university of north carolina at chapel hill - web view · 2003-12-22school of dentistry....

MOTOR: MATERNAL ORAL THERAPY TO REDUCE OBSTETRIC RISK

CENTER FOR ORAL AND SYSTEMIC DISEASES

MOTOR : Maternal Oral Therapyto Reduce Obstetric Risk

Clinical Protocol

A University of North Carolina at Chapel Hill Multi-Center Clinical Trial Protocol

Three Clinical Performance Sites : University of Alabama1

University of Texas at San Antonio2

University of North Carolina at Chapel Hill3/Duke University Medical Center4

Data and Statistics Coordinating Center:University of North Carolina at Chapel Hill School of Public Health5

PREPARED BY STEVEN OFFENBACHER, DDS, PHD3, JAMES D. BECK PHD3

AND THE MOTOR INVESTIGATORS

MOTOR: Clinical Protocol, October 1, 2003

UNIVERSITY OF NORTH CAROLINA AT CHAPEL HILLSCHOOL OF DENTISTRY

CHAPEL HILL, NC 27599-7450

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UNC: LLOYD CHAMBLESS5, DAVID COUPER PHD5, DAWN STEWART, PHD5, HEATHER L. JARED, RDH, MS3,4

DUKE: PHIL HEINE MD4, AMY P. MURTHA MD4

UAB: JOHN HAUTH MD1, MARJORIE JEFFCOAT DMD1

UTHSCSA: DAVID COCHRAN DDS, PHD2, DONALD DUDLEY MD2

OCTOBER 1, 2003

MOTOR: Clinical Protocol, October 1, 2003 2


MOTOR Investigators

DATE OF PROTOCOL: October 1, 2003

PRINCIPALINVESTIGATOR: Steven Offenbacher, DDS, PhD, MMSc

Professor and DirectorCenter for Oral and Systemic Diseases and Department of PeriodontologyUniversity of North Carolina School of DentistryChapel Hill, NC 27599-7450Telephone: 919-962-7081Fax: 919-966-3683E-mail: [email protected]

CO-PRINCIPALINVESTIGATOR: James D. Beck

Professor and Co-DirectorCenter for Oral and Systemic Diseases and Department of PeriodontologyUniversity of North Carolina School of DentistryChapel Hill, NC 27599-7450Telephone: 919-962-7081Fax: 919-966-3683E-mail: [email protected]

THREE INVESTIGATIVE CENTERS

PERIODONTAL PI OBSTETRIC PI CONTACT PERSON Steven Offenbacher, DDS, PhD, MMSc Dr. R. Phillips Heine Heather Jared, RDH, MsProfessor and Director Director Maternal Fetal Medicine Dental Research CenterCenter for Oral and Systemic Diseases Associate Professor UNC Chapel HillDepartment of Periodontology DUMC (919) 962-7081UNC School of Dentistry P O Box 3967 (919) 966-7537Chapel Hill, NC 27599-7450 Durham, NC 27710Telephone: 919-962-7081 Telephone: (919) 681-5220Fax: 919-966-3683 Fax: (919) 681-7861E-mail:[email protected] Email: [email protected]

Dr. Marjorie K. Jeffcoat* Dr. John Hauth Eliz G. BoltonChairman, Dept. of Periodontology UAB School of Medicine UAB School of Dentistry


mailto:[email protected]



School of Dentistry OHB 458 (205) 934-4506University of Alabama at Birmingham Birmingham, AL 35294-7333 fax-(205) 934-79011919 7th Avenue, S. (205) 934-5611 Birmingham, AL 35294-7333Birmingham, AL [email protected]

Dr. David L. Cochran Dr. Donald Dudley N.J. (Jodie) HarrisonUTHSCSA/Periodontics-7894 7703 Floyd Curl Dr. UTHSCSA/Periodontics-78947703 Floyd Curl Dr. Mail Code 7831 7703 Floyd Curl Dr.San Antonio, TX 78284-3900 San Antonio, TX 78284-3900 San Antonio, TX [email protected] [email protected] (210) 567-3601 fax-(210) 567-

6299 [email protected]

* Dr. Jeffcoat has relocated to University of Pennsylvania where she is serving as Dean. She will remain study PI at UAB maintaining a joint appointment, but will be replaced locally by Dr.

Michael Reddy at UAB who will be the on-site periodontal investigator.

Data and Statistics Coordinating Center

Lloyd (Woody) E.. Chambless, PhD [email protected] of North Carolina137 E. Franklin Street, Suite 400Mail Station: 8030, Room 28Chapel Hill, NC 27514Phone: 919-962-3264 Fax: 919-962-3265

David Couper, PhD (Co-PI) [email protected] of North Carolina137 E. Franklin Street, Suite 400Mail Station: 8030, Room 12Chapel Hill, NC 27514Phone: 919-962-3229 Fax: 919-962-3265

Dawn Stewart, MS [email protected] of North Carolina137 E. Franklin Street, Suite 400Mail Station: 8030, Room 34Chapel Hill, NC 27514Phone: 919-962-3029 Fax: 919-962-3265








mailto:%[email protected]


Abstract

STUDY OBJECTIVES: The principal objective is to conduct a 5 year, multi-centered, clinical trial enrolling mothers with periodontal disease prior to 236

weeks gestational age, randomizing to one of two treatment arms: Group 1- Scaling and root planning with a subgingival polish, by 236

gestational age or Group 2- post-partum scaling and root planning with a subgingival polish (delayed treatment control). The overall goal of this clinical trial is to satisfy the following specific aims: 1) to determine the effects of maternal periodontal treatments during the second trimester on the rate of preterm birth at <37 weeks gestational age (GA) 2) to determine the effects of maternal periodontal treatments during the second trimester on mean birth weight adjusting for gestational age among neonates GA<37 weeks and on neonatal morbidity, and 3) to collect and archive biological samples during the conduct of trial to enable future studies regarding mechanism of infectious pathogenesis.

STUDY DESIGN: The intervention is designed as a multi-center, randomized, controlled, clinical trial to determine the effects of periodontal therapy on the rate of preterm birth. Study participants will be assigned to one of two study arms. All pregnant women who present to the designated OB clinics are potential subjects for this study. A total of 1800 patients will be enrolled at 3 performance sites, enrolling about 600 subjects at each site at a rate of about 171 subjects/year at each site, randomly assigning these subjects to one of 2 treatment arms. Each performance site will enroll about 300 subjects into each treatment group using the intent-to-treat principle, obtaining follow-up on all subjects.

In treatment Group 1 participants will be assigned to standard localized periodontal therapy of scaling and root planning with subgingival polishing between three and six months of gestation. Group 2 will receive the same local periodontal therapy immediately following delivery.

MASKING: The dental examiner will not be aware of the randomization treatment assignments of participants until after a complete baseline periodontal examination has been conducted. The study protocol allows the dental examiner to know the treatment assignment of participants but this knowledge will not affect the assessment of the primary obstetric outcome of the study. OB personnel or individuals collecting OB data will be masked as to dental treatments. At delivery the second dental exam will be made without the examiner knowing the pregnancy outcome.



SAMPLE SIZE: The target sample size is to enroll 1800 pregnant moms (600 at each performance site) who meet the inclusion criteria at study entry and consent to participate in the study.

METHOD FOR SUBJECTASSIGNMENT: All pregnant women who present to the designated OB clinics are

potential subjects for this study. Patients will be consented, screened for eligibility and randomized to one of two study arms. Randomization will be performed using a computer-generated assignment scheme designed and performed in a masked manner by the data coordinating center.

PRIMARY and SECONDARY OUTCOME MEASURES:

The primary outcome is preterm delivery at less than 37 weeks gestational age, as determined by ultrasound dating. Secondary outcomes include (1) preterm delivery less than 35 weeks, (2) weight for gestational age, and (3) neonatal morbidity/mortality. It is our central hypothesis that mothers with periodontitis that receive periodontal treatment during the second trimester of pregnancy will experience a lower rate of preterm delivery at <37 weeks and secondarily <35 weeks; that periodontal treatment of these pregnant mothers will result in an increase in the weight for gestational age of deliveries occurring less than 37 weeks gestational age and reduce neonatal morbidity and mortality. We will determine the effects of periodontal therapy on the rate of preterm birth at GA<37 weeks as the principal outcome and on mean birth weight among neonates with GA<35 weeks, as a secondary outcome adjusting for race, gender and gestational age.

POTENTIAL CONFOUNDERS AND COVARIATES:

There are many potential risk factors that relate to preterm birth and growth restriction that need to be considered in this investigation. There are also exposures, effect modifiers and covariates that influence periodontal disease status and preterm birth. Data will be collected on the major variables of interest to include race, age, smoking, previous preterm delivery, first births, bacterial vaginosis, chorioamnionitis, STDs, antibiotic usage, SES and substance abuse. In addition we will measure fetal fibronectin and collect vaginal smears to examine for potential subclinical vaginosis. Detailed information will be collected on these potential factors and used to assure that randomization has effectively balanced risk between treatment arms and to permit post-hoc assessments.



PLAN FOR MONITORING: There will be an administrative Steering committee consisting of the

Obstetric and Periodontal PI from each clinical site, the NIDCR co-investigators and the Data and Statistical Coordinating Center (DSCC) investigators. The Steering committee will meet twice the first year and once a year thereafter. Study coordinators will also attend one of the two annual meetings. Data will be collected on dental, obstetric and neonatal outcomes by the data & statistical coordinating center, monitoring weekly for adverse events. The DSCC will be collating adverse events and safety data centrally to provide safety assessment reports to the DSMB. The DSMB will monitor outcomes and adverse events and assure maternal and infant safety and provide feedback to NIDCR every 6 months or as needed.

ADVERSE EVENTS: The dental examiner will conduct a comprehensive oral soft tissue (cancer screening) and periodontal examination at baseline and at post partum. Following enrollment mothers will be followed up by, OB surveillance through parturition, a post-delivery dental follow-up and neonatal surveillance that includes chart review after discharge. All of these provide an opportunity to detect and monitor adverse events. All reported and observed serious adverse events will be documented on an adverse event case report form describing the onset, duration, severity, assessment of causality and relationship to treatment intervention. This will be followed until resolution. A member of the investigative team will review subject’s OB charts on a weekly basis to note any adverse events or treatment provided (outside of routine). In addition all neonatal discharge summary findings will be collected to monitor any adverse neonatal morbidity such as neonatal sepsis and necrotizing enterocolitis. Any dental treatment will be noted in the subject’s clinical record to be reviewed by the dental examiner.

PLAN FOR DATA ANALYSIS: The details of the analysis plan appear in the body of the protocol,

and are summarized here. The incidence of preterm birth as the principal outcome will be evaluated using a chi-square test. Approximately 240 cases are expected at GA<35 weeks. Success of randomization for possible confounders will be evaluated by logistic regression models. Significance will be indicated by an alpha level of 0.05. Mean birth weight among preterm babies will be analyzed for correlations and significant differences between study arms using a non-parametric test (Kruskal-Wallis test) Parametric (regression) models will be used to adjust for gestational age and other factors.]. Analyses will be conducted using the intent to treat philosophy. Data will be collected on a



series of potential risk factors, covariates, confounders and effect modifiers that may influence the primary and secondary outcomes or periodontal status. Any unbalanced distribution of risks or exposures will be included in the regression model analysis. Adverse event data will be reported regularly. Interim analyses for efficacy will be conducted after 600 and 1200 completed pregnancies.



