Steven Offenbacher DDS, PhD, MMSc James D Beck, PhD Center for Oral and Systemic Disease Comprehensive Center for Inflammatory Disorders University of North Carolina at Chapel Hill 2001 Frances Glenn Mayson Symposium “Pre-term Labor: Is the Fetus Trying to Tell Us Something November, 2001

What are the major pregnancy complications? n Preterm : <37 wks completed gestational age n Low birth weight <2500g n PPROM : Preterm premature rupture of membranes n PTL : Preterm labor n Preeclampsia : Pregnancy induced hypertension and proteinuria Key Terms (outcomes) Partus & Neonate Obstetric

Why is this important? n About 10% all births are preterm low birth weight n Preterm birth accounts for two-thirds of all infant mortality n Each gram of fetal weight under 2500g costs 75$ in neonatal unit expenses n USA NICU costs in excess of 5.5 billion dollars a year as consequence of preterm births Preterm Births

Infant Health Issues n Improvements in perinatal care have dramatically improved survival rates of premature births n This has resulted in improvement in neonatal mortality n BUT There has been no decrease in the rate of preterm births. n Actual costs and number of low birth weight survivors with disabilitites has increased.

Low-birthweight live births (Percent of live births less than 2,500g in US during selected years according to maternal race)

Infant Health Issues  Since 1981, percent born (survivors) < 37 weeks has increased 17%  Significant disparities by race and ethnicity  Preterm infants are – 7x more likely to die prior to 1st birthday – at increased risk for: neuro-developmental, respiratory disorders, learning disabilities, delayed development, cerebral palsy

KNOWN RISK FACTORS nTobacco, drug or alcohol usage. n Previous preterm delivery n Concurrent genitourinary tract infection: Bacterial vaginosis, chorioamnionitits, STDs MAJOR

KNOWN RISK FACTORS n Maternal age, weight, stature, cervical length n Nulliparous n Stress – physical & social, familial support, SES n Familial history n Level of prenatal care MINOR 25-50% of preterm births occur in absence of significant risk factors

Cervical dilation Fetus Amnion Cervix Vagina Fetus Amnion Vaginal Infection [BV, bacterial vaginosis] Uterine contraction Pathogenesis of bacterial vaginosis induced prematurity ? Fetus Amnion Uterus Placenta & Membranes IL-1 , TNF , PGE 2 MM

PGE2, IL-1b, TNFa, IL-6, MMPs Lymphocytes Monocytes Gram- Flora Leukocyte Wall Epithelial Ulceration Bacterial Invasion, Bacteremia Periodontitis: Intra-Oral to Extra-Oral Pathway

Biochemical Mediators of Prematurity IL IL-1  PGE 2 IL-1 TNF TNF  LPS Can induce uterine contraction, cervical dilation, labor or abortion, placenta damage, inhibit fetal growth, mediate preterm delivery, low birth weight and neonatal morbidity. IL-6 IL IL-6

Biochemical mediators and indicators of fetal stress n Fetal antibody to bacteria : e.g. Rubella IgM n sICAM: soluble Intracellular adhesion molecule, a marker of vascular (endothelial) stress n Fetal CRP (C-reactive protein), acute phase response

Prospective Study of Pregnant Mothers Maternal Periodontitis Models that adjust for obstetric risk factors: e.g. Demographic, smoking, SES, infection, parity Clinical periodontal disease as exposure for abnormal pregnancy outcome Risk Prematurity (gestational age <37 weeks) Low Birth Weight (<2500g) Impaired Fetal Growth (low weight for getational age)

Prospective Study of Pregnant Mothers Prematurity Low Birth Weight Impaired Fetal Growth Maternal Periodontitis Biological Mechanisms Microbial Infection Maternal Antibody Fetal Exposure Pregnancy Outcome

OCAP- Oral Conditions and Pregnancy- NIDCR funded Preliminary Analyses Prospective study of pregnant women to determine contribution of periodontal disease to pregnancy complications. Exclusions included HIV, drug abuse, diabetes, hypertension, STD Currently enrolled over 1200 subjects, expected total of about Data presented reflect 814 deliveries

