atraumatic caries Saliva              trimester)           S. mutans              Culturing       between saliva         Fair
 (2011)27             tive cohort
                                         excavation and                         T2: 1 week after                                            samples before
                                         fillings + extrac-                     treatment                                                   and after oral
                                         tion of retained                                                                                   environment
                                         roots)                                                                                             stabilization

Scientific Reports |       (2021) 11:16870 |                      https://doi.org/10.1038/s41598-021-96495-1                                                                      11


                              Country, study   Groups (no. of                          Measurement         Microorganisms      detection                         Quality
           Author (year)      design           subjects)           Sample source       interval            evaluated           methods        Study findings     assessment
                                                                                                                                              The detection
                                               Pregnant women                                              P. gingivalis, P.
                                                                                                                                              of assessed
                                               with preeclamp-                                             intermedia, P.
                                               sia (57)                                                    nigrescens, T.
                                                                                       T1: Before treat-                                      did not decrease
           Jaramillo et al.                    Treatment group                                             forsythia, C. rectus,
                              Colombia RCT​                        Subgingival fluid   ment                                      PCR          following peri-   See Fig. 2
           (2012)29                            (26): SRP                                                   E. Corrodens, D.
                                                                                       T2: Postpartum                                         odontal treatment
                                               Control group                                               pneumosintes, A.
                                                                                                                                              in control group
                                               (31): Supragingi-                                           actinomycetem-
                                                                                                                                              and intervention
                                               val prophy                                                  comitans
                                                                                                                                              Salivary S. mutans
                                                                                                                                              was reduced after
                                               Pregnant women
                                                                                                                                              the atraumatic
                                               with a minimal of
                                                                                                                                              restorative treat-
                                               3 decayed teeth
                                                                                       T1: Before treat-                                      ment (p < 0.001)
                                               Treatment group
           Asad et al.                                                                 ment                                                   Salivary S.
                              Pakistan, RCT​   (32): atraumatic   Stimulated saliva                        S. mutans           Realtime PCR                      See Fig. 2
           (2018)28                                                                    T2: 1 week after                                       mutans remained
                                               restorative treat-
                                                                                       treatment                                              the same level
                                                                                                                                              between the two
                                               Control group
                                                                                                                                              study time point
                                               (32): no treatment
                                                                                                                                              in the control
                                                                                                                                              group (p = 0.29)
                                                                                                                                              No difference in
                                                                                                                                              S. mutans among
                                                                                                                                              the pregnant
                                                                                                                                              women who used
                                                                                                                                              xylitol toothpaste
                                                                                                                                              compared to those
                                                                                                                                              who used tooth-
                                               Treatment group                         T1: Before the
                                                                                                                                              paste without
                                               (23): toothpaste                        use of xylitol
                                                                                                                                              xylitol (p = 0.062)
           Escalante-Medina                    with 10% xylitol                        toothpaste
                            Peru, RCT​                             Saliva                                  S. mutans           Culturing      Both toothpastes, See Fig. 2
           et al. (2019)62                     Control group                           T2: 14 days after
                                                                                                                                              with and without
                                               (22): toothpaste                        the use of the
                                                                                                                                              xylitol, were effec-
                                               without xylitol                         toothpaste
                                                                                                                                              tive to decrease
                                                                                                                                              the count of S.
                                                                                                                                              mutans in the
                                                                                                                                              saliva of pregnant
                                                                                                                                              women (p = 0.001
                                                                                                                                              and p = 0.005,

                                               Table 3.  Oral microbial differences responding to prenatal dental treatment.