Table of Contents

1.0 INTRODUCTION......................................................................................................................................11

2.0 BACKGROUND AND RATIONALE......................................................................................................11

2.1 Healthcare significance of research problem.............................................................................11

2.2 Infection, inflammation and preterm birth...................................................................................12

2.3 Summary of previous findings of periodontal infection associated preterm birth..........14

3.0 AIMS AND HYPOTHESES.....................................................................................................................15

4.0 STUDY DESIGN........................................................................................................................................16

4.1 Overview...............................................................................................................................................16

4.2 Human Subjects..................................................................................................................................17

4.3 Inclusion Criteria...............................................................................................................................17

4.4 Exclusion Criteria..............................................................................................................................17

4.5 Informed Consent..............................................................................................................................18

4.6 Patient Incentives...............................................................................................................................18

4.7 Study Flowchart and Schedule of Visits......................................................................................18

4.8 Study Visit 1: OB screening visit, oral screening and enrollment........................................19

4.9 Study Visit 2: Baseline periodontal and baseline OB/GYN exam and randomization.....204.9.1 OB/GYN Baseline Visit 2A.........................................................................................204.9.2 Dental Baseline Visit 2B.............................................................................................204.9.3 Patient Randomization...............................................................................................204.9.1 Patient Histories and Tracking..................................................................................21

4.10 Study Visit 3: Post-partum exam, obstetric and neonatal outcomes................................................21

4.11 Study Visit 4: Group 2 treatment....................................................................................................21

5.0 PERIODONTAL DISEASE MEASUREMENTS...................................................................................21

5.1 Clinical indices.......................................................................................................................................22

6.0 DENTAL EXAMINER CALIBRATION PROCEDURE......................................................................23

6.1 Introduction...........................................................................................................................................23

6.2 Summary................................................................................................................................................23

7.0 PLAQUE, SERUM, PLASMA AND LEUKOCYTE BUFFY COAT SAMPLING.................................24

8.0 OBSTETRIC MEASURES AND SAMPLES..........................................................................................25

8.1 Fetal Fibronectin...................................................................................................................................25

8.2 Vaginal Microbial Flora........................................................................................................................25





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8.3 Vaginal pH Determination................................................................................................................26

8.4 Vaginal Gram Stain............................................................................................................................26

8.5 Maternal Blood Samples...................................................................................................................26

9.0 NEONATAL MEASURES........................................................................................................................26

10.0 PARTICIPANT IDENTIFICATION.......................................................................................................27

11.0 TREATMENT PROCEDURES...............................................................................................................27

11.1 Periodontal Treatments........................................................................................................................2711.1.1 Investigational Materials...........................................................................................28

12.0 POST-PARTUM EXAMINATION AND BIOLOGICAL SAMPLE COLLECTION.........................28

13.0 POST-PARTUM SCALING AND ROOT PLANING (GROUP 2 ONLY)........................................28

14.0 SAFETY EVALUATIONS........................................................................................................................28

14.1 Oral safety............................................................................................................................................29

14.2 OB Safety..............................................................................................................................................29

14.3 Oral Rescue treatments....................................................................................................................29

14.4 Neonatal Safety...................................................................................................................................29

15.0 OBSTETRIC & NEONATAL MONITORING, DATA CLOSURE AND ANALYSES....................29

16.0 EXAMINER CALIBRATIONS.................................................................................................................30

17.0 CONDITIONS FOR DISCONTINUING SUBJECTS...........................................................................30

18.0 ADVERSE EXPERIENCE REPORTING...............................................................................................30

19.0 DATA ANALYSIS AND STATISTICS...................................................................................................31

19.1 Sample Size Determination..............................................................................................................31

19.2 Power for varying event rates in the two treatment groups and sample sizes.................32

19.3 Data analysis........................................................................................................................................32

19.4 Analysis of endpoints........................................................................................................................32

19.5 Interim Analyses for Efficacy & Safety.........................................................................................33

20.0 ETHICAL AND REGULATORY REQUIREMENTS FOR PROTOCOLS INVOLVING HUMAN SUBJECTS.............................................................................................................................................................34

20.1 Study Conduct.....................................................................................................................................34

20.2 Informed Consent...............................................................................................................................34

20.3 Institutional Review Board or Ethical Review Committee.......................................................34

20.4 Protocol Amendments and Emergency Deviations..................................................................34

20.5 Monitoring of the Study....................................................................................................................35

20.6 Study Records.....................................................................................................................................35

20.7 Inspection of Records.......................................................................................................................35

21.0 INVESTIGATOR’S STATEMENT..........................................................................................................35

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22.0 HUMAN SUBJECTS................................................................................................................................37

22.1 Risks to the Subjects........................................................................................................................3722.1.1 Human Subjects Involvement and Characteristics:...............................................3722.1.2 Sources of Materials :.............................................................................................3822.1.3 Potential Risks:............................................................................................................39

22.2 Adequacy of Protection against Risks.........................................................................................4022.2.1 Recruitment and Informed Consent.............................................................................4022.2.2 Protection against Risk..................................................................................................40

22.3 Potential Benefits of the Proposed Research to the Subjects and Others.........................41

22.4 Importance of the Knowledge to be Gained................................................................................41

22.5 Inclusion of Women...........................................................................................................................42

22.6 Inclusion of Minorities.......................................................................................................................42

23.0 REFERENCES..........................................................................................................................................43

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1.0 INTRODUCTION

Case control and prospective cohort studies support the concept that maternal periodontal infections are associated with an increased risk of premature delivery and low birth weight infants, adjusting for traditional obstetric risk factors. Human data also support the concept that maternal periodontal infections are associated with an increased risk for fetal growth restriction, pre-eclampsia, perinatal stress as well as increased fetal morbidity and mortality. The long term goal of our research is to understand the mechanisms by which maternal periodontal infections mediate these abnormal pregnancy outcomes and whether treating periodontal disease in pregnant women improves pregnancy outcomes. This protocol describes procedures and guidelines for conducting a clinical intervention trial entitled MOTOR “Maternal Oral Therapy to Reduce Obstetric Risk” It is our central hypothesis that mothers with periodontitis who receive periodontal treatment during the second trimester of pregnancy will experience a lower rate of preterm birth at gestational age less than 37 weeks. We also hypothesize that periodontal treatment of these pregnant mothers will result in a higher mean birth weight of the premature infants of gestational age less than 37 weeks. We have organized three clinical research centers with joint periodontal and obstetric expertise at the University of Alabama (Jeffcoat/Hauth), the University of Texas at San Antonio (Cochran/Dudley) and the University of North Carolina/Duke (Offenbacher & Beck/ Heine & Murtha) that will collaborate to conduct a multi-centered clinical trial utilizing a Central Coordinating Center at the UNC School of Public Health (Chambless, Couper and Stewart). Our principal objective is to conduct a multi-centered, clinical trial enrolling mothers with periodontal disease prior to 236 weeks gestational age randomized to two treatment arms: Group 1) Scaling and root planning plus oral prophylaxis (tooth polish), prior to 236 weeks gestational age or Group 2) Post-partum scaling and root planning plus oral prophylaxis (delayed treatment control).

2.0 BACKGROUND AND RATIONALE

2.1 Healthcare significance of research problem Each year about half a million babies born in the United States, which is about one in ten births, arrive too early and too small 1,2. Preterm birth that occurs at less than 37 weeks gestation and associated low birth weight (LBW) of less than 2500 grams (about 5 1/2 lbs) represent the major cause of neonatal mortality and, among survivors, a major contributor to long-term disability 3. There are tremendous disparities in the prevalence of preterm birth and attendant neonatal complications. For example, pregnant African American women experience a rate of premature delivery approximately three-fold that of Whites or Hispanics. Furthermore, the incidence of LBW has not changed significantly over the last several decades. Despite advances in maternal prenatal care and increased public awareness, the incidence of preterm birth has not changed significantly over the last 40 years. It has been suggested 4 that the inability of the healthcare system to decrease the occurrence of these unfavorable pregnancy outcomes is likely due to fact that we have not as yet identified all of the contributing causes of preterm birth and therefore fail to appropriately target and manage relevant risk factors. Thus far, smoking and alcohol

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consumption are generally accepted as two major modifiable risk factors 4,5. Other potential risk factors appear to be less informative from a clinical management standpoint. Some of these reported risk factors 6-8 include parity (that is, the number of previous births, since the rate is higher among first births), short cervical length, short maternal stature, low maternal weight, high physical and psychological stress, low socioeconomic status and education, and poor maternal nutrition. These factors are not consistently confirmed by epidemiological studies and prospective treatment studies targeting these risk factors have not been encouraging 5,7,8.

Currently, the best single predictor of risk for preterm birth among pregnant women is whether the mother has already experienced a preterm delivery – a clinical history finding that increases the risk almost 3-fold 2. Thus the risk of preterm birth tends to remain with the mother throughout multiple pregnancies, even with increased levels of prenatal surveillance and preventive interventions. This finding and the coupled observation that there is a familial tendency for preterm birth has lead to the suggestion that there may be genetic components to risk 9. Whatever the genetic contribution, penetrance is far from complete, as it is not uncommon for a multiparous mother to have both normal full-term deliveries, as well as prematurities in her pregnancy history. Thus, intermittent, short-term exposures during the pregnancy may also play an important role in determining the outcome of a specific partus.

2.2 Infection, inflammation and preterm birth

One of the more important acute exposures that has been implicated in preterm birth is an acute maternal genitourinary tract infection at some point during the pregnancy 2,10. Bacterial vaginosis (BV) is a gram negative, predominantly anaerobic infection of the vagina, usually diagnosed from clinical signs and symptoms. It is associated with a decrease in the normal lactobacillus-dominated flora and an increase in anaerobes and facultative species including Gardnerella vaginalis, Mobiluncus curtsii, Prevotella bivia and Bacteroides ureolyticus. BV is a relatively common condition that occurs in about 10% of all pregnancies. It may ascend from the vagina to the cervix and even result in inflammation of the maternal-fetal membranes (chorioamnionitis). Extending beyond the membranes, the organisms may appear in the amniotic fluid compartment that is shared with the fetal lungs and/or may involve placental tissues and result in exposure to the fetal hematogenously. Despite the observed epidemiological linkage of bacterial vaginosis (BV) with preterm birth, the results from randomized clinical trials to determine the effects of treating BV with systemic antibiotics on incident preterm birth are equivocal (see 10 for review). Several potential explanations for the failure of clinical trials to show a beneficial effect of treatment have been suggested 11,12, including problems associated with target population selection, diagnostic criteria, as well as the efficacy of the intervention strategies. Nonetheless, there are compelling molecular data linking maternal infection and concomitant inflammation to preterm birth as well as neonatal complications. It appears that inflammation of the uterus and membranes represents a common effector mechanism that results in preterm birth, and that either clinical or subclinical infection remains a logical and highly suspect candidate as a stimulus for increased inflammation.

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Thus, BV remains an important putative risk factor or lower genital tract marker of subsequent preterm birth.

