PROF. JANNEKE H.H.M.

VAN DE WIJGERT (Orcid ID : 0000-0003-2728-4560)

Article type : Systematic review

Accepted Article
Lactobacilli-containing vaginal probiotics to cure or prevent bacterial or
fungal vaginal dysbiosis: a systematic review and recommendations for
future trial designs

Janneke H.H.M. van de Wijgert1,2 and Marijn C. Verwijs1

Affiliations:
1. Institute of Infection and Global Health, University of Liverpool, Ronald Ross Building,
8 West Derby Street, Liverpool L69 7BE, UK
2. Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht,
Utrecht University, Stratenum Huispost nr STR 6.131, PO Box 85 500, 3508 GA
Utrecht, The Netherlands

Correspondence to:
Professor Janneke van de Wijgert, MD PhD MPH
Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht,
Utrecht University, Stratenum Huispost nr STR 6.131, PO Box 85 500, 3508 GA Utrecht,
The Netherlands
ORCID: 0000-0003-2728-4560
E-mail: j.vandewijgert@liverpool.ac.uk

Running title: Systematic review of vaginal probiotics

Abstract

Background: Vaginal probiotics claiming to cure and/or prevent bacterial and/or fungal

vaginal dysbiosis are available on the market but did, until recently, not have to be approved

as drugs for human use.

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/1471-0528.15870
This article is protected by copyright. All rights reserved.
 Objectives: We evaluated the impact of vaginal probiotics on bacterial vaginosis (BV) and

vulvovaginal candidiasis (VVC) cure and/or recurrence, as well as vaginal microbiota (VMB)
Accepted Article
composition and vaginal detection of probiotic strains.

Search strategy: We performed a systematic literature search in MEDLINE and Embase up

to 15 January 2019.

Selection criteria: There were no restrictions in probiotic strains/formulations, study

populations, and designs. BV had to be diagnosed by Nugent or Ison-Hay Gram stain scoring,

VVC by culture, wet mount, or PCR, and VMB composition/detection by molecular

techniques.

Data collection and analysis: The authors independently extracted data.

Main results: All 22 vaginal probiotics evaluated in the 34 eligible studies contained

Lactobacillus strains, and some contained additional active ingredients. The probiotics hold

promise for BV cure and prevention, but much less so for VVC cure and prevention. No

major safety concerns were reported in any of the studies. Vaginal detection of probiotic

strains never lasted long beyond the dosing period, suggesting that they did not colonise the

vagina. However, findings are not definitive because heterogeneity was high and the quality

of most studies suboptimal.

Conclusions: Availability of vaginal probiotics for vaginal health indications will likely

decline in 2020 because of regulatory changes. We urge the field to invest in clinical

evidence-based product development, and to conduct future trials more rigorously.

Funding: None.

Keywords: Probiotics, live biotherapeutic products, lactobacilli, bacterial vaginosis,

vulvovaginal candidiasis, vaginal microbiota

This article is protected by copyright. All rights reserved.

 Prospero registration: CRD42017075717
Accepted Article
Tweetable abstract: Lactobacilli-containing vaginal probiotics hold promise for bacterial

vaginosis cure and prevention, but not for vulvovaginal candidiasis.

Introduction

Understanding of the vaginal microbiota (VMB) has advanced significantly in the last two

decades due to the increased availability of next generation sequencing.1 Optimal VMBs are

dominated by lactobacilli, which do not cause immune activation or inflammation, and are

not associated with adverse outcomes such as HIV acquisition and preterm birth.2 Of the five

most common lactobacilli in the vaginal niche (Lactobacillus crispatus, L. iners, L. gasseri,

L. jensenii, and L. vaginalis), L. crispatus-domination is most efficient at keeping bacterial

vaginosis (BV)-associated anaerobes at bay. L. crispatus produces more lactic acid than the

other lactobacilli, and produces several other antimicrobial compounds.3

BV is the most common bacterial vaginal dysbiosis, and is usually characterised by a highly

diverse community of anaerobes including Gardnerella vaginalis, but sometimes by G.

vaginalis-domination.1 G. vaginalis is thought to play an important role in biofilm formation,

and G. vaginalis-domination may indicate the presence of a biofilm.4 Vaginal dysbiosis by

bacterial pathobionts with a higher pathogenicity potential than BV-anaerobes (such as

streptococci, staphylococci, enterococci, and Enterobacteriaceae) is much less common, but

clinically relevant.2 Almost all cases of fungal vaginal dysbiosis are caused by Candida

species, predominantly C. albicans.2 In contrast to BV-anaerobes, bacterial pathobionts and

Candida species often co-exist with lactobacilli in the VMB, perhaps because they can

tolerate high concentrations of lactic acid.5

This article is protected by copyright. All rights reserved.

 This increased VMB knowledge has led to a renewed interest in using exogenous lactobacilli
Accepted Article
(probiotics or live biotherapeutic products)6 to optimise the VMB as part of curative or

preventive vaginal dysbiosis interventions. There are multiple reasons for this: antibiotic BV

treatment (with oral or vaginal metronidazole or clindamycin) and antifungal treatment of

vulvovaginal candidiasis (VVC; with oral or vaginal –azoles) result in suboptimal cure

fractions and high recurrence rates;7 BV biofilms are not penetrated sufficiently by

antibiotics;4 and prolonged use increases the probability of side effects and drug resistance.

Possible mechanisms by which probiotic strains might exert positive effects include niche

occupation so that other bacteria cannot expand and biofilms cannot be established, increased

local production or release of lactic acid and other antimicrobial compounds, modulation of

local cervicovaginal mucosal immune responses, and/or inhibition of C. albicans hyphae

formation.3,8,9

We performed a systematic review to examine the impact of vaginally-applied lactobacilli-

containing probiotics on the VMB in clinical studies. We also offer recommendations for

future trial designs based on this review and our own experiences.10

Methods

The systematic review focussed on BV and VVC cure and/or recurrence as defined by

conventional diagnostic methods, as well as VMB composition and vaginal detection of

probiotic strains as assessed by molecular methods. It was conducted according to the

PRISMA 200911 guidelines and registered on PROSPERO (CRD42017075717).12 Patients or

the public were not involved in this review and a core outcome set was not available. We

searched MEDLINE13 and Embase14 up to 15 January 2019 (search terms in Appendix S1),

This article is protected by copyright. All rights reserved.

 and also included a trial completed by our group that had been fully analysed, and presented

at an international conference, but not yet published, on that date.10 No filters were used
Accepted Article
except for ‘English language’. Duplicates were manually removed. Two researchers

independently screened titles and abstracts (with JvdW as tiebreaker) and additional articles

were identified by screening the reference lists of selected articles, relevant reviews, and

expert opinion pