Routine screening of abnormal vaginal flora during pregnancy reduces the odds of preterm birth: a systematic review and meta-analysis

Prematurity is the leading cause of perinatal mortality and the morbidity among children under the age of 5. The prevalence of preterm birth is between 5 and 18% worldwide. Approximately 30% of preterm deliveries occur as a consequence of fetal or maternal infections. Bacterial vaginosis can increase the risk of ascending infections. However, there is no recommendation or protocol for screening of abnormal vaginal flora. The aim of this systematic review was to investigate the effectiveness of routine screening of abnormal vaginal flora during pregnancy care. We conducted our systematic search in the following databases: MEDLINE via PubMed, Embase, and Cochrane Library. Studies reporting on pregnant women with no symptoms of bacterial vaginosis were included in our analysis if they provided data on the outcome of their pregnancy. The intervention group went through screening of abnormal vaginal flora in addition to routine pregnancy care. Odds ratio (OR) with 95% confidence intervals (CIs) was used as effect size measure. From each study the total number of patients and number of events was extracted in both the intervention and control arm to calculate OR. Altogether we included 13 trials with 143,534 patients. The screening methods were Gram stain, pH screening, pH self-screening and pH screening combined with Gram stain. Regular screening of vaginal flora compared to no screening significantly reduces the odds of preterm birth before 37 weeks (8.98% vs 9.42%; OR 0.71, CI 0.57–0.87), birthweight under 2500 g (6.53% vs 7.24%; OR 0.64, CI 0.50–0.81), preterm birth before 32 weeks (1.35% vs 2.03%; OR 0.51, CI 0.31–0.85) and birthweight under 1000 g (0.86% vs 2.2%; OR 0.33, CI 0.19–0.57). In conclusion, the routine screening of abnormal vaginal flora might prevent preterm birth, extreme preterm birth, low birthweight deliveries and very low birthweight deliveries. Further research is needed to assess the problem more accurately.

The random-effects model was used to calculate odds ratio (OR) with a 95% confidence interval (CI).Forest plots were used to graphically summarize the results irrespective of the number of studies included in the pooled analysis.Forest plots with less than three studies were interpreted with limitations.
Where applicable, we reported the prediction intervals (i.e., the expected range of effects of future studies) of results following the recommendations of IntHout et al. 26 .
Cochrane's Q test was used to assess the statistical heterogeneity with a p-value < 0.1 as a threshold for a statistically significant difference, and the I 2 index was used to quantify between-study heterogeneity.In addition, Egger's test and funnel plots were applied to report and visualize publication bias if at least ten studies were involved in the analysis.To assess the source of heterogeneity we performed multiple sensitivity analyses (leave-one-out meta-analysis, Baujat plot, and influence diagnostics).
A subgroup analysis was carried out with data on study design (RCT and non-RCT) and the type of screening method.
Besides heterogeneity, a p-value < 0.05 was considered statistically significant.Further details of the synthesis are included in the eMaterial.

Ethical approval.
No ethical approval was required for this systematic review with meta-analysis, as all data were already published in peer-reviewed journals.In addition, no patients were involved in our study's design, conduct, or interpretation.The datasets used in this study can be found in the full-text articles included in the systematic review and meta-analysis.
Baseline characteristics of included studies.Baseline characteristics of the enrolled studies are detailed in Table 1.Of the 13 included articles, nine were published from Europe, three from Asia, and one from North America.The screening methods used were different in the studies.The Nugent score evaluated the results in studies where Gram stain was used.In addition, the screening procedures' timing and frequency among the studies were not uniform.In the intervention group, the preterm birth rate before 37 weeks ranged from 2.96 to 21.8%, while in the control group, between 5.1 and 22.3%.In most cases, oral or local Lactobacillus, Clindamycin, or Metronidazole was used to treat abnormal vaginal flora.
Two studies included multiple pregnancies 19,20 .However, in these studies, the author reported the same rate of multiple pregnancies in the intervention and control groups.Furthermore, we performed a leave-one-out (sensitivity) analysis to investigate whether these studies influenced our result in a significant way, and we found no differences (eFigs.9-14).Further details are summarized in Tables S2 and S3.
Analyzing different types of screenings, Gram stain compared to control significantly reduces the odds of birthweight under 2500 g (7.94% vs. 14.79%;OR 0.55, CI 0.41-0.73;eFig.11).Based on two studies, pH selfscreening and based on one study, combined pH and Gram stain screening did not significantly differ from the control group.
Analyzing the different types of screenings, Gram stain based on three studies significantly reduces the odds of birthweight under 1000 g (1.21% vs. 5.27%; OR 0.22, CI 0.18-0.26;substantial heterogeneity).Based on one study, pH self-screening showed a similar effect.
Publication bias and heterogeneity.Outlier and influence analyses did not show a significant difference for any of the above mentioned results.S4 and S5.The overall risk of bias shows some concerns.On the other hand, the quality of evidence was very-low for most results.A summary of the assessment is included in Table S6.

Discussion
The available evidence from our results shows that routine screening of abnormal vaginal flora during pregnancy can decrease the odds of preterm delivery.Our study is the most comprehensive on the topic, including thirteen studies with 143,534 patients worldwide.
There was a significant decrease in the odds of preterm birth before 37 and 32 weeks in the intervention group.Similarly, there was a significant decrease in the odds of delivering a newborn under 2500 g, 2000 g, and 1500 g in the intervention group.Finally, we found the most robust decrease in the odds of birthweight under 1000 g.This result has the most important clinical relevance because the chance of complications and mortality is the highest among these neonates.However, the prediction interval for most of the outcomes was wide and crossed the level of significance.
Although our study was not aimed at finding the most effective screening method, we included trials with any screening type.Based on our data, Gram stain was effective in each subgroup.Furthermore, pH self-screening, pH screening by healthcare professionals, or a combination of screening methods seemed to be effective.Interestingly, according to our data, the earlier the preterm delivery occurs, the more likely to have bacterial vaginosis in the background.In eFig.27.we can see that as the number of weeks of gestation decreases, the greater the reduction of the odds of preterm delivery.
In different guidelines, controversial information can be found on the recommendation for routine screening of abnormal vaginal flora and treatment of bacterial vaginosis.The ACOG Practice Bulletin No. 234, published at the end of 2021, does not recommend routine screening 13 .This recommendation is based on a meta-analysis from 2013 14 .According to their results, antibiotic therapy effectively treated bacterial vaginosis.However, it did not reduce the risk of preterm birth before 37 weeks (OR 0.88; CI 0.71-1.09).On the other hand, according to the US Preventive Services Task Force Recommendation Statement, routine screening and treatment can benefit pregnant patients with a history of preterm birth 15,16 .However, the certainty of this evidence is moderate, which is why this data is insufficient for a recommendation of routine screening and treatment.It is important to mention that the ACOG recommendations against the routine screening of vaginal flora are based only on studies that investigated the efficacy or effectiveness of the treatment of bacterial vaginosis, while this review takes into account pregnant women with abnormal vaginal flora, which is why the ACOG and task force recommendations are not included in clinical trials like those of Kiss 28 .
We need to mention that the screening methods, routine pregnancy care, and treatment protocols were different in the studies as they differ from country to country.However, all screening protocols are accepted diagnostic methods.On the other hand, we performed the necessary subgroup analysis and multiple sensitivity analyses (leave-one-out meta-analysis, Baujat plot, and influence diagnostics) to exclude the potential biasing studies (eFigs.4-6, 8-10, 14-16, 18-26.) Another important point is the inclusion of cohort studies in our meta-analysis.Because of the low number of articles, we also decided to include both RCTs and cohort studies.Unfortunately, only a few RCTs were eligible for our study.Although we did not get significant results (OR 0.74, CI 0.53-1.03eFig. 1) for preterm delivery (< 37 weeks), the data still suggest that routine screening might be beneficial in preventing preterm deliveries.In the case of low birthweight (< 2500 g) we got significant results in the analysis of RCTs.(OR 0.71, CI 0.54-0.93eFig.12).
According to the National Institute of Child Health and Human Development (NICHD), the definition of high-risk pregnancy is a pregnancy with any of the following maternal risk factors: existing health condition, malnutrition, overweight or obesity, age < 18 or > 34 years, tobacco smoking, alcohol abuse, and any condition of pregnancy such as gestational diabetes or pregnancy-induced hypertension, previous caesarean delivery, miscarriage, or preterm birth.In the study of Farr et al. 12 a high-risk population was examined.We could see a more significant reduction in the odds of preterm birth than in low-risk population-based studies.This study seemed to be an outlier during the analysis because of the high-risk population.However, as an implication for practice, it is important to note that we can significantly reduce the odds of preterm deliveries and very or extreme preterm deliveries among high-risk pregnant women.Furthermore, we performed a leave-one-out-analysis, and we could not see a major or significant change in the results with the exclusion of this study.
It is worth mentioning that the recommendation for screening abnormal vaginal flora seems as effective as screening cervical length during the second trimester in some populations 13 .This shows that an easily executable screening method can help prevent preterm delivery 35,36 .In the case of shortened cervical length, we can choose progesterone, pessary, or cerclage as a therapy 13,37 .If we cannot postpone delivery by weeks, at least we can be prepared for preterm labor and apply prophylactic steroids to avoid the respiratory distress of premature neonates 38,39 .In the case of abnormal vaginal flora, we can use easily executable and low-cost screening and treatment methods.
Preterm birth and prematurity heavily burden the health service and the economy 40,41 .One of the included studies examined the screening for cost-effectiveness.They found that 46 euros spent on screening and treatment per person could save 56,228 euros per each prevented preterm birth 29 .Each study used different treatment protocols (Table 1).As we talk about asymptomatic infection, some studies compared medical treatment to placebo.The general therapy for bacterial vaginosis is local or oral metronidazole or clindamycin 42,43 .
During the search process we found several important and valuable RCTs 42,44,45 .However these studies did not match our question based on the PICO framework.In these articles the efficacy of treating abnormal vaginal flora was compared to no treatment.Two of these studies 42,45 found that treatment of abnormal vaginal flora (with oral clindamycin or metronidazole) can reduce the risk of preterm deliveries, while the third study 44 did not prove the efficacy of oral clindamycin therapy.Therefore, there is no consensus what agent to use for the treatment of abnormal flora, or to treat it at all.