Early reports of an association between maternal periodontal disease and preterm birth 13

immediately raised the question as to whether these mothers with an oral anaerobic infection might also have a greater prevalence of clinical or subclinical vaginosis. Early results based upon either clinical definition of BV or microscopic determinations do not seem to indicate that the association of periodontal disease with preterm birth is due to a concomitant vaginosis or chorioamnionitis 13,14. Studies by Jeffcoat et al. 15 suggest that incidence of BV by vaginal Gram stain scoring was not different between patients with periodontitis as compared to patients without periodontitis (p=0.56) Although more studies are needed to clarify these associations, the data thus far indicate that the effects of periodontal disease on preterm birth appear to be independent of vaginal and reproductive track infections. Thus, the effects of periodontal disease are an additional burden on the maternal-fetal unit.

Acute infections involving distant organ systems, other than the genitourinary track, have been clearly shown to be capable of ultimately targeting the fetal-placental unit. The literature is replete with diverse examples of maternal primary distant infections that result in an abnormal pregnancy outcome, including rubella (endocrine), shigellosis (gastrointestinal), encephalitis (neurological) and pneumonia (pulmonary) 16. Most induce maternal cytokinemia and resultant fever that threatens the pregnancy and some of these infectious agents have been shown to target the placenta inducing local inflammation and necrosis. Some infectious agents that are abortofacient, such as Rubella and Campylobacter are capable of crossing the fetal placental barrier and result in direct exposure into the fetal circulation. Thus, maternal systemic infections can elicit an inflammatory response that results in inflammation of the maternal-fetal-placental unit including the uterus, the chorioamniotic membranes, the placenta, the amniotic fluid, the fetal lungs and the fetal circulation. These inflammatory stimuli induce hyperirritability of the smooth muscle of the uterus enhancing contractility, cervical dilation and premature labor. Inflammation of the chorioamniotic membranes results in thinning and premature rupture. Infection and the resulting inflammatory response can also elicit damage to the placenta. Placental damage can cause areas of focal hemorrhage and necrosis and that results in poor fetal perfusion, fetal growth restriction and distress. Most of the obstetric predisposing conditions that result in preterm birth and growth restriction are thought to be orchestrated by a common biochemical effector pathway that has been initially described by Romero 17. An elegant review of these effector mechanism concepts with special application to periodontal disease has been recent provided by Curtis and colleagues in (Williams reference 18 ).

Infectious exposure to the mother during pregnancy is currently believed to be a significant factor that triggers in utero fetal stress that ultimately contributes to long-term growth and development problems that begin with the neonate and extend throughout the lifetime of the individual. The quality of the environment, as well as the duration of the gestation in utero has been suggested to be a critical determinant of long-term well being of the individual ranging from cognitive and learning skills to susceptibility to heart disease 1,19-21.

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Maternal infections during pregnancy and attendant inflammatory responses have been linked to specific neonatal problems including periventricular leukomalacia (white matter necrosis), respiratory distress (often leading to chronic lung disease of preterm birth) and cerebral palsy 1,19-21 Furthermore, increasing evidence suggest that the molecular and cellular inflammatory effector pathways that underlie the pathogenesis of preterm birth are also involved in growth restriction and developmental problems ranging from respiratory distress to cognitive and learning disabilities. For example, fetal neurological tissues are especially susceptible to damage via cytokines, such as interferon gamma, that induce apoptosis and impair synapse development of embryological neurons 22,23. Thus, the threat of maternal infectious exposures during pregnancy does not appear to be solely limited to effects on the duration of the pregnancy but also to perinatal growth and development. Growth impairment that occurs during gestation can result in a neonate that is small for gestational age (SGA, typically defined as a birth weight that is below the 10th percentile of weight for that gestational age). Thus, current evidence suggests that maternal stressors result in both a maternal and a fetal inflammatory response that impairs fetal growth and development as well as expediting parturition by weakening and rupturing membranes and inducing labor. The relative stability or slight increase in the incidence of these neonatal conditions in the population over the last several decades suggests that current strategies to identify and manage maternal infection and inflammation during pregnancy remain to be optimized.

2.3 Summary of previous findings of periodontal infection associated preterm birth

The role of maternal periodontitis as a potential maternal-fetal stressor that has detrimental effects on the pregnancy outcome is a relatively new field of investigation. Early work with pregnant rodent models demonstrated that low-grade challenges with oral organisms during pregnancy resulted in impaired fetal growth. This was demonstrated using a chronic subcutaneous infection model with Porphyromonas gingivalis and also in a model of experimental periodontitis. In both models the infectious challenge was associated with an inflammatory challenge to the fetus, as measured by amniotic fluid PGE2 and TNF and attendant growth restriction 24. Since there are no animal models of preterm birth, these data provided important proof-of-concept experiments that raised the possibility that distant, low grade oral infections might also trigger inflammation of the human maternal-fetal unit in a manner analogous to that seen with reproductive tract infections. The first reported human case-control study suggested that mothers with premature (<37 weeks gestational age), low birth weight babies (under 2500g) had more severe periodontal disease than mothers with full term deliveries and that periodontitis appeared to confer considerable risk independent of other traditional obstetric risk factors (12). However, this investigation was a relatively small study of 124 cases and this design does not permit the establishment of temporality of exposure (periodontal disease) as it relates to the outcome (preterm birth). Nonetheless, the potential magnitude of the effect of periodontal disease was surprisingly large (adjusted odds ratio 6.7, p=0.003) and provided impetus for further study prompting the conduct of prospective studies to appropriately measure attributable risk and cross-sectional studies to confirm this association.

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Importantly, a separate NIDCR-funded, prospective study of 1313 mothers conducted at the University of Alabama by, Hauth, Jeffcoat and colleagues has, in parallel, confirmed that maternal periodontitis is an independent risk factor for preterm birth 15. These investigators report that with increasing severity of periodontal disease as an exposure there is an increased risk for preterm birth with odds ratios in the range of 4-7 for severe periodontitis, adjusting for age, race, smoking and parity. Thus, maternal periodontitis as a potential risk factor for preterm birth appears to be gaining considerable supportive evidence, although other case-control studies on low risk populations in Europe have not seen a relationship 25. Furthermore, two small independent studies have reported that providing periodontal treatments to pregnant women with periodontal disease may reduce the risk of preterm birth 26,27. In an observational study Mitchell-Lewis 26 suggested that mothers in a dental school environment receiving care experienced a lower rate of prematurity than a community-based control group. However, an intervention trial by Lopez 27 provides a more direct test of the association. In this study, 390 mothers were randomly assigned to one of two groups - periodontal treatment prior to 22 weeks vs delayed, antepartum treatment. The observed rate of preterm birth (<37 weeks) was 10.2% in the untreated and 1.8% in the periodontal treatment group (p<0.001). This suggestion is supported by recent pilot data generated by our colleagues at the University of Alabama.

In a paper entitled “Periodontal Disease and Preterm Birth: Results of a Pilot Intervention Study” (J Periodontology 2003;74:1214-1218, Jeffcoat MK, Hauth JC, Geurs NC, Reddy MS, Cliver SP, Hodgkins PM and Goldenberg RL) the rate of prematurity at <35 weeks was 6.3% in the reference group and 0.8% in the scaling and root planning group. The effects of periodontal treatment were strong, but in this pilot of 366 mothers assigned to 3 treatment arms, the findings did not achieve statistical significance comparing scaling and root planning to the prophylaxis group. In total, these preliminary findings are encouraging and build a strong rationale for conducting a prospective cohort study to provide a more robust measure of attributable risk as well as a multi-center, randomized controlled clinical trial to test the hypothesis that treating periodontal disease reduces risk of preterm birth.

3.0 AIMS AND HYPOTHESES

The objective of this study is to conduct a multi-center, randomized, delayed treatment-controlled clinical trial to determine the effects of periodontal therapy on the rate of preterm birth at gestational age (GA) <37 weeks as the principal outcome and GA<35 weeks, mean birth weight among neonates with GA< 37 weeks, adjusting for race, baby’s gender, and gestational age, and neonatal morbidity/mortality, as secondary outcomes.

The specific aims of this intervention study are:Specific Aim 1: to determine the effects of periodontal therapy consisting of scaling and root planing (S&RP) and oral hygiene instructions among pregnant women with periodontal disease on the rate of preterm delivery at GA<37weeks, as compared to a delayed treatment group. It is our central hypothesis that mothers with periodontitis that receive periodontal treatment during the second trimester of pregnancy will experience a lower rate of preterm delivery.

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Specific Aim 2: to determine the effects of periodontal therapy consisting of S&RP and oral hygiene instructions among pregnant women with periodontal disease on the rate of preterm delivery at GA<35weeks, mean birth weight of premature neonates at GA <37 weeks and neonatal morbidity/mortality as compared to a delayed treatment group. It is hypothesized that periodontal treatment will result in a lower rate of preterm delivery, an increase in the mean birth weight of the premature infants adjusting for race, baby gender and gestational age, and decreased neonatal morbidity/mortality

Specific Aim 3: To collect and archive biological samples during the conduct of the clinical trial to enable future studies regarding mechanisms of infectious pathogenesis of prematurity and growth restriction, including assessments of maternal and fetal inflammatory and immune responses and studies of intrauterine fetal exposure to oral and vaginal pathogens of maternal origin

4.0 STUDY DESIGN 4.1 Overview

This study involves the coordinated efforts of both a dental team and an obstetric team at each of the three field centers. All patients presenting for routine obstetric care early in pregnancy will potentially be eligible to be enrolled. Patients will be screened for potential inclusion or exclusion by medical and obstetric history. Once a patient has been determined to potentially meet the inclusion/exclusion criteria she will be approached for informed consent. Once informed consent has been obtained, further eligibility will be determined by a periodontal screening exam to identify those with periodontal disease, and an obstetric screening exam with ultrasound to enable study personnel to confirm eligibility. Eligible patients will then be enrolled and randomized to one of the two treatment arms Group 1) a periodontal treatment arm providing care by 236 weeks gestation or Group 2) a post-partum periodontal treatment that is delayed until the mother leaves the hospital. At baseline all mothers will receive an oral cancer screening, full-mouth measurements of periodontal disease status and samples of dental plaque will be collected. This baseline visit will be performed by dental staff, generally within or adjacent to the OB clinic using a dental chair. At a baseline prenatal OB visit samples will be collected for this study during the vaginal exam (microbial samples to test for vaginosis) and blood tests (additional serum and plasma tubes). A detailed OB history will be collected. Group 1 mothers will have periodontal care by 236 weeks gestational age consisting of scaling and root planing, using local anesthesia as needed, with oral prophylaxis and oral care instructions. These procedures will usually be completed in one to two dental treatment visits, however up to four visits may be provided. Mothers assigned to Group two will have no dental visits until after delivery. All mothers and babies will be followed from enrollment through delivery and discharge to determine outcomes and to monitor for adverse events (periodontal, obstetric and neonatal). At delivery, birth weight and gestational age will be determined for primary and secondary outcome analyses. At

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post-partum, there will be a repeat oral examination and plaque sampling performed prior to discharge. Neonatal status will also be monitored through discharge. After delivery Group 2 mothers will be scheduled for their periodontal treatment post-partum. During the course of this study, all mothers will receive routine obstetric care through their obstetric clinic, independent and unobstructed by this study protocol. Thus, in terms of obstetric and neonatal management of care, this study serves only to superimpose an additional dental treatment procedure upon routine obstetric care. In addition, the periodontal care is non-experimental and within the current standards of care for the management of periodontal disease among pregnant women.

