Limitation of funding

The trial intends to evaluate the efficacy of specially designed probiotics to prevent premature birth and related neonatal morbidity associated to intra-uterine infection. The tested probiotics are efficacious to decrease the prevalence of bacterial vaginosis. The study hypothesis is that the early administration of those probiotics to pregnant women with bacterial vaginosis can prevent premature birth through antibiotic activity and modulation of the immune response to infection.

The study is designed to be a randomized placebo-controlled double-blind trial that will screen asymptomatic low risk pregnant women without indication of elective premature birth admitted before the 20th week of pregnancy (Date of LMP and ultrasound) in prenatal care public services involved in the project in the city of Rio de Janeiro. Relevant prognostic factors will be registered: history of premature birth, race and low body index.

After clinical screening will be followed by a further screening process:

1. vaginal pH exam will be determined by taking a swab and rolling it on a glass slide that will be touched by a pH stick . This procedure will immediately exclude patients with pH < 4,5 from the next step;
2. The slides from patients with a vaginal pH > 4.4, will be Gram stained and interpreted according to the criteria of Nugent et al in order to select patients with BV or intermediate Nugent score (4-10). Patients with Nugent score below 4 will be excluded.

Pregnant women who are excluded from any of the above steps will be reassessed every 4 weeks, for new cases of BV/Intermediate score, during the next prenatal care visits, until they reach the 19th week.

BV/Intermediate patients will be randomized (centralized blocked randomization process), after signed informed consent, to receive either probiotics or placebo capsules twice a day (each capsule with probiotics shall contain > 1 million bacilli of each of 2 selected strains of lactobacillus: Lactobacillus rhamnosus GR1 and Lactobacillus reuteri RC-14).

Women shall take the capsules until they reach approximately the 24th week of gestation, the minimum duration of the treatment being 6 weeks (for those women randomized to the trial by the 20th week). Capsules will be maintained in refrigerators throughout the trial.

Pregnant women will receive the usual prenatal care at their institutions, which should follow the related evidence-based municipal guidelines.

Vaginal pH and Nugent scores will be assessed after the end of the treatment for both treatment groups, so to assess changes in those parameters as partial results regarding the efficacy of the study probiotics.

Randomized patients, whether compliant with the treatment regimens or not, will be followed up for the assessment of the trial endpoints: spontaneous premature birth (<37, <35, <32 weeks of pregnancy) and related neonatal events: early sepsis, bronchopulmonary dysplasia, periventricular leukomalacia and necrotizing enterocolitis, besides death and average hospital stay. Study personnel will abstract that information from delivery and neonatal records using a pre-tested questionnaire. Definitions and diagnostic methods used for identifying such conditions will be similar to those recommended by the Vermont- Oxford Network, issued in 2002.

The occurrence of adverse events will be monitored with a specific questionnaire.

The adherence to the treatment will be assessed with specific questioning of the pregnant woman and by counting the capsules contained in the bottles returned by them at each prenatal care visit. Reasons for non adherence, including the occurrence of adverse events, will be looked into in order to propitiate adherence whenever possible.

Special attention will be devoted to avoid undesirable psychological effects derived from the eventual labeling of women as high risk for premature birth.

Procedures to assure the concealment of the randomization and the blinding of the pregnant women (identical placebo) and study personnel, including caretakers and the technician who will be performing the pH and Nugent exams, are described in the protocol.

In order to add further explanatory power to the present trial, regarding the mechanisms of action of the study probiotics, a nested case-control study (1 case and 3 controls) will be carried out to determine the level of selected cytokines (IL-1B, IL1-ra and IL-6) at the vaginal fluid, before and after treatment, and the occurrence of TNF an IL 6(308 and 174) polymorphisms, and their association, individually and in interaction, with the study endpoints (cases). Special vaginal swabs (cytokines) and oral samples (polymorphisms) will be collected for those purposes and processed accordingly. Written informed consent will be also sought for such procedures.

The trial was approved by an Institutional Review Board which has sent it for final approval by the National Review Board.

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Sample size to show efficacy of study probiotics for preventing premature birth less than 35 and 32 weeks (significance level of 0.05 and power of 0.80):

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Considering:

• that using the pH <4.5 as a cutoff point for the Nugent assessment will result in excluding around 40% of the clinically pre-selected women (Hauth et al,2003) but will only exclude a small percentage of women with Nugent´s Intermediate score and related preterm births,
• a non-adherence rate to the treatment regimens of about 5%,
• that the incidence in the placebo group of premature birth less than 35 weeks will be around 6%;
• and an efficacy rate of 50% for the study main end-point (premature birth),

the number estimated to be necessary for the randomization phase of the present trial, is about 1500 women.

To assess efficacy regarding premature birth less than 32 weeks, same significance level and power, the corresponding number is nearly 3000.

Inclusion Criteria:

-Asymptomatic pregnant women with no indication of elective preterm delivery, before the 20th week of pregnancy

Exclusion Criteria:

• major malformations in present pregnancy
• cervical cerclage
• symptomatic vaginosis
• insulin dependent diabetes
• arterial hypertension
• Multiple gestation
• Antibiotic therapy in present pregnancy
• Syphilis or gonorrhea in present pregnancy
• asthma requiring chronic or intermittent therapy
• corticotherapy(recent or chronic)
• perinatal hemolytic disease
• Systemic Erit. Lupus