Strengths and limitation.
Regarding the strengths of our analysis, we followed a pre-defined protocol, which was registered in advance.We also applied a rigorous methodology.Another strength of our study is the low risk of bias.Additionally, the number of enrolled patients was high, and we had precisely defined outcomes.Finally, the examined screening methods are easily executable.
Considering the limitations of our study, a low number of randomized controlled studies were eligible to be enrolled.In addition, different types of screening methods were examined.Furthermore, different treatment protocols were applied for abnormal vaginal flora in the included studies, and pregnancy care protocols were different in each country.Therefore, the inclusion criteria of each study were slightly different.The different screening methods can also be considered a limitation due to their heterogeneity and different effects.However, it is important to mention that despite the different protocols, the results of the studies were very similar, which favors the hypothesis of the benefit of screening of abnormal flora.Due to the low number of studies, we could not analyze publication bias.

Conclusion
Our systematic review found that the routine screening of abnormal vaginal flora might have a beneficial effect on the prevention of preterm birth and low birth weight deliveries.Furthermore, our results suggest that the odds of extreme preterm birth and very low birth weight delivery can also be reducedby using this method.
Moreover, our results show that any investigated screening methods, especially Gram stain, might be effective.
Implications for practice and research.We suggest the routine screening of abnormal vaginal flora during pregnancy care based on our results.Furthermore, clinicians might consider the routine screening in the abovementioned high-risk populations.
Further randomized controlled trials are recommended for all screening methods to assess the question more accurately.The efficacy of screening methods should be compared to find the most effective one.Establishing the exact screening protocol and matching it to local pregnancy care in each country needs further investigation.

Figure 2 .
Figure 2. Forest plot representing the odds of preterm birth before 37 weeks.

Figure 3 .
Figure 3. Forest plot representing the odds of preterm birth before 32 weeks.

Figure 4 .
Figure 4. Forest plots representing the odds of bithweight under 1500 g (A) and 1000 g (B).

Table 1 .
Basic characteristics of the included studies.RCT randomized controlled trial, I/C internvention/ control.

Risk of bias assessment and level of evidence. The
risk of bias assessment results is summarized in Tables