4.2 Human Subjects

A total of 1800 subjects assigned to one of two treatment arms will be selected based on the following inclusion and exclusion criteria. Eligibility will be determined by a medical history, periodontal screening examination and an obstetric screening that includes an ultrasound for gestational age determination and to rule out the potential presence of multiple gestation and other obstetric exclusions.

4.3 Inclusion Criteria

Willing to be randomized and complete treatment protocols and provide informed consent

Planning on prenatal care and delivery at the enrollment center Pregnant and able to complete periodontal treatment prior to 236 weeks gestation At least 16 years old at enrollment Minimum of 20 teeth present Three (3) or more periodontal sites with > 3mm clinical attachment loss

4.4 Exclusion Criteria

Multiple gestation Positive history of HIV infection, AIDS, autoimmune disease, diabetes (gestational

diabetes is acceptable) Any medical contraindication to periodontal probing or periodontal treatment that

would require antibiotic prophylaxis, (e.g., congenital heart disease, use of Phen-fen for weight loss without a clear echocardiogram, mitral valve prolapse)

Rampant decay, symptomatic teeth or any other dental finding such as periodontal abscess or endodontic fistula that would preclude enrollment.

Chronic use of medications that cause gingival enlargement such as phenytoin, cyclosporin -A, or calcium channel antagonists

Chronic use of steroids Concomitant use of orthodontic appliances (braces) Any obstetric finding that precludes enrollment in the study Participation in another randomized controlled trial or not agreeing to refrain from

participation in another randomized controlled trial during the current pregnancy.

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4.5 Informed Consent

This process involves a HIPPA certification of a waiver for chart screening and clinic surveillance to identify potential study subjects. Patients will be approached in the OB/GYN clinic by study personnel for obtaining informed consent for screening. Patients will usually be first approached by obstetric personnel to ascertain eligibility and patient interest followed by explanation of the study and obtaining informed consent for screening. Once consent has been obtained there will be both an oral and obstetric screening to determine study eligibility. If the patient is eligible and willing to participate informed consent for participation in the entire study will be obtained. Two of the sites plan to have one consent form that covers screening and study enrollment. In compliance with federal guidelines, all informed consent processes and HIPPA regulations will be applied in accordance with the requirements of the local IRB Committee for the use of Humans Subjects for Research Purposes at each of the individual clinical performance sites.

4.6 Patient Incentives

Participants will receive periodontal therapy at no cost and a monetary sum of approximately $5.00 in cash at each study visit to facilitate transportation to and from the clinic.

4.7 Study Flowchart and Schedule of Visits

This section describes the patient flow through the clinical protocol that consists of four or more study visits. These four visits include 1) a dental and obstetric screening, 2) an OB and dental baseline 3) periodontal treatment visits (1-2 typically, but up to four) and 4) a post-partum visit. A patient typically schedules an appointment for the baseline and treatment visits. These visits are depicted in the following flow chart and described below.

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4.8 Study Visit 1: OB screening visit, oral screening and enrollment

A total of 1800 subjects will be randomized into the study, 600 in each of 3 centers from an estimated total patient pool of about 29,000 subjects. We conservatively assume that only 32% will meet the medical and dental criteria, and that only 50% will agree to participate. Using these estimates, to enroll 1800 subjects we would need to screen approximately 11,250 patients over 42 months. If one center has difficulty recruiting, we should still have an ample pool of patients to recruit from other centers to meet our goal after 42 months of enrollment. All pregnant women who present to the OB clinics for a prenatal care visit who meet the medical inclusion and exclusion criteria in time to be randomized and treated by 236 weeks gestation will be asked to participate by the physician. The study will be explained by the OB nurse and study hygienist and with the physician; informed consent for oral screening and OB screening will be obtained. Once consent has been obtained, the patient will be screened for periodontal status in a dental chair located within the OB clinic, and if eligible an OB screening (ultrasound) will be conducted to assure OB eligibility. If both dental and medical eligibility criteria are met according to the inclusion/exclusion criteria listed above, informed consent for the full study will be obtained and an accession number is assigned to that study subject. Patients will be scheduled for Study Visit 2 that will actually involve two appointments – one OB/GYN (Visit 2A) baseline and one dental baseline (Visit 2B). Under most clinical situations the dental screening is

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conducted first followed by OB screening. If the patient is eligible then the OB baseline samples are completed at the end of the OB screening. Thus, Visit 2A occurs immediately after ultrasound eligibility is confirmed and the vaginal and blood samples needed for this study are collected during that OB examination visit. The patient is then scheduled for the dental baseline visit and randomization occurs at the end of that dental baseline visit. Patients will not be randomized until both dental and OB baselines are completed, but they can occur in either order, depending on the availability of the ultrasound data. Patients will be scheduled for both the Dental and OB visits usually during the same day, but no more than 14 days apart.

4.9 Study Visit 2: Baseline periodontal and baseline OB/GYN exam and randomization

4.9.1 OB/GYN Baseline Visit 2A

During the OB visit a speculum exam will be performed to collect vaginal and cervical samples for microbiological determinations, inflammatory mediator measurements and fetal fibronectin quantification. Antepartum maternal blood for cotinine assessments, as well as vaginal and cervical samples (for fetal fibronectin and bacterial vaginosis testing) is also collected at this first OB baseline visit.

4.9.2 Dental Baseline Visit 2B

Dental Baseline Visit 2B: Full mouth periodontal exam, as described above, will be performed on all subjects within the dental unit. A licensed dental hygienist or dentist, not involved in the periodontal treatment, will perform an exam of the oral cavity. The same examining hygienist will also perform the postpartum examination at Study Visit 3. Oral procedures include a cancer screening, a periodontal exam, and the collection of plaque samples. These microbiological samples will be collected and stored for future studies. Four plaque samples will be collected, selecting the deepest site in each quadrant and stored separately for future analyses. After oral examination, patients will be randomized to one of the two treatment groups.

4.9.3 Patient Randomization

Randomization: The DSCC will develop and implement an automated randomization system and will also provide a telephone contact at the DSCC in case the automated system fails. In order to ensure at an early stage in the study an approximately equal distribution of subjects across the treatment groups a permuted block randomization scheme will be used, stratifying subjects by clinical center. The Steering Committee, as described in the Manual of Procedures, may decide to add other stratifying factors. To minimize the possibility and effect of an investigator being able to predict some treatment assignments, a random mixture of block sizes will be used within each center’s strata. The DSCC will provide the Steering Committee with weekly reports on the number of subjects

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recruited and randomized. Once randomized to treatment groups, then intent-to-treat methods will be followed. Patients assigned to treatment Group 1 will have treatments previously described begun at this appointment, Group 2 patients will have treatments performed postpartum. If needed additional dental treatments will be scheduled to complete the scaling and root planing.

4.9.1 Patient Histories and Tracking

Patient Histories and Tracking: Each study subject will complete a demographic form. This form collects the subject's address, employment status, education level, current medications, alcohol/tobacco exposures, and psychosocial exposures. For each subject enrolled, the study personnel will complete a medical history form. This form collects the subject's address, insurance provider, past obstetrical and gynecologic history, present obstetrical history, and general medical history. After enrollment, the subject is tracked and followed throughout the pregnancy, ensuring the collection of necessary data and biological samples. Each subject is listed on an active list of study patients and on a data completion checklist sheet. These forms give details of which data or biological sample has been collected and which still are needed. This sheet is updated after each contact with the subject. During the subject's pregnancy, obstetric surveillance will be followed, assuring the collection of all necessary data.

4.10 Study Visit 3: Post-partum exam, obstetric and neonatal outcomes

After the subject delivers the baby, oral and perinatal clinical data and biological samples will be collected while she is still in the hospital. Samples collected in labor and delivery include maternal blood, placental biopsies, and fetal cord blood that are collected, processed and shipped to UNC for processing. At the post-partum visit additional oral plaque samples will be collected bedside. Bedside exams will be performed to collect periodontal clinical data. Additional maternal interviews are conducted and neonatal data are collected until the baby is discharged. The study personnel will conduct a postpartum interview that includes questions about exposures prior to and during the current pregnancy, support provided by family members during the current pregnancy and contact information for future use. Mothers who were randomized to Group 2 will be scheduled for their periodontal treatment (Study Visit 4).

4.11 Study Visit 4: Group 2 treatment

This visit is to provide the treatment that Group 2 patients did not receive during Study Visit 2. This visit completes the patient treatment protocol and patients are advised of their dental needs and treatment options.

5.0 PERIODONTAL DISEASE MEASUREMENTS

Periodontal disease will be measured at baseline and at postpartum. Periodontal disease status will be expressed based upon clinical signs, as described below. Plaque will also be collected for future analyses to assess microbial burden, as determined by quantification of

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plaque composition by DNA macroarray (checkerboard). All of these measures are to determine the efficacy of periodontal therapy and provide baseline disease level characterization to use as a control variable in secondary analyses. Specifically these measures will be used to determine 1) whether baseline periodontal status relates to periodontal therapeutic response and pregnancy outcome 2) whether certain oral organisms that are suspected as fetal pathogens (specifically Campylobacter rectus and Porphyromonas gingivalis) are eliminated or persist following therapy. None of these measures are end-points of therapy but will be considered as they specifically relate to the outcome as potential effect modifiers of preterm birth and growth restriction.

5.1 Clinical indices

Clinical periodontal parameters will be collected for all teeth. Clinical parameters that include probing will be measured using a manual University of North Carolina (UNC-12) periodontal probe at six sites per tooth (i.e. mesiobuccal, direct buccal, distobuccal, mesiolingual, direct lingual, and distolingual) on all teeth including third molars. The clinical parameters to be recorded include the following:

Soft Tissue Examination : A visual intraoral soft tissue cancer screening will be performed.

Plaque scores (Silness and Loe H, 1967) for three buccal and the mid-lingual surfaces of each tooth will be assigned using a 0 to 3 scale. Absence of plaque or stain = 0, deposits covering less than one-third of crown surface = 1, less than two-thirds = 2, more than two-thirds = 3.

Gingival index score (Loe and Silness, 1963 ) using a 0 to 3 scale where normal = 0, mild inflammation = 1, moderate = 2 with bleeding on probing, and severe inflammation = 3 with tendency to spontaneous bleeding.

Probing Pocket Depth (PD) will be measured from the free gingival margin to the base of the pocket, and will be recorded in whole millimeters. When taking probe readings, the probe will be positioned parallel to the long axis of the tooth and inserted to the base of the sulcus at the line angle. The probe will be walked in each of the six sextants (mesiofacial, midfacial, distofacial, mesiolingual, midlingual and distolingual) and the deepest reading will be recorded for each tooth sextant. For the interproximal sextants, the probe will be angled slightly in order to insert into the col space as close to the mid-interproximal area as possible. If a PD reading falls between two-mm readings, the rule shall be to round up to the nearest mm.