                                                   Some ­studies12,39,51 indicated significant differences from pregnancy to the postpartum period. A total bacte-
                                               rial count reduced significantly after delivery (p < 0.01)51. Several species, like S. mutans and Parvimonas micra,
                                               showed significant differences in postpartum compared to the early stages of ­pregnancy51. This finding was also
                                               noticed in another study where A. actinomycetemcomitans, P. gingivalis, Tannerella forsythia, P. micra showed an
                                               abrupt decline after ­delivery39. A. actonomycetemcomitans, especially, dropped significantly in its amount after
                                               delivery (p = 0.039)39. A significant decline in the abundance of pathogenic species from pregnancy to postpartum
                                               period was observed as ­well12.

                                               Impact of prenatal dental treatment on maternal oral flora.                       Four ­studies27,28,58,62 revealed lower
                                               S. mutans carriage in the group with oral health care intervention during pregnancy compared to the control
                                               group. Fluoride and chlorhexidine treatment as a caries-preventive regimen during pregnancy showed a statisti-
                                               cal difference in the salivary S. mutans levels between the study and control groups by the end of the 3-month
                                               treatment ­period58. At the end of the pregnancy, the reduction in S. mutans level was still significant in the study
                                               group (p < 0.01)58.
                                                   Two ­studies27,28 which conducted oral environmental stabilization, including atraumatic restorative treat-
                                               ment, revealed statistically significant decrease in S. mutans (p < 0.000127 and p < 0.00128) before and after the
                                               intervention. Comparatively, there was no significant reduction in salivary S. mutans count in the group who
                                               did not get the treatment (p = 0.29)28. Interestingly, children of treated group mothers had significantly lower
                                               salivary S. mutans levels than those of untreated group mothers (p < 0.05)58.
                                                   Periodontal pathogenic microbiomes did not reveal consistent results. Three s­ tudies29,60,61 performed SRP as
                                               treatment. Some microbiomes had significantly greater reductions where counts of P. gingivalis, P. intermedia, T.
                                               denticola, T. forsythia, and C. rectus was significantly lower in treated women (p < 0.01)61. A similar result was also
                                               found with detection of P. intermedia and P. nigrescens reduced significantly in the treatment group (p < 0.05)60.
                                               Yet, the study by Jaramillo et al.29 did not detect a significant decrease in the levels of bacterial species between
                                               treated and untreated groups. Quality of evidence and strength of recommendation by GRADE assessment
                                               is described in ESM Appendix 4. Quality of evidence was assessed with the study design and factors to either
                                               increase or reduce the quality for clinical interventional studies. Strength of recommendation was evaluated based
                                               on whether all individuals will be best served by the recommended course of action. Depending on whether the
                                               course is conditional or discretionary, the recommendation was given either strong or weak.

          Scientific Reports |     (2021) 11:16870 |                    https://doi.org/10.1038/s41598-021-96495-1                                                            12