19 Flow Chart of Oral Conditions and Pregnancy Study (OCAP) University of North Carolina Chapel Hill and Duke University Medical Center Presentation at Prenatal Clinic Refuse Ineligible Eligible Women < 26 weeks gestation Refer for dental cleaning at UNC School of Dentistry Obtain samples at initial prenatal or enrollment visit Consent Monitor for PTL, PROM, bacterial vaginosis and other infections Obtain samples during immediate postpartum period

OCAP Measurements OB Risk profile: Demographic, obstetric, medical, behavioral & socioeconomic parameters Maternal Blood Antepartum IgG & IgM to Periodontal & Vaginal Organisms Inflammatory Mediators IL-1, TNFa, CRP, sICAM, IL-6, total 8-iso PGF2a Interleukin SNPs Vaginal & Cervical Vaginal and Periodontal Organisms Inflammatory Mediators IL-8, IL-1, TNFa, IL-6 Periodontal Plaque Gingival Crevicular Fluid Periodontal Exam

OCAP Measurements OB Risk profile: Patient interview, chart abstraction Repeat: Maternal Blood Postpartum Periodontal Plaque Gingival Crevicular Fluid Periodontal exam Fetal cord blood Neonatal data IgM to Periodontal Organisms (and vaginal) Inflammatory Mediators IL-1, TNFa, CRP, sICAM, IL-6, total 8-iso PGF2a

OCAP Pregnancy Outcomes 1.Prematurity (Gestational Age <37 weeks). Excluding elective abortion 2.Birth weight (<2500g) 3.Weight for gestational age (small for gestational age, growth restriction) Weight Gestational Age (weeks) Prematurity 2500g Growth Restriction (lowest 10%)

Health= no PD>3mm or CAL>2mm Mod-severe= 4+ sites 5+mm PD and 2+mm C AL Incidence/Progression = 4+ sites with PD increase 2+mm

Maternal Periodontitis and Gestational Age Adjusted for race, smoking, food stamps, marital status, previous preterm birth, first time birth, bacterial vaginosis, chorioamnionitis,

Maternal Periodontitis and Birth Weight Adjusted for race, smoking, food stamps, marital status, previous preterm birth, first time birth, bacterial vaginosis, chorioamnionitis,

Distribution of time to premature delivery among 767 births based upon maternal antepartum periodontal status Gestational Age (weeks) % Pregnant Health = absence of any PD>3mm and no sites with AL>2mm Mod-Severe = 5mm PD and 2mm AL at 4 or more sites

Adjusted* Prevalence of Gestational Age at Delivery by Antepartum Maternal Periodontal Disease Status % of Births Maternal Antepartum Periodontal Status * Maternal age, race, smoking, marital status, food stamps, bacterial vaginosis, and chorioamnionitis. Health = absence of any PD>3mm and no sites with CAL>2mm Mod-Severe = 5+mm PD and 2+mm CAL at 4 or more sites

Adjusted* Prevalence of Birth Weight at Delivery by Antepartum Maternal Periodontal Disease Status % of Births Maternal Antepartum Periodontal Status * Maternal age, race, smoking, marital status, food stamps, bacterial vaginosis, and chorioamnionitis. HealthMildMod/Severe

Effects of maternal periodontal disease incidence/ progression * during pregnancy on mean birth weight by gestational age adjusting for maternal race, sex of baby and parity * 4+ sites with 2+mm PD increase

Perinatal Mortality Health: 1.97% Mild: 3.16% Mod-severe: 16.3% 34/ 967, (p<0.0001)

Antepartum maternal periodontal disease status and progression on birth weight for all births of GA<37 weeks

Prospective Study of Pregnant Mothers Prematurity Low Birth Weight Impaired Fetal Growth Maternal Periodontitis Biological Mechanisms Microbial Infection Maternal Antibody Fetal Exposure Pregnancy Outcome

YES P.g. placental infection NO P.g. placental infection Runted fetus Normal weight fetus MW Fetal Memb. Placenta Liver Positive control Fetal Memb. Placenta Spleen 1,500 bp 400 bp 1,000 bp Fetus #63-L3 Fetus #63-R2 P.g. PCR signal in pregnant mouse with 2 small/5 normal fetus

P. gingivalis B. forsythus T. denticola E. corrodens C. gingivalis C. sputigena C. ochracea A. actino. a S. mitis S. oralis S. sanguis S. gordonii S. intermedius V. parvula A. odontolyticus P. intermedia P. nigrescens P. micros F. nucleatum F. periodonticum E. nodatum S. constellatus C. showae C. rectus C. gracilis S. noxia A. actino. b Socransky et al * * * * * * * * * * * *** * *