Bleeding upon Probing (BOP) will be recorded as presence or absence during probing measures

CEJ Measure will be measured from the cementoenamel junction (CEJ) to the gingival margin and will be recorded in whole mm. If a reading falls between two-mm readings, the rule shall be to round up and record the higher of the two readings. If the gingival margin occurs coronal to the CEJ, the CEJ measure will be recorded as a positive value. If the gingival margin occurs apical to the CEJ, the CEJ measure will be recorded as a negative value. CEJ measurements will be taken at the same site as the recorded deepest sulcus depth at each site.

Clinical attachment level (CAL) will be calculated as PD minus CEJ and shall reflect the linear distance from the CEJ to the base of the pocket. CAL shall not be

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recorded on case report forms, but rather will be calculated during the computer analysis of data.

6.0 DENTAL EXAMINER CALIBRATION PROCEDURE

6.1 Introduction

Periodontal determination of plaque (PI), gingival inflammation (GI), gingival bleeding (BOP), pocket probing depths (PD) and clinical attachment levels (CAL) are customarily used in clinical studies to measure the disease status of periodontitis and to measure effects of periodontal therapy. The measurement of these periodontal parameters poses several limitations and technique-sensitive sources of variability. Probe penetration and depth may vary with the degree of inflammation, probing force, angulation, position and instrument tip diameter. Other confounding factors include patient discomfort, accuracy of probe markings, anatomical differences in tooth crown and roots, and technique variability within and between examiners. Thus, all of these measurements are important response variables for inclusion in periodontal clinical trials.

Studies evaluating the efficacy of therapeutic interventions for periodontal disease require stringent control of measurement error. In studies involving more than one examiner, use of these assessment parameters must be based on acceptable levels of inter-examiner variability. The levels of intra- and inter-examiner reliability should be determined for each examiner and center involved in a clinical trial by the performance of a calibration session. A training exercise with a standard examiner (SE) will help to standardize to a common measurement technique and to a common definition of measurement parameters. An interactive calibration session will enable the investigators to quantify the measurement variability among and between examiners and enable the optimization of the measurement process. Prior to the start of any clinical trial investigating a periodontal therapeutic, a calibration session will be conducted according to the guidelines set forth in this document.

6.2 Summary

The purpose of an examiner calibration session is to quantify intra- and inter-examiner reliability of measuring periodontal soft and hard tissue parameters. Chairside and bedside calibrations will be conducted. Examiner dental chairside calibrations take place at the University of North Carolina School of Dentistry, GO Health Center. Dental calibrations at bedside are conducted at the Caviness General Clinical Research Center at the University of North Carolina School of Medicine and UNC Hospitals. A sample of persons with evidence of adult periodontitis are recruited to serve as calibration subjects. Each examiner will assess PI, GI, BOP and PD and CAL using the manual University of North Carolina (UNC) probe. Prior to arrival in Chapel Hill, examiners will review the study manual. The training session will begin with a review seminar. The standard examiner (SE) will go over the criteria being used for each aspect of the examination and will answer questions. The structure of the calibration session will be described. The seminar will also include a review of the clinical procedure manual, use of the specific indices, instrumentation, case scenario slides, and demonstration models. A review of the use of

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the direct data entry system (DDES) will also be conducted. During the calibration session, data will be entered directly into the computer to allow for a comparison of data calls. Thus, examiners will have an opportunity to become familiar with the data entry program during the calibration. In addition, laboratory procedures for the collection, shipping, and handling of biological specimens will be addressed. One practice calibration subject will be examined by each examiner with the SE observing and providing direct and immediate feedback on examiner technique. Examiners will practice taking a plaque sample using the standard protocol described in the manual. After the practice calibration subject has been examined, an opportunity is provided for discussion of the practice session. Immediately following completion of data collection, scores will be printed and compared for agreement within one millimeter. All disagreement calls will be discussed and the call repeated on the subject in the presence of the SE. For PD and CEJ, disagreement will be defined as a difference of more than 1 millimeter. At this point, adjustments to the examiners' technique will be made. By the end of the second clinical training day, we usually find that the SE and examiners are in close agreement. Acceptable reliability for PD and CEJ measures are inter-rater/inter-examiner reliability scores of at least .60 or above and intra-rater or intra-examiner reliability scores of at least .75 or above. In our experience, most examiners exceed these minimum scores.

Examiners will return to their respective field center and will examine 5-10 “trial patients”. They will then ship the examination data and biological specimens to study investigators at the UNC School of Dentistry, Chapel Hill, where the data will be evaluated. When the investigators are satisfied that the examiners can consistently follow the protocol, the examiners will be given written permission to begin examinations in the field. Centralized training sessions will be repeated annually.

7.0 PLAQUE, SERUM, PLASMA AND LEUKOCYTE BUFFY COAT SAMPLING

Four plaque samples will be collected during the periodontal exam visit. Plaque will be sampled from the deepest probed site in each quadrant. The site is isolated with a cotton roll and gross supragingival plaque or debris is removed. Samples are taken by placing a sterile curette (preferably with a long straight shank) at the base of each sulcus, adapting the cutting edge to the surface of the tooth and using a single upward exploratory stroke. Each plaque sample will be stored separately at -40 or lower in a labeled vial containing TE and sodium hydroxide will be added after collection and the sample vortexed by hand. Samples are then shipped to UNC School of Dentistry to be stored for future microbiological analysis.

Blood will be collected for future analysis for serum, plasma and leukocyte buffy coat. Two 5-7ml vacutainer tubes of peripheral blood obtained by venipuncture of the antecubital area will be collected for the purposes of this study. Usually this is done during a routine obstetric blood draw and does not require an additional venipuncture. If needed, however it can be collected during the dental baseline visit. All blood samples will be processed, labeled and stored in –80 freezers until processed. Details appear in Manual of Procedures

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8.0 OBSTETRIC MEASURES AND SAMPLES

An extensive medical history, OB history, medications history, and OB progress notes summary and discharge data set are collected. It is recognized that BV, STD and chorioamnionitis are major clinical and subclinical risk factors that must be considered as potential confounders. Thus, in addition to clinical signs and symptoms and treatment indications, clinical samples will be collected for the post-hoc assessment of subclinical disease. During the first OB visit a smear for gram stain will be taken and read for Nugent scoring for bacterial vaginosis will be taken. Furthermore accurate gestational age determination is critical to the outcome measure and will be obtained by ultrasound, obtained as early as possible, but no later than 24 weeks gestational age.

Among the current best clinical indicators of preterm birth risk include cervical length and fetal fibronectin, especially among those with previous preterm deliveries. Thus, fetal fibronectin will be obtained by sampling the vaginal fluids in the region of the posterior fornix and quantifying of fetal fibronectin by ELISA methods. Dacron vaginal swabs for fibronectin will be collected on all mothers. Because costs prohibit performing transvaginal ultrasounds on all study subjects, this information will not be collected.

8.1 Fetal Fibronectin

Fetal fibronectin samples must be collected prior to any other vaginal sample, manipulation of the cervix or vaginal tract. During a sterile speculum examination a Dacron swab will be lightly rotated around the external cervical os for approximately 15 seconds and then across the posterior vaginal fornix for an additional 15 seconds to absorb cervicovaginal secretions. Subsequent attempts to saturate the swab may invalidate the test. Only a Dacron swab should be used for the sample collection, cotton swabs are not acceptable. While collecting the sample care should be used to avoid obvious mucus aggregates and the swabs should be lightly rotated to prevent collection of unnecessary cellular materials. Once the swab has been saturated immerse the Dacron tip in the buffer. The shaft will be aligned with the top of the tube and broken at the score. The shaft will then be aligned with the hole inside the tube cap and the pushed down lightly while securing the cap. It is important to align the shaft to prevent leakage. The sample will be stored for future analysis.

8.2 Vaginal Microbial Flora

Vaginal samples for microbial analysis will be collected from the posterior vaginal fornix and the cervical os. These samples will be obtained after the fetal fibronectin samples and before the vaginal pH measurement. Sterile paper points should be attached to ring forceps and inserted into the vaginal tract at the posterior vaginal fornix. The paperpoints should be gently rotated across the posterior vaginal fornix for approximately 15 seconds. Remove the ring forcep insert the paperpoints into a microtube containing TE. A second set of sterile paperpoints should be attached to the ring forceps and inserted into the vagina at the cervical os. Once inserted the paperpoints should be gently rotated across

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the cervical os for approximately 15 seconds. Remove the ring forcep and insert the paperpoints into a separate microtube containing TE. Once the samples have been collected, sodium hydroxide should be added to both tubes, the lids secured and the tubes vortexed. The samples will then be stored for future analysis.

8.3 Vaginal pH Determination

Vaginal pH samples will be collected after the vaginal microbial sampling. Using a dry Dacron swab with a plastic shaft, take a vaginal secretion specimen from the upper third of the vaginal sidewalls. Touch the swab to the reagent block on the ColorpHast indicator strip. The pH must be read after the color has stopped changing but, before the paper dries completely. Record the vaginal pH as indicated in the MOP. Only ColorpHast strips, stored away from light, should be used to obtain vaginal pH. If the color falls between two values on the chart, round up. Do not interpolate between two values.

8.4 Vaginal Gram Stain

A Gram stain from the vaginal sidewalls will be collected after the vaginal pH. Either using a dry Dacron swab or the swab from the vaginal pH, take a vaginal secretion from the upper third of the vaginal sidewalls. the swab should be gently rolled across the entire glass slide, from end to end, avoiding blobbing and in a thin layer. The slide should be fixed by allowing to air. A barcode with the patient identification code will be fixed to the slide. The slide will then be stored for future analysis.

8.5 Maternal Blood Samples

Maternal blood samples for the study will be drawn at the baseline OB appointment. These are performed only once, prepartum, to provide a serum tube and a CPT tube for buffy coat and plasma. Details appear in the Manual of Procedures.

9.0 NEONATAL MEASURES

The principle and secondary outcomes for this study require the collection of gestational age as determined by ultrasound and confirmed at birth by Dubowitz and delivery birth weight. APGARS at one minute and at five minutes will also be determined. We also will employ a composite neonatal outcome. This composite outcome is consistent with a composite neonatal outcome used by the Maternal and Fetal Network and is contains the following conditions:

Fetal demise subsequent to randomization Infant death prior to discharge from hospital to home or a chronic care

facility Respiratory Distress Syndrome (RDS) Documented neonatal sepsis Significant intraventricular hemorrhage (IVH grades 3-4)

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Necrotizing enterocolitis (NEC stages 2-3)

Neonatal histories will be monitored by baby discharge summaries and daily surveillance of the neonatal intensive care unit (NICU). The primary flag is admission to neonatal intensive care. This will be followed on a daily basis for any infant entering the NICU, and the event will be reported within 48 hour to the data coordinating center and forwarded to the DSMB every 3 months. Critical issues that will be addressed by abstraction of the neonatal discharge summary are the need for O2, any chronic lung disease of preterm birth, sepsis, GBS infection, neonatal periventricular leukomalacia from ultrasounds on very low birth weight infants and any events of necrotizing enterocolitis. As stated before, these are important safety measures.