                       Country, study    Groups (no. of                         Measurement        Microorganisms         detection                              Quality
 Author (year)         design            subjects)          Sample source       interval           evaluated              methods            Study findings      assessment
                                                                                                                                             -Detection of
                                         Pregnant women                                                                                      T. forsythia was
                                         (88)                                                      A. actinomyce-                            significantly higher
                                         Threatened pre-                                           temcomitans,                              among Threatened
 Hasegawa et al.       Japan, cross-                        Subgingival
                                         mature labor                           Not specified      P. gingivalis, P.      PCR                premature labor      Fair
 (2003)63              sectional                            plaque
                                         Full term (22)                                            intermedia, T.                            preterm delivery
                                         Preterm (18)                                              forsythia                                 group than the
                                         Healthy (48)                                                                                        full-term group
                                                                                                                                             (p < 0.05)
                                                                                                                                             Detection of
                                                                                                                                             pathogens in
                                                                                                                                             orange and red
                                                                                                                                             clusters of sub-
                                                                                                                                             gingival plaque
                                                                                                                                             samples was lower
                                                                                                                                             in full-term group
                                                                                                                                             (16.7%) compared
                                                                                                                                             to preterm group
                                                                                                                                             (83.3%) (p < 0.01)
                                                                                                                                             Carriage of
                                                                                                                                             pathogens orange
                                                                                                   Red cluster                               and red clusters
                                         Women at risk
                                                                                                   P. gingivalis, T.                         of subgingival
                                         for miscarriage
                                                                                                   forsythensis, T.                          plaque samples
                                         or preterm deliv-
                                                            Amniotic fluid,                        denticola                                 was higher in
 Dörtbudak et al.      Austria, cross-   ery (36)
                                                            vaginal smears      15–20 weeks GA     Orange cluster:        Culturing, PCR     preterm group        Poor
 (2005)21              sectional         Preterm delivery
                                                            and dental plaque                      F. nucleatum, P.                          (p < 0.01)
                                                                                                   intermedia, P.                            The levels of
                                         Full-term delivery
                                                                                                   nigrescens, C.                            Amniotic IL-6
                                                                                                   rectus                                    and PGE2 were
                                                                                                                                             higher in women
                                                                                                                                             delivering pre-
                                                                                                                                             term (p < 0.001);
                                                                                                                                             Amniotic IL-6
                                                                                                                                             (r = 0.56, p < 0.01)
                                                                                                                                             and PGE2
                                                                                                                                             (r = 0.50, p < 0.01)
                                                                                                                                             cytokine levels
                                                                                                                                             were correlated
                                                                                                                                             with subgingival
                                                                                                                                             bacterial counts
                                                                                                                                             Postpartum bacte-
                                                                                                                                             rial carriage dif-
                                                                                                                                             ference between
                                                                                                                                             preterm and
                                                                                                                                             full-term groups
                                                                                                                                              P. gingivalis, T.
                                                                                                                                             forsythensis, P.
                                                                                                                                             intermedia, and
                                                                                                                                             P. nigrescens
                                                                                                   P. gingivalis, P.
                                         Women with                                                                                          (p < 0.05)
                                                                                                   intermedia, P.
                                         periodontal                                                                                          T. denticola
                                                                                                   nigrescens, T. for-
                                         disease (31)                                                                     Checkerboard       and C. rectus
                       USA, nested case–                    Subgingival         T1: 22 weeks GA    sythensis, T. den-
 Lin et al. (2007)64                     Preterm delivery                                                                 DNA–DNA            (p < 0.065)        Fair
                       control                              plaque              T2: Postpartum     ticola, C. rectus,F.
                                         (14)                                                                             hybridization      Patients with
                                                                                                   nucleatum, A.
                                         Full-term delivery                                                                                  a high level of
                                         (17)                                                                                                C. rectus at T1
                                                                                                                                             showed a non-sig-
                                                                                                                                             nificant tendency
                                                                                                                                             to have a higher
                                                                                                                                             risk for preterm
                                                                                                                                             births (odds
                                                                                                                                             ratio [OR] = 4.6;
                                                                                                                                             95% confidence
                                                                                                                                             interval [CI]
                                                                                                                                             Preterm group
                                                                                                                                             had lower level
                                         Pregnant women
                                                                                One time point at                                            of Lactobacilli
                                                                                recruitment (from                         Culturing using    (p = 0.009)
 Durand et al.                           Preterm delivery                                         S. mutans, Lacto-
                       USA, case–control                    Saliva              1st trimester to                          commercially kit   No difference in   Fair
 (2009)65                                (34)                                                     bacilli spp.
                                                                                8 weeks postpar-                          (CRT bacteria®)    S. mutans carriage
                                         Full-term delivery
                                                                                tum)                                                         between preterm
                                                                                                                                             and full-term
                                                                                                                                             groups (p = 0.053)

Scientific Reports |        (2021) 11:16870 |                   https://doi.org/10.1038/s41598-021-96495-1                                                                    13