P.g. P.n. P.i. B.f. C.r. A.a. E.c. F.n. T.d. P.m. C.o. V.p. S,s. S.o. S.s. A.v. Plaque samples Pooled bacterial standards Maternal Plaque Plaque lanes

Maternal serum Maternal serum Human IgG (200-50ng/ml) Protein A Maternal IgG P.g. P.n. P.i. B.f. C.r. A.a. E.c. F.n. P.m. C.o. V.p. S.s. T.d. E.n. S.i. S.o. M.c. P.b. B.v. G.v. Vaginal Oral

Fetal IgM Fetal IgM Human IgM (200-50ng/ml) P.g. P.n. P.i. B.f. C.r. A.a. E.c. F.n. P.m. C.o. V.p. S.s. T.d. E.n. S.i. S.o. M.c. P.b. B.v. G.v. Vaginal Oral Protein A

Fetal IgM Fetal IgM Human IgM (200-50ng/ml) P.g. P.n. P.i. B.f. C.r. A.a. E.c. F.n. P.m. C.o. V.p. S.s. T.d. E.n. S.i. S.o. M.c. P.b. B.v. G.v. Vaginal Oral Protein A

Prevalence of Orange and Red Organisms in Maternal Plaque From 337 Term and 106 Preterm Deliveries Organism C. rectus F. nucleatum P. micros P. nigrescens P. intermedia P. gingivalis B. forsythus T. denticola Term Preterm Orange Red Prevalence Orange>Red Microbes Prevalence of O&R for Term ~ Preterm (n=337) (n=106) Maternal Plaque

# Orange Cluster Organisms Present % Distribution of Red Cluster (1+ Positive) Frequency Distribution of Red Microbial Cluster Organisms as Function of Orange Cluster Organisms within Maternal Plaque DNA Microbial Macroarray “Checkerboard” Red cluster organism detection is enhanced by or “requires” Orange cluster organisms

# Orange Cluster Organism IgG Positive % Distribution of Red Cluster Positive IgG Frequency Distribution of Maternal IgG Antibody Responses to Red Microbial Cluster Organisms as Function of Orange Cluster IgG Responses Maternal IgG (n=390) Red seropositity is enhanced by or “requires” Orange seropositivity

Prevalence of Orange and Red Organism Positive IgG in Maternal Sera from 289 Term and 101 Preterm Deliveries Organism C. rectus F. nucleatum P. micros P. nigrescens P. intermedia P. gingivalis B. forsythus T. denticola Term Preterm Orange Red Prevalence of positive maternal IgG responses for O&R Organisms among preterm has a non-significant trend to be generally less than that of full term (n=289) (n=101) Maternal IgG (n=390)

Prevalence of Orange and Red Fetal IgM responses among 271 Term and 80 Preterm Neonates Organism C. rectus F. nucleatum P. micros P. nigrescens P. intermedia P. gingivalis B. forsythus T. denticola Term Preterm Orange Red (n=271)(n=80) Fetal IgM (n=351) P= P=0.02 NS NS P=0.03 P=0.015 Prevalence of Fetal IgM seropositive responses to Orange and Red Cluster organisms is signficantly higher among preterm newborns

# Orange Cluster Organism IgM Positive % Distribution of Red Cluster Positive IgM Frequency Distribution of Fetal IgM Responses to Red Microbial Cluster Organisms as Function of Fetal Orange Cluster IgM Responses Fetal Red cluster IgM response appears in absence of Orange cluster organisms, [Orange IgM requires Red IgM response]

Rate of Prematurity as related to Maternal and Fetal Seropositivity to Orange and Red Cluster Microbes (n=287) Maternal IgG Red Cluster Seroreactivity Fetal IgM Orange Cluster Seroreactivity A-C, P=0.03 B-C, P< D-C, P= % 20.9% 17.5% 32.8% 33.3% 16.2% 66.7% 27.3% A B C D Column test: P<0.0001, OR=4.3, ( ) Row test: P=0.0003, OR=2.37, ( ) Combined: OR=10.3