10.0 PARTICIPANT IDENTIFICATION

At screening, participants will be assigned a unique sequential 6-digit identification registration number and accompanying digits or letters that comply with a bar coding scheme to identify study, location, participant visits and samples acquired. This unique identifier will be used for patient randomization assignment to treatment group, to assure confidentiality, as well as patient and sample blinding.

11.0 TREATMENT PROCEDURES

Periodontal treatment procedures will be performed according to the randomization assignment. The study site will institute a patient and specimen tracking system that will accomplish the aims of the study but not interfere with the procedures of the OB clinic or impede medical care. There are no OB treatment implications for this study, as prenatal care will be provided as indicated by the primary OB physician and staff without regard to the conduct of this study. All OB prenatal procedures will be in accord with the standard of care and there are no OB treatments that would make any patient ineligible or censured. Furthermore, the OB physicians and staff will not know the periodontal treatment group assignment of the mother. All periodontal treatments are consistent with the current standard of care for periodontal treatment of pregnant women.

11.1 Periodontal Treatments

Scaling and root planing will be performed using anesthesia, if indicated. This will be performed one quadrant at a time and finishing all four quadrants to completion in 1 to 4 visits. All scaling and root planing procedures must be completed prior to 236 weeks gestational age. Hand instruments and ultrasonic devices may be used. Patients are also give an oral prophylaxis with a rubber cup and prophy paste and provided with oral care instructions and materials as described in the Manual of Procedures.

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11.1.1 Investigational Materials

All subjects will receive a new manual toothbrush. Toothbrushes will be provided for distribution to all subjects. Subjects will be instructed to brush as they would normally. Appropriate quantities toothpaste will be provided for distribution to all subjects in both groups. Sufficient product shall be provided for the subject and all family members in the household.

12.0 POST-PARTUM EXAMINATION AND BIOLOGICAL SAMPLE COLLECTION

After the subject delivers the baby, oral and perinatal clinical data and biological samples will be collected while she is still in the hospital. Samples collected in labor and delivery include maternal blood, placental biopsies, and fetal cord blood that are collected, processed and shipped to UNC for processing. At the post-partum visit additional samples will be collected bedside and include oral plaque samples. Bedside exams will be performed to collect periodontal clinical data. Additional maternal interviews are conducted and neonatal data are collected until the baby is discharged. The study personnel will conduct a postpartum interview that includes questions about exposures prior to and during the current pregnancy, support provided by family members during the current pregnancy and contact information for future use. Mothers who were randomized to Group 2 will be scheduled for their periodontal treatment (Study Visit 4).

13.0 POST-PARTUM SCALING AND ROOT PLANING (GROUP 2 ONLY)

A scaling and root planing appointment will be conducted approximately 4 weeks after delivery.

The dental health care provider will perform an oral soft tissue screening for adverse events and will evaluate periodontal inflammatory status through visual examination.

The study dental health care provider will perform conventional scaling and root planing periodontal therapy incorporating clinical judgment for the use of local anesthesia. Hand instrumentation will be supplemented with ultrasonic scaling per standards of care. If more than 1 visit is required for the periodontal treatment, additional appointments will be scheduled as needed. Full mouth prophy polishing will follow the scaling and root planing.

The dental health care provider will monitor for safety and adverse events.

14.0 SAFETY EVALUATIONS

Patient demographics, medical history, vital signs, medications and infections will be reviewed from the OB patient record. At every study visit, patient vital signs will be assessed including pulse, respiration, and blood pressure.

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14.1 Oral safety

At each dental study visit an assessment of the subjects' extra- and intra-oral structures will be conducted. Each examination will include a survey of the face, lymph nodes, lips, buccal mucosa, floor of the mouth, tongue, hard and soft palates, gingiva, edentulous ridges and teeth. Findings will be recorded as normal or abnormal. In particular, oral soft tissues will be evaluated as part of safety monitoring and documentation will be given to the participant for referral purpose. The attending dentist will be available to all patients for urgent dental care.

14.2 OB Safety

Data will be collected and recorded at each visit regarding adverse events reported by subjects. OB charts will be monitored weekly by the research RN and adverse events documented. Any patient who fails to make a scheduled appointment will be contacted by phone within one week. Additional details of OB surveillance appear in the Manual of Procedures.

14.3 Oral Rescue treatments

The attending dentist will see any patient with oral complaints for evaluation and management. Appropriate urgent care treatments will be offered to all patients with dental emergencies.

14.4 Neonatal Safety

As described previously, neonatal intensive care unit admissions will be monitored daily and findings from neonatal discharge summaries reviewed.

15.0 OBSTETRIC & NEONATAL MONITORING, DATA CLOSURE AND ANALYSES

Patient enrollment will end at approximately 48 months. If the last mothers are enrolled at 48 months then the last mothers should deliver at approximately 54 months, providing 6 months from the last delivery to check, clean and close the database. A postpartum maternal database will have been constructed to collect interview data and data abstracted from the subject's chart (written and electronic) at the completion of the pregnancy. These data include delivery information for the current pregnancy, updated maternal medical history, changes to previous and current pregnancy history and documents any obstetric or neonatal complications that were diagnosed perinatally. For any subject whose placenta is sent to pathology for diagnosis, a pathology report will be generated.

A neonatal database will have been created to collect information on each infant beginning at delivery. This database will collect gestational age (based upon ultrasound), birth weight, infant length, and head circumference for all infants. For

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infants needing intensive care attention additional information will be collected. These data include morbidity and mortality outcomes that will be used to create: a composite perinatal outcome assessment score including at least one of the following: fetal demise subsequent to randomization, infant death prior to discharge, type 1 respiratory distress syndrome (includes Hyaline membrane disease and respiratory insufficiency of prematurity) and documented neonatal sepsis, Grade 3-4 IVH (intraventricular hemorrhage) and Stage 2-3 NEC. For infants admitted to the intensive care unit, information will be collected until the infant is discharged from the hospital. Spontaneous abortions, neonatal deaths, neonatal admissions to the ICU, respiratory distress, NEC, sepsis and GBS will be recorded to assess the potential effects of periodontal therapy on these events. These parameters will be considered in the context of gestational age and obstetric complications. These data will be reported to the DSMB for evaluation.

16.0 EXAMINER CALIBRATIONS

All clinical examiners will be calibrated and certified prior to commencement of the study for training of study procedures and for documentation of acceptable intra- and inter-examiner measurement reliability. Both chair side and bedside calibration will be performed. Calibration for cervical length determinations will be conducted via a training session and certification process. These calibration procedures are described in the Manual of Procedures.

17.0 CONDITIONS FOR DISCONTINUING SUBJECTS

Subjects in this clinical study may be discontinued for the following reasons: 1) death and 2) withdrawal of consent.

18.0 SERIOUS ADVERSE EVENT REPORTING

A serious adverse event is “any untoward medical occurrence that results in death, islife threatening, requires or prolongs hospitalization, causes persistent or significantdisability or incapacity, results in congenital anomalies or birth defects or, in the opinionof the investigators, represents other significant hazards or potentially serious harm toresearch participants or others (From Section 3.1 of “Guidelines for Developing aManual of Operations (MOP)”, National Institute of Dental and Craniofacial Research,drafted 15 November 2002.)

All serious adverse events will be recorded although a distinction will be drawn between reactions normally experienced in conjunction with conventional periodontal therapy and experiences not usually encountered. Details of monitoring schedule appear in Chapter 10 of the Manual of Procedures and include a two week follow-up after periodontal therapy that increases to weekly during the last trimester.

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Unanticipated adverse events will be evaluated by weekly OB patient chart reviews by the OB research nurse. The onset, duration, treatment, and outcome of all such unanticipated adverse experiences must be recorded on the appropriate case report forms.

In the event of a serious adverse experience the site’s principal investigator will contact the DSCC within 5 days. The DSCC will report to the DSMB and NIDCR according to the guidelines established by those groups. Sites are responsible for their own IRB reporting.Any adverse experience that is determined to be reportable to the Regulatory Authorities should be promptly reported to the Institutional Review Board (IRB).

19.0 DATA ANALYSIS AND STATISTICS

19.1 Sample Size Determination

The sample size for the study was determined using Specific Aim 2. At the time the grant was awarded, the current Specific Aim 2 was Specific Aim 1. At the first DSMB meeting the Board members expressed concern that the assumptions used were optimistic and recommended that Specific Aims 1 and 2 be interchanged, with the sample size left unchanged. This section describes how the sample size was determined using the original Specific Aim 1 (now Specific Aim 2). The next section discusses the power for the current Specific Aim 1 for the chosen sample size

Specific Aim 2 involves comparing the proportions of neonates having gestational age (GA) <35 weeks across the two treatment groups. Power calculations were performed using a test of equality of the two proportions, assuming a total sample size of 1800 split equally across the two groups. All statistical tests are assumed to use = 0.05. Power calculations were done using nQuery Advisor 4.0.

In the UAB pilot trial that has been recently published (J of Periodontology 2003; 74:1214-1218) the rate of GA<35 weeks presenting in the untreated group was 6.4%.and the rate among women treated using scaling and root planning was 0.8%, an 87% reduction. We base our sample size on a rate of 6% in group 2 (delayed periodontal therapy) versus 2% in group 1 (periodontal therapy during pregnancy). These rates are somewhat more conservative than those observed in the UAB pilot trial and include an allowance for women to receive a treatment other than the one to which they were assigned. On top of these rates we assume a loss to follow-up rate of 3% in each group. This 3% rate is conservative as the rate in the pilot trial was 0.5% (2 out of 368). In addition we are adding the 3% loss to follow-up rate to each group under an intent-to-treat assumption, with the further assumption that all individuals lost had a preterm birth. (If there is differential loss to follow-up, analyses will be conducted with and without the latter assumption and both results would be reported. Adding this 3% yields rates of GA<35 week of 9% versus 5%. In this situation a sample size of 900 per treatment group will give power of 91% to detect such a difference between the two treatment groups. The table below shows the effect on the power of deviations from our assumptions about rates in the two groups and of recruiting fewer subjects.

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19.2 Effect size detectable with study sample size

We determined the effect size detectable with 90% power for the same sample size and statistical and loss to follow-up assumptions as the previous section. Without periodontal treatment during pregnancy, the rate of GA<37 weeks is likely to be about 20%. If it is 20%, then we will have 90% power to detect a 6.3 percentage point treatment effect (that is, a GA<37 rate of 13.7% in the women treated during pregnancy. If the GA<37 rate in the delayed treatment group is just 16% we will have 90% power to detect a 5.8 percentage point difference.

The second part of Specific aim 2 involves comparing birth weights across treatment groups among neonates with GA <37 weeks. In the UAB pilot study the rates of GA <37 weeks were 13.7% and 4.0% for treatment groups 1, and 2, respectively. We assumed a standard deviation of 0.55kg for birth weight among neonates with GA <37 weeks. This estimate was obtained using data from the Tasmanian Infant Health Survey. Under these assumptions and using a two-sample t-test with 900 subjects per group, we have 90% power to detect a difference of 0.34kg between the mean weights in the two treatment groups. If the standard deviation of birth weights within each group is 0.6kg rather than 0.55kg the difference detectable with 90% power increases to 0.37kg. If the proportions with GA<37 weeks are 10% and 5% and the standard deviation of birth weights is 0.55kg then we have 90% power to detect a difference of 0.33kg. Truncation at GA <37 weeks is likely to result in skewed distributions of birth weight within each treatment group. Thus our primary analysis for this aim will use a non-parametric test rather than a t-test. This will reduce the power somewhat from the estimates given here.