                                Country, study       Groups (no. of                          Measurement       Microorganisms      detection                                Quality
           Author (year)        design               subjects)            Sample source      interval          evaluated           methods             Study findings       assessment
                                                                                                                                                       P. gingivalis was
                                                                                                                                                       detected in saliva
                                                                                                                                                       among 7 out
                                                                                                                                                       the 15 low birth
                                                                                                                                                       weight group, and
                                                                                                                                                       3 of the 8 normal
                                                     High risk (hospi-                                                                                 delivery group
                                                     talized) Pregnant                                                                                 P. gingivalis was
                                                     women (23)                                                                                        detected in plaque
           Hasegawa et al.      Japan, cross-                          Saliva and Subgin-
                                                     Normal birth                         2nd trimester        P. gingivalis       PCR                 among 8 out        Fair
           (2011)66             sectional                              gival plaque
                                                     weight (8)                                                                                        the 15 low birth
                                                     Low birth weight                                                                                  weight group, and
                                                     (15)                                                                                              4 of the 8 normal
                                                                                                                                                       delivery group
                                                                                                                                                       No report on
                                                                                                                                                       statistical data
                                                                                                                                                       regarding oral
                                                                                                                                                       P. gingivalis and
                                                                                                                                                       birth weight
                                                                                                                                                       A significant sta-
                                                                                                                                                       tistical difference
                                                                                                                                                       between the mean
                                                                                                                                                       of gram-negative
                                                                                                                                                       cocci and intrau-
                                                                                                                                                       terine fetal death
                                                                                                               and negative cocci,
                                                     Pregnant women                                                                                    cases (p = 0.04)
                                                     (243)                                                                                             A significant
                                                                                                               and negative
           Sadeghi et al.       Iran. prospective    Premature deliv-                                                              Culturing, Bacte-   relationship in
                                                                        Saliva               20–30 weeks GA    bacilli, Spirilla,                                          Fair
           (2011)67             cohort               ery (10)                                                                      ria gram staining   the presence of
                                                     Full-term delivery                                                                                spirochetes in
                                                                                                               Fusiform bacteria,
                                                     (233)                                                                                             saliva between
                                                                                                                                                       premature and
                                                                                                                                                       normal delivery
                                                                                                                                                       (p < 0.05)
                                                                                                                                                       No significant
                                                                                                                                                       relationship for
                                                                                                                                                       other bacteria
                                                                                                                                                       The amount of
                                                                                                                                                       subgingival P.
                                                                                                                                                       gingivalis of pre-
                                                     Pregnant women                                            A. actinomycetem-                       term women was
                                                     (80)                                    14–30 weeks GA    comitans, P. gingi-                     higher than that
           Cassini et al.       Italy, prospective   Preterm delivery                        (One time point   valis, T. forsythia,                    of term women
                                                                        plaque, vaginal                                             Realtime PCR                            Fair
           (2013)22             cohort               (8)                                     for microbial     T. denticola,                           None of assessed
                                                     Full-term delivery                      analysis)         F. nucleatum, P.                        periodontopatho-
                                                     (72)                                                      intermedia                              gen resulted as
                                                                                                                                                       correlated to
                                                                                                                                                       preterm low
                                                                                                                                                       P. gingivalis
                                                                                                                                                       detection was
                                                     Pregnant women                                                                                    more frequently
                                                     (95)                                                                                              detected among
                                                     Threatened prema-                                                                                 preterm group
                                                     ture labor (TPL)                                                                                  than full-term
                                                     Preterm delivery                                                                                  group among TPL
                                                                          Subgingival                          A. actinomyce-
                                                     (13)                                                                                              women
                                Japan, cross-                             plaque, unstimu-                     temcomitans,
           Ye et al. (2013)23                        Full-term delivery                      26–28 weeks GA                        ELISA               No significant dif- Good
                                sectional                                 lated saliva and                     P. gingivalis, T.
                                                     (34)                                                                                              ference in detec-
                                                                          peripheral blood                     denticola
                                                     Healthy women                                                                                     tion frequency
                                                     Preterm delivery                                                                                  of A. actinomy-
                                                     (1)                                                                                               cetemcomitans, P.
                                                     Full-term delivery                                                                                gingivalis and T.
                                                     (47)                                                                                              denticola between
                                                                                                                                                       TPL and healthy