Incident Periodontal Disease Progression (% mothers with 4+ sites, 2+mm PD increase) as related to Maternal and Fetal Seropositivity to Orange and Red Cluster Microbes (n=287) Maternal IgG Red Cluster Seroreactivity Fetal IgM Orange Cluster Seroreactivity % 31.0 % 61.1 % 28.0 % A B C D B-C, OR=3.5, P=0.015

Maternal Periodontal Disease GA<37 weeks Fetal Orange Complex IgM Response A B Prevalent, OR=2.44, 95% CI = 1.18, 5.04 Seropositive, OR= % CI = 2.22,11.3 C Incident Progression, OR=2. 34, 95% CI = 1.001, 5.4 Models of Interrelationships among Maternal Periodontal Disease, Fetal Exposure to Oral Pathogens and Prematurity

Postpartum Maternal Periodontal Disease (4+ sites with PD> 5mm) and Mean Fetal Cord Serum CRP by Gestational Age (n=186) Gestational Age in Weeks Mean Fetal CRP (ug/mL)

Fetal IgM C. rectus and Mean Fetal Cord Serum CRP by Gestational Age (n=186) Gestational Age in Weeks Mean Fetal CRP (ug/mL)

Patterns of Maternal Colonization, maternal IgG, fetal IgM and prematurity Maternal Infection Maternal Antibody

Patterns of Maternal Colonization, maternal IgG, fetal IgM and prematurity Maternal Infection Maternal Antibody

Patterns of Maternal Colonization, maternal IgG, fetal IgM and prematurity Maternal Infection Maternal Antibody No protective Red Complex IgG Fetal Exposure Fetal IgM to Red Complex

Patterns of Maternal Colonization, maternal IgG, fetal IgM and prematurity Maternal Infection Maternal Antibody No protective Red Complex IgG Fetal IgM to Red & Orange Complexes Increased Fetal CRP Fetal Exposure Red and Orange Abnormal Pregnancy Outcome Maternal Periodontal Disease Progression ?

Conclusions 1.Results suggest that antepartum periodontal disease as well as incidence/ progression of periodontal disease is a significant independent risk factor for prematurity, low birth weight and fetal growth impairment. 2.Orange & Red complex organisms are similar at post-partum in preterm and full-term mothers. 3.Fetal IgM response to Red complex organisms occurs in absence of orange IgM, indicating early or initial exposure to maternal organims of the red complex

Conclusions 4.Maternal antibody to Red complex appears to “prevent” fetal exposure to maternal oral microbes. 5.Fetal IgM seropositive responses coupled with seronegative maternal red complex IgG confers greatest risk for prematurity OR=10.3.

New findings: University of Alabama Study of Prematurity (Jeffcoat & Hauth) *Adjusted for smoking, parity, age and race [p<0.05, as compared to no periodontal disease, full term deliveries] Mild-moderate Severe N=1313, Examined wks

New findings : Nestor Lopez (Santiago, Chile) Effects of Periodontal Therapy on rate of prematurity 850 pregnant women <20 weeks 390 women with periodontal disease ( 1 or more sites with PD>4mm and >3mm attachment loss). Random assignment to 2 groups; periodontal scaling & root planing vs delayed treatment (post- partum). Attrition low:24 and 15, respectively

Nestor Lopez (Santiago, Chile) Risk Factor Distribution Untreated 28 (4.5) 1.2 (0.9) Treated 27 (4.3) 1.4 (1.1) Age Parity %Primiparous % previous preterm %previous miscarriage P Value

Nestor Lopez (Santiago, Chile) Risk Factor Distribution (%) Untreated Treated Smokers UTI Antibiotic Tx Vaginosis P Value

Nestor Lopez (Santiago, Chile) Results: Untreated 3344(598) 10.1%(n=19) Treated 3501 (429) 1.8%(n=3) Mean birthweight Incident GA<37* P Value *Univariate RR =5.49, 95%CI=

Nestor Lopez (Santiago, Chile) Results: (logistic regression) Odds Ratio % CI No periodontal Tx Previous GA<37 Low # Prenatal visits Mother underweight P Value Variable Also controlled for age, parity, smoking, vaginosis, first prenatal visit

Nestor Lopez (Santiago, Chile) Conclusion: Early studies by Lopez, as well as those reported by Papapanou, suggest that periodontal therapy during pregnancy may reduce the risk of prematurity.