19.3 Data analysis

The Data and Statistical Coordinating Center will have primary responsibility for performing data analysis for the writing groups developing core manuscripts and presentations. It will also be responsible for preparing detailed data reports at regular intervals for the Steering Committee. The CSCC has been producing analyses for writing groups, DSMBs, Steering Committees, and policy boards of collaborative studies for more than 30 years (including the LRC, SOLVD, ARIC, ACAS, STILE, DAIS, and COMBINE studies). We are experienced in producing statistically sound tables and graphs that are easily understood by non-statisticians. For details of the analyses to be conducted, see the Analysis of Endpoints sub-section later in this section.

19.4 Analysis of endpoints

Specific Aim 1: The primary endpoint will be evaluated in an intent-to-treat analysis using a test of equality of two proportions. If the number of subjects in each cell is sufficient a chi-squared test will be used, else Fisher's exact test will be used. Success of the randomization and even distribution of possible confounders will be evaluated. If there appears to be an unequal distribution of important confounders then as a secondary

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analysis a logistic regression model will be used to adjust for these factors. Potential confounders that will be considered are discussed below.

The first part and third parts of Specific Aim 2 (comparisons of rates of GA<35 weeks and of neonatal morbidity/mortality, respectively) will be analyzed in the same way as Specific Aim 1. The second part of Specific Aim 2 is to determine whether birth weight among those infants with GA<37 weeks differs by treatment group. The truncation of gestational age at 37 weeks is likely to yield a skewed distribution of birth weights within each treatment group. Thus we propose using a non-parametric test (Kruskal-Wallis test) as the primary analysis for this specific aim. Secondary analyses will use parametric models. In order to adjust for covariates in secondary analyses we will investigate transforming the birth weight to improve the normality of residuals in a linear model. The first such model will include an adjustment only for gestational age.

Analyses will be conducted using the intent-to-treat philosophy. The length of gestation is relatively short and information on the primary outcomes of interest should be obtainable from maternity clinics, so even if subjects drop out of the study the number with missing outcome information should be negligible. More problematic are pregnancies that do not end in a live birth (such as miscarriages). These will be reported to the DSMB as adverse events but also need to be considered in the analyses of the primary outcomes. For the primary outcome we intend to regard such events as having GA<37 weeks. For Specific Aim 2 assessing birth weight, all deliveries whether live or stillbirths will be used for analyses.

Analyses for specific aims will also include investigation of possible effect modification by race/ethnicity. We do not have evidence to suggest such effect modification and the study is not powered to test for it. However, regardless of whether there is significant effect modification by race/ethnicity, we will also report results within each race/ethnic group.

19.5 Interim Analyses for Efficacy & Safety

There are three potential reasons for ending a trial early: 1) harmful effects of the treatment being used may be discovered, 2) efficacy of the treatment may be proved, or 3) there may be no remaining hope for a reasonable evaluation of the proposed hypothesis. The DSCC will report regularly to the DSMB on adverse events, irrespective of whether there is any reason to suspect they may be harmful effects of the treatments.

We propose conducting two interim analyses for efficacy, with these analyses being conducted after 600 and 1200 completed pregnancies. The primary outcome is length of gestation and the maximum length of gestation is known and relatively short. Thus it is feasible to specify doing the interim analyses after specified numbers of completed pregnancies rather than at specified times. We propose using O’Brien-Fleming type boundaries 38 as the decision criteria at the interim analyses for efficacy. To allow for extra interim analyses to be requested by the DSMB or variation in the exact timing of the analyses, we will use the Lan-DeMets alpha-spending function modification of the O’Brien-Fleming boundaries 39. In addition, conditional power as proposed by Halperin 40, will be

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used in the decision to stop the trial if the difference between treatments is small. This method computes the conditional probability of rejecting the null hypothesis given a specific alternative and the data already collected at the time of the analysis. If this probability is too small, one may choose to discontinue the trial. Computationally, we will use a generalization of the method of Lan and Wittes 41.

20.0 ETHICAL AND REGULATORY REQUIREMENTS FOR PROTOCOLS INVOLVING HUMAN SUBJECTS

20.1 Study Conduct

The investigator agrees that the study will be conducted according to the principles of Good Clinical Practices (GCP) and the Declaration of Helsinki. The investigator will conduct all aspects of this study in accordance with all national, state, and local laws of the pertinent regulatory authorities.

The principal investigator must sign the Investigator’s Statement (see section 11.0) and provide a copy of a current Curriculum Vita for each study team member

20.2 Informed Consent

The study's informed consent document must be provided to the respective institutional Investigational Review Boards for final approval. Before recruitment and enrollment, each prospective patient candidate will be given a full explanation of the study, and allowed to read the approved informed consent form. Once the investigator is assured that an individual understands the implications of participating in the study, the subject will be asked to give consent to participate in the study by signing the informed consent form. The investigator will provide a copy of the signed informed consent form to the subject.

20.3 Institutional Review Board or Ethical Review Committee

Before initiation of the study, the investigator must obtain approval of the research protocol and informed consent form from an IRB complying with the provisions specified in 21 CFR Part 56 or applicable pertinent governmental regulations. The investigator must assure IRB compliance with the applicable regulations.

The investigator is responsible for obtaining continued review of the clinical research at intervals not exceeding one year or otherwise specified by the IRB.

20.4 Protocol Amendments and Emergency Deviations

Changes to the research covered by this protocol must be implemented by formal protocol amendment. Amendments to the protocol may be initiated at the request of the investigator. In either case, a formal amendment cannot be initiated until it has been signed by the investigator and approved by the IRB.

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20.5 Monitoring of the Study

This will be performed by the coordinating center, as described in the Manual of Procedures.

20.6 Study Records

All study records will be stored centrally at each clinical performance site maintaining patient confidentially. Primary medical source documents will be abstracted to create a separate obstetrical dataset that will augment the questionnaire information. A standardized data instrument will be used to collect medical and dental data on all subjects.

20.7 Inspection of Records

This will be performed by the coordinating center, as described in Manual of Procedures.

21.0 INVESTIGATOR’S STATEMENT

I agree to conduct the trial as outlined in the protocol in accordance with the NIDCR's guidelines and all applicable government regulations including proposed Part 54: Obligations of Clinical Investigators of Regulated Articles. These guidelines and regulations include, but are not limited to:* Permission to allow the NIDCR or other regulatory agencies to inspect study facilities

and pertinent records at reasonable times and in a reasonable manner that ensures patient confidentiality

* Submission of the proposed clinical investigation including the protocol and the consent form to a duly constituted IRB for approval and acquisition of written approval for each prior to the use of the test article.

* Use of written informed consent that is obtained prior to administration of test articles containing all the elements of consent as specified in 21 CFR 50.25 of the federal regulations and that has been previously approved by the NIDCR and the IRB.

* Submission of any proposed change in or deviation from the protocol to the IRB using a signed formal amendment document prepared by the NIDCR. Any proposed changes or deviations from the protocol require that the informed consent also reflects such changes or deviations and that the revised informed consent be approved by the IRB.

* Documentation and explanation of individual protocol deviations on the appropriate CRF page or in letters to the NIDCR.

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* Submission of reports of serious adverse events as outlined in the protocol to the IRB and NIDCR within 10 working days.

* Submission of timely progress reports to the IRB and NIDCR at appropriate intervals on a schedule determined by the IRB.

* Record keeping; federal regulations (21 CFR 31 2.62) require an investigator to prepare and maintain adequate and accurate case histories designed to record all observations and other data (such as test article accountability) pertinent to the investigation on each individual enrolled in the study. The investigator must maintain these records for a period of at least two years following completion of the study report.

I agree that all information provided to me by the NIDCR including pre-clinical data, protocols, CRFS and verbal and written information will be kept strictly confidential and confined to the clinical personnel involved in conducting the trial. It is recognized that this information may be related in confidence to the IRB. I also understand that no reports or information about the trial or its progress will be provided to anyone not involved in the trial other than the NIDCR, or in confidence to the IRB or the NIDCR or other legally constituted authority.

______________________________ ___________________

Principal Investigator (Signature) Date

______________________________

Principal Investigator (Print)

______________________________ ____________________

______________________________

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22.0 HUMAN SUBJECTSThe following details regarding the use of Human subjects applies to all three performance sites.

22.1 Risks to the Subjects22.1.1 Human Subjects Involvement and Characteristics:

All pregnant women presenting for prenatal care between 160 and 236 weeks gestational age will be invited to enroll in the periodontal treatment study, but is limited to women the age of 16 and over (age of legal consent). All patients will be seen by their primary care OB nurses and physicians who will manage their comprehensive obstetric care in a routine manner using protocols recommended by the American College of Obstetricians and Gynecologists. This study does not in any manner, influence, or modify the obstetric, perinatal or neonatal care received by the subjects. The experimental trial is to determine whether providing periodontal care during pregnancy decreases the overall incidence of pregnancy complications. Thus, the intervention component is periodontal care, but we are collecting data by observing the obstetric outcomes. The dental component will be provided by licensed dental professionals and all treatments are in accordance with ADA (American Dental Association) and AAP (American Association of Periodontology) guidelines for examining and treating periodontal conditions in pregnant women. None of the periodontal examination or treatment procedures are experimental as they reflect the procedures and standard of care that might be delivered in the private dental sector in the second trimester of pregnancy. After obtaining informed consent pregnant mothers will have a short oral screening exam that consists of periodontal probing measures, checking for the presence of gum disease. If disease is present, mothers will be asked to consent to participate in the full MOTOR study (Maternal Oral Therapy to Reduce Obstetric Risk). Subjects must be willing to be randomized to one of two treatment arms. The two treatments are procedurally identical in nature, but one group (Group 1) receives periodontal treatment early in the second trimester and the other group (Group 2) receives care post-partum. The timing of the therapy for Group 1 is in accordance with standard ADA guidelines which recommend that the periodontal care should be delayed until the second trimester to be beyond the organogenesis embryonic stage and to be prior to the third trimester to minimize both maternal and fetal stress. The diagnostics performed are routine manual examinations with periodontal probes and will not necessitate radiographs. The care provided may include local anesthesia, the use of hand and ultrasonic instruments, but are not generally regarded as invasive or surgical procedures. At post-partum a repeat oral examination is performed in the hospital, prior to discharge. Mothers will be inconvenienced by the additional dental examinations, the oral interviews and dental treatments, but will gain improved oral health. We recognize that certain mothers will also receive systemic antibiotics to manage OB/Gyn. Conditions, such as bacterial vaginosis (BV) or STDs and that these may impact our periodontal status or effects of therapy. We will monitor these treatments only and will not have any impact on the clinical decisions regarding the clinical management of those conditions. It is expected that the effect of systemic antibiotic usage should be randomized across treatment groups, but we will be able to correct for any unbalanced treatments post-hoc. Also, any research findings that are generated as part of this study, such as the independent reading of vaginal

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smears for the determination of subclinical BV, will not be conducted as CLIA laboratory analyses, therefore these results will not be used for the clinical management of the patients and the data will be separate from the patient’s clinical chart.