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                        Country, study      Groups (no. of                         Measurement          Microorganisms         detection                            Quality
 Author (year)          design              subjects)           Sample source      interval             evaluated              methods         Study findings       assessment
                                                                                                                                               A sevenfold
                                                                                                                                               higher risk of
                                                                                                                                               development of
                                                                                                                                               preterm delivery
                                                                                                                                               in women with
                                                                                                        P. gingivalis, P.
                                                                                                                                               anaerobes in sub-
                                                                                                        intermedia, F.
                                            Pregnant women                                                                                     gingival plaque
                                                                                                        nucleatum, Bacte-
                                            (70)                                                                                               than women
                                                                                                        roides sp., Veillon-
 Andonova et al.        Croatia, case–      Preterm delivery   Subgingival         28–36 + 6 weeks                                             without
                                                                                                        ela sp., P. micros,    Culturing                            Fair
 (2015)24               control             (30)               plaque              GA                                                          Levels of P.
                                                                                                        S. intermedius,
                                            Full-term delivery                                                                                 gingivalis, F.
                                                                                                        A. actinomyce-
                                            (40)                                                                                               nucleatum, A.
                                                                                                        temcomitans E.
                                                                                                                                               comitans were
                                                                                                                                               statistically sig-
                                                                                                                                               nificantly higher
                                                                                                                                               in preterm births
                                                                                                                                               compared to full-
                                                                                                                                               term deliveries
                                                                                                                                               A. meyeri and
                                                                                                                                               C. bifermentans
                                                                                                        P. oralis, V.                          were significantly
                                                                                                        parvula, P.                            associated with
                                           Pregnant women                                               melanionogenica,                       higher odds of
                                           (94)                                                         P. anaerobius, P.                      preterm birth
 Hassan et al.          Saudi Arabia, Pro- Preterm delivery   Subgingival                               asaccharolticus,                       (11.2 and 5.1),
                                                                                   2nd trimester                            Culturing                             Fair
 (2016)68               spective cohort    (22)               plaque                                    C. subterminate,                       with the estimate
                                           Full-term delivery                                           C. perfringens, C.                     of C. bifermentans
                                           (72)                                                         clostridioforme, C.                    showing greater
                                                                                                        bifermentans, E.                       precision (95%
                                                                                                        lenta, A. meyeri                       confidence inter-
                                                                                                                                               val = 1.5, 17.5)
                                                                                                                                               (p < 0.05)
                                            Pregnant women
                                                                                                                                               P. gingivalis and
                                                                                                        P. gingivalis, P.                      T. denticola were
                                            Preterm low birth
                                                                                                        intermedia, T.                         significantly more
                        Argentina, cross-   weight delivery     Subgingival
 Usin et al. (2016)69                                                              3rd trimester        forsythia, T. denti- PCR               prevalent in Full- Fair
                        sectional           (18)                plaque
                                                                                                        cola, A. actinomy-                     term normal birth
                                            Full-term normal
                                                                                                        cetemcomitans                          weight delivery
                                            birth weight
                                            delivery (116)
                                            Pregnant women                                                                                     Higher peri-
                                                                                                        P. gingivalis, P.
                                            (330)                                  T1: During preg-                                            odontopathogenic
                                                                                                        intermedia, F.
 Costa et al.           Brazil, case–       Preterm delivery   Gingival crevicu-   nancy                                       DNA-DNA         bacteria burden
                                                                                                        nucleatum, A.                                            Fair
 (2019)25               control             (110)              lar fluid, blood    T2: at the time of                          hybridization   (PBB) did not
                                            Full-term delivery                     delivery                                                    increase the risk
                                            (220)                                                                                              of preterm birth
                                                                                                                                               P. gingivalis-
                                                                                                                                               related placenta
                                                                                                                                               infection with
                                            Pregnant women                                          P. gingivalis, T.
                                                                                                                                               adverse pregnancy
                                            (94)                                                    forsythia, T. denti-
                                                              Subgingival                                                                      outcome group
 Gomez et al.           Colombia, case–     Adverse birth                                           cola, E. nodatum,
                                                              plaque, placental    During pregnancy                      PCR                   reflects high levels Good
 (2020)70               control             outcome (23)                                            A. actinomyce-
                                                              samples                                                                          of IFN-γ with
                                            Non-adverse birth                                       temcomitans, F.
                                                                                                                                               a significative
                                            outcome (17)                                            nucleatum
                                                                                                                                               decreasing of NK-
                                                                                                                                               related cytokines
                                                                                                                                               (p < 0.05)
                                                                                                                                               Quantity of P.
                                                                                                                                               gingivalis and
                                                                                                                                               T. forsythia in
                                           Pregnant women
                                                                                                                                               plaque samples
                                                                                                                                               and detection
                                                                                                   P. gingivalis, P.                           frequency of P.
                                           preterm labor
                                                                                                   intermedia, T.                              intermedia in
                                           (TPL) (Low birth     Saliva, Subgingival
                        Japan, prospective                                                         forsythia, T. denti-                        saliva were higher
 Ye et al. (2020)71                        weight) (9)          plaque, placental During pregnancy                      qPCR, ELISA                               Good
                        cohort                                                                     cola, A. actinomy-                          in TPL- Low
                                           Threatened           samples
                                                                                                   cetemcomitans, F.                           birthweight deliv-
                                           preterm labor
                                                                                                   nucleatum                                   ery than those
                                           (Normal weight
                                                                                                                                               in TPL-Healthy
                                           delivery) (19)
                                                                                                                                               delivery group
                                           Control (36)
                                                                                                                                               and/or in control-
                                                                                                                                               healthy delivery