Clinical Implications of OCAP Findings  Antepartum mild or moderate-severe periodontal disease and increases in PD during pregnancy appear to increase the risk for fetal exposure and pregnancy problems.  The magnitude of periodontal disease impact is significant with Odds ratios 2-10 depending on baseline status and worsening of condition during pregnancy  Evidence of fetal exposure to periodontal pathogens raises bacteremia risk questions  Maternal antibody appears to be protective  Prevention would appear to be prudent clinical management strategy

Implications for Health Care  Association may not be causal in nature.  The potential benefits of treatment have not yet been established.  Periodontitis is both preventable and treatable.  Periodontal health is necessary for overall health and periodontal disease may be as deleterious to pregnant mother as smoking, or alcohol consumption.  We need to educate the public, our patients and health care professionals to promote the need for good oral health as an important part of a healthy lifestyle.

OCAP Team  UNC Epidemiologists  Susi Lieff (Ecol)  Rosemary McKaig (Ecol)  Pierre Beukins (SPH)  Gary Slade (Ecol)  UNC Clinical Researchers  Heather Jared (Hyg)  Tracy Kachold (Hyg)  Marsha Black (Tech)  Sally Timlin (Nurse)  Karen Dorman (Nurse)  Sacha Singh (Perio)  UNC Clinical Scientists  Ken Moise (MFM)  Kim Boggess (MFM)  Bob Strauss (MFM)  Carl Bose (Ped)  Phoebus Madianos (perio)  John Gilmore (Psych)  Janet Southerland (Ecol)  Duke Clinical Scientists  Phil Heine (MFM)  Amy Murtha (MFM)  Rick Auten (Ped)  Ricki Goldstein (Ped)

OCAP Team  UNC Lab Scientists  Catherine Champagne  Estelle Riche  UNC Lab Technicians  Frances Smith  Russ Levy  Sandra Elmore  Jeremy Eissens  Jermaine Fuller  Christine Downey  UNC Computer Specialists  Kevin Moss  Morris Worley  UNC Students & Fellows  Mark Suttle  Dongming Lin  Alvin Yeo  Julie Hofheimer  UNC Administrative  Gail Plaisance

Colleagues Epidemiologists  Sam Arbes  Jim Beck  Paul Eke  John Elter  Susi Lieff  Rosemary McKaig  Jim Pankow  Gary Slade Basic Scientists  Catherine Champagne  Sara Geva  Sid Kalachandra  Estelle Riché  Chitpol Siddhivarn Clinical Scientists  Kim Boggess  Elisa Ghezzi  Alison Lohman  Phoebus Madianos  Sally Mauriello  Steve Offenbacher  David Paquette  Rocio Quinonez  Janet Southerland  Ray Williams Behavioral Scientists  Julie Hofheimer

Clinical Research  Peggy Allen  Marsha Black  Heather Jared  Tracy Kachold  Sue Riggsbee  Barbara Oliver  Betsi Petway Research Students  Nadine Brodala  Waka Kadoma  Dongming Lin  Nora Rooney  Diane Sitki  Mark Suttle Valuable Assistants Administration  Gail Plaisance Computer Specialists  Kevin Moss  Morris Worley Laboratory  Christine Downey  Jeremy Eissens  Kendra Floyd  Jermaine Fuller  Russ Levy  Frances Smith

Collaborators UNC Epidemiology  Gerardo Heiss  Jim Pankow  HA Tyroler UNC Cardiology  Wayne Cascio  Efthymios Deliargyris  Alan Hinderliter  Sid Smith UNC Ob/Gyn  Kim Boggess  Karen Dorman  Bob Strauss  Steve Wells UNC Psychiatry  John Gilmore UNC Biostatistics  Woody Chambless  David Couper  Gary Koch UNC Pediatrics  Carl Bose  Diane Marshall Duke Ob/Gyn  Elizabeth Livingston  Amy Murtha Duke Pediatrics  Rick Auten

Acknowledgements NIH & NIDCR Grants :  DE R01HD26652, DE08289, DE  Contracts from NHLBI  R01 DE 11551, P60 DE Dental Organizations :  AAP, ADA Corporate sponsors :  Procter and Gamble, Block Drug, Colgate, Orapharma, Interleukin Genetics, Sunstar, Merck, Glaxo-Wellcome, Phillips

Steven Offenbacher & James Beck S t e v e n O f f e n b a c h e r & J a m e s B e c k