As this is a study on pregnancy outcomes in women with periodontal disease, all members of the study population will be women who are pregnant. Members of minority racial groups will be encouraged to participate so that our study population mirrors the minority composition of the three communities.

All women will have the study explained to them orally. Women agreeing to participate in the study protocol will sign a consent form agreeing to participate and will be given a copy of the consent. The protocol will be submitted to the Institutional Review Board for the protection of human subjects at each of the respective institutions for review and approval. In addition the UNC Go Health Center will be calibrating examiners for MOTOR. This training is administratively under a separate clinical protocol that involves the use of patients who participate in the oral examination for initial certification of examiners and periodic recalibration. These subjects who are examined in both dental units and hospital settings are covered under a separate Human subjects IRB protocol and informed consent. Finally, there is also a truncated “study screening only” informed consent that is used to check these mothers for the presence or absence of periodontal disease for eligibility purposes. This also includes an oral cancer screening done only for the benefit of the subject. For the purposes of this application and human subjects discussion, these latter two protocols and informed consent are not discussed, as they are covered under existing approved protocols.

22.1.2 Sources of Materials :

Human specimens will include maternal plaque, maternal blood for DNA, vaginal fluid samples and maternal serum. At the time of delivery placental biopsies will be collected and fetal cord blood will be processed for serum. Maternal blood will be drawn for obstetric purposes for clinical reasons and an additional aliquot collected during the same blood draw will be utilized for research purposes. During the routine speculum examination at the prenatal visit additional swabs of the vaginal fornix will be performed for research purposes. Maternal plaque will be sampled from the teeth during the oral examination and stored for research purposes. Placental biopsies are routinely taken for histology for all preterm deliveries. Additional biopsy fragments will be collected for research purposes. Fetal cord blood is normally discarded, but will be collected for research purposes in this investigation. Clinical and history data will be collected by oral examination, patient interview and medical chart abstraction. These will involve the creation of secondary computerized source documents for the study which will be maintained separate from the patients’ medical records and identified only by study participant ID number. All biological samples are also identified by the patient ID number only.

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22.1.3 Potential Risks:

This is a non-invasive protocol. Periodontal examinations are performed post-partum in accordance with the ADA and American Academy of Periodontology recommendations for the clinical examination and management of periodontal patients. There may be some minor discomfort during the periodontal examination, but it is minimal and is identical to the types of diagnostic procedures carried out during a routine dental examination. Blood will be collected from mothers during their routine blood draw and will not require an additional phlebotomy. Cord blood samples are collected from the placenta postpartum and provide no risk to mother or newborn.

Periodontal therapy is performed during the second trimester to minimize the obstetric risk in accordance with the ADA and American Academy of Periodontology recommendations for the clinical management of periodontal conditions during pregnancy. There is some clinical risk associated with scaling and root planning (e.g. discomfort) venipuncture (e.g. bruising), and the collection of vaginal samples (e.g. discomfort), but procedures are within the normal risks associated with these dental and obstetric procedures.

Group 2 subjects will be identified for post-partum periodontal therapy. These individuals will have delayed treatment of their periodontal condition. It is important to note that patients who would have any acute severe or symptomatic dental problems that should be addressed prior to delivery are specifically excluded from this study. That places the level of oral disease within that which would be considered elective and would typically be deferred until after delivery. If there should be an acute episode in either treatment group the patient will be seen and receive urgent care by the study dentists.

Scaling and root planing with or without the use of local anesthesia as performed during the second trimester of pregnancy is the current standard of care to treat periodontal disease in pregnant women. The risks include mild discomfort during or after the procedure. Excessive bleeding, sensitivity or post-operative discomfort is not anticipated. There is a transient and acute mild bacteremia induced by scaling and root planning, but this has not been perceived to represent significant risk. It is important to note that the UAB pilot included this treatment arm and there were no adverse events reported for the almost 200 subjects who had this procedure. Other clinical trials either in the literature or underway (Moms 2B) are not experiencing any AEs. However, the data are far from complete regarding this important issue of maternal and fetal safety. Therefore, it will be an important contribution of this study to provide evidence regarding the safety of periodontal treatment during pregnancy as it is possible that treatment may result in a transient bacteremia that may influence the pregnancy outcome or the health of the neonate. For this reason we have a surveillance plan in place that will be coordinated through the DSCC and reported to the DSMB formed for this study that will perform oversight for the expressed purposes of protecting human subjects. The group 2 subjects will have delayed treatment. It is estimated that the treatment delay will be approximately 20 weeks. This time frame is well within what would be acceptable delay especially among women were not seeking dental care. These women are seeking obstetric care, not periodontal care.

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Within our own university-based periodontal clinics, a 6 month waiting list is not unusual for initial admission for screening examinations. Thus, a short delay is not normally perceived to place the patient at risk for significant periodontal destruction, since this is a chronic, slow-progressing condition. If however, the clinician perceives that the periodontal condition is sufficiently problematic, that is either symptomatic or placing the periodontal tissues at significant risk during the pregnancy, then the subject is not eligible for the study and will be referred for care.

22.2 Adequacy of Protection against Risks

22.2.1 Recruitment and Informed Consent

Pregnant women arriving for their first prenatal visit will be approached by their physician in the OB clinic and informed of the study. If they express interest, then the study personnel (research nurse or dental hygienist) will describe the study to them. If the potential subject remains interested, then informed consent for a screening exam will be requested and the subject will be provided a copy of the signed consent form. If the results of the screening exam indicate that the subject meets the periodontal inclusion criteria as well as the other inclusion and exclusion criteria, the subject will be told that they meet the criteria for the study, an accession number is generated, and an appointment is made for the initial dental visit. At the first dental visit, detailed informed consent is provided by the dental personnel and the subject is asked to sign the study consent form. Informed consent will be maintained in the patient’s chart and a copy stored in the study coordinator’s office. All confidentiality and anonymity will be maintained by using an ID system that provides patient data and links to biological samples but is not linked to patient identifiers.

22.2.2 Protection against Risk

The study biological sampling protocols do not confer significant additional clinical risk. The subjects may withdraw from the study at any time and may also elect to have their biological samples discarded at any time. Our bar-coding system that catalogues sample location greatly facilitates the logging out of the study subject’s samples, retrieval from storage and permanent destruction of biologicals. This function will be managed by the DSCC and the UNC laboratory.

Safety is a critical concern and there are extensive safeguards in the study design that have been discussed in Section C and detailed in the clinical manual in Appendix C. It is noteworthy that Drs Jeffcoat and Hauth have already enrolled almost 400 subjects in this treatment protocol and have had no adverse events and no significant problems with patient tolerance of the periodontal treatments or antibiotic usage.

Mothers will have safety obstetric monitoring and antepartum evaluation to determine the effects of periodontal therapy on the gestational age and weight of the newborn, as an adjunctive therapy to the routine obstetric standard of care. The Coordinating Center will

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provide reports to the NIDCR-appointed Data Safety Monitoring Board (DSMB) that will periodically assess the progress and safety outcomes to assure that periodontal therapy is not placing any mothers or neonates at unacceptable risk. These assessments will include neonatal health problems including NEC (necrotizing enterocolitis), GBS (Group B Streptococcal infection) and respiratory distress as an indicator of perinatal safety.

22.3 Potential Benefits of the Proposed Research to the Subjects and Others

We anticipate that periodontal treatment will significantly reduce the rate of preterm birth at less than 37 and 35 weeks gestation and increase the mean fetal weight for those less than 37 weeks gestational age. The UAB intervention trial has experienced a significant 87% reduction in preterm births at less than 35 weeks gestational age with no adverse events, enrolling and treated 366 subjects of which 2/3 had scaling and root planing. This clinical trial should provide practical new information to guide the proper management of this common clinical problem and may eventually result in a dramatic change in the standard of care to reduce the incidence and severity of abnormal pregnancy outcomes.

Mothers will benefit from having an oral examination that includes an oral cancer screening and periodontal assessments. There are minimal risks; only possibility of slight discomfort during the oral examination and treatments and this risk of discomfort is minimal compared to the social, economic and emotional trauma that is a consequence of preterm delivery. It has been our experience that women are anxious to contribute to any study that may lead to improving our identification of potential causes of pregnancy complications. At the end of the study, all patients are seen by a staff dentist for treatment recommendations and appropriate referral for care. Thus, mothers will have an improvement in oral health as a consequence of participation and possibly have beneficial impact on the pregnancy.

Mothers will also receive the benefits of oral care to reduce periodontal disease and they will receive reimbursement for visits that include parking and other transportation expenses. Additional study participation incentives may include free oral hygiene instructions and oral care aids such as toothpaste and toothbrushes.

22.4 Importance of the Knowledge to be Gained

Despite improvements in prenatal care and in our ability to improve the survival and the health of preterm infants, there has been no decrease in the overall rate of preterm delivery in the US in the last 4 decades. The causes of this problem have not been adequately identified to enable successful interventions. Periodontal disease represents a preventable and treatable condition, which if demonstrated to be the cause of disparities among those at highest risk- including the underserved minority populations, then these data may have a significant impact on reducing the number of preterm deliveries among this group. Each gram of neonatal birth weight under 2500g costs 75$ in NICU costs. Our data in OCAP indicate that infants born between 28-32 weeks gestational age are on average 400g smaller among mothers with periodontal disease as compared to gestational age matched infants from mothers without periodontal disease. This translates into approximately $30,000 of NICU costs and higher rates of long-term disability. Thus, the

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identification of a modifiable risk factor among these API pregnant women would have the potential to have tremendous health and quality of life benefits.

22.5 Inclusion of Women

All subjects in this study are women. The earliest age of eligibility has been lowered from 18 to 16, which is in response to study section recommendations and consistent with local laws regarding age of consent. It is important to note that these populations of younger individuals who are at high risk for preterm delivery also have a much higher prevalence of periodontal disease that predicted, based upon national studies of periodontal health among adolescents and young adults, such as the NHANES study. It is our hypothesis that this disparity may account for the increased risk for prematurity. It is also important to point out that this periodontal disease in these individuals is not due to a clinical diagnosis of juvenile periodontitis, as it does not follow the clinical presentation of this rather uncommon condition. We propose to enroll women for 42 months beginning November 1, 2003 and ending May 1, 2007.

22.6 Inclusion of Minorities

Our performance sites have been selected to provide representation for Whites, African Americans, Hispanic/Latino and Others who are seeking prenatal care. No special efforts are planned for outreach to enrich these populations within the clinical centers as the proposed enrollment distribution of the clinics meets our need for racial and ethnic diversity. The MOTOR study is being conducted within tertiary care facilities that are normally enriched for high risk patients via the community referral system. Thus, mothers with previous preterm deliveries and African Americans tend to be over-represented relative to their proportion within the community at large. Since this sample represents a high risk target group, the applicability of the findings may not be strictly generalizable to all pregnant women. However, as with all intervention trials designed to test efficacy, the study is targeted to those with the highest risk to provide the largest possible therapeutic margin of benefit. We propose to enroll women for 42 months beginning November 1, 2003 and ending May 1, 2007.

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