Scientific Reports |         (2021) 11:16870 |                      https://doi.org/10.1038/s41598-021-96495-1                                                                   15


                                Country, study     Groups (no. of                      Measurement       Microorganisms     detection                              Quality
           Author (year)        design             subjects)           Sample source   interval          evaluated          methods             Study findings     assessment
                                                                                                                                                The detection fre-
                                                                                                                                                quency of P. gin-
                                                                                                                                                givalis in plaque
                                                                                                                                                and placenta
                                                                                                                                                were significantly
                                                                                                                                                correlated with
                                                   Pregnant women
                                                                                                                                                low birthweight
                                                                                                                                                delivery in TPL
                                                   Threatened pre-
                                                                                                                                                group. In the
                                                   term labor (TPL)
                                                                                                                                                receiver operating
                                                   (Low birthweight) Saliva, Subgingival
                                Japan, prospective                                                                                              characteristic
           Ye et al. (2020)72                      (14)               plaque, placental 26–28 weeks GA   P. gingivalis      qPCR                                     Good
                                cohort                                                                                                          curve analysis, an
                                                   Threatened         samples
                                                                                                                                                amount of P. gin-
                                                   preterm labor
                                                                                                                                                givalis in plaque
                                                   (Healthy delivery)
                                                                                                                                                ≥ 86.45 copies
                                                                                                                                                showed a sensitiv-
                                                   Control (48)
                                                                                                                                                ity of 0.786 and a
                                                                                                                                                specificity of 0.727
                                                                                                                                                (AUC 0.792)
                                                                                                                                                for predicting
                                                                                                                                                low birthweight
                                                                                                                                                delivery in TPL
                                                                                                                                                There was no sig-
                                                                                                                                                nificant difference
                                                                                                                                                in periodontal
                                                                                                                                                parameters and
                                                                                                         P. gingivalis, P.
                                                                                                                                                serum IgG levels
                                                                                                         intermedia, T.
                                                                                                                                                for periodon-
                                                                                                         forsythia, T. den-
                                                   Pregnant women                                                                               tal pathogens
                                                                                                         ticola, A. actino-
                                                   (90)                                                                                         between PLBW
                                                   Preterm low birth                                                                            and healthy deliv-
                                China, prospec-                                                          F. nucleatum,       Culturing, qPCR,
           Ye et al. (2020)73                      weight (PLBW)       Saliva          2nd trimester                                            ery (HD) groups Good
                                tive cohort                                                              E. saphenum,        ELISA
                                                   (22)                                                                                         The amount of
                                                                                                         Fretibacterium sp.,
                                                   Healthy delivery                                                                             E. saphenum in
                                                                                                         R. dentocariosa
                                                   (68)                                                                                         saliva and serum
                                                                                                         Human oral taxon
                                                                                                                                                IgG against A.
                                                                                                         (HOT) 360, TM7
                                                                                                         sp. HOT 356
                                                                                                                                                were negatively
                                                                                                                                                correlated with

                                                  Table 4.  Association between oral microorganisms during pregnancy and adverse birth outcome—preterm

                                                  Impact of periodontal disease on oral microorganisms during pregnancy.                       Three ­studies75,79,80 did
                                                  not identify any significant findings that the clinical periodontal condition and the levels of subgingival micro-
                                                  biome during pregnancy are related to pregnancy complications.
                                                      However, when subgingival plaque in women with threatened premature labor was assessed, P. gingivalis was
                                                  found in the half of patients with periodontal ­disease74. The presence of Eikenella corrodens and Capnocytophaga
                                                  spp. were significantly related to preterm birth and low birth weight respectively (p = 0.022 and p = 0.008)75. No
                                                  statistical significance was found in overall microbiome diversity in comparison of healthy gingiva and gingivitis
                                                  groups. However, bacterial taxa like Mogibacteriaceae and genera Veillonella and Prevotella were more prevalent
                                                  in the gingivitis g­ roup79.

                                                   Association between oral microorganism during pregnancy and adverse birth outcome. Five
                                                  ­studies22–24,71,72 showed that the amount of P. gingivalis in subgingival plaque was significantly higher in women
                                                   with preterm birth than women with term birth. Also, CFU counts of red and orange complex pathogens,
                                                   in which P. gingivalis belongs, from dental plaque in women with preterm delivery was significantly higher
                                                   (p < 0.01)21. The levels of Fusobacterium nucleatum, T. forsythia, Treponema denticola, and A. actinomycetem-
                                                   comitans were highly related to the preterm births compared to term d    ­ eliveries22,24.
                                                       However, higher periodontopathogenic bacteria burden did not increase the risk of preterm birth, despite the
                                                   increase in periodontal disease ­activity25. The levels of microorganisms like P. gingivalis, T. forsythensis, T. denti-
                                                   cola, P. intermedia, and F. nucleatum were not significantly higher in the preterm group than in the term ­group64.

                                                  Impact of systemic diseases on oral microorganism during pregnancy. Gestational diabetes mel-
                                                  litus (GDM). Two ­studies82,85 did not find significant differences in either clinical periodontal disease nor in
                                                  the diversity and richness between women with GDM and non-GDM. The detection rate and the number of
                                                  oral bacteria in women with GDM were higher than in non-GDM women, especially in the second trimester
                                                  of ­pregnancy84. Oral bacterial detection rate and total number in several species, such as black-pigmented bac-
                                                  teria, were significantly higher in pregnant women with GDM than those in non-diabetic pregnant ­women84.
                                                  Conversely, oral bacterial detection of oral streptococci and lactobacilli did not show any significant d
                                                                                                                                                          ­ ifferences84.

          Scientific Reports |       (2021) 11:16870 |                     https://doi.org/10.1038/s41598-021-96495-1                                                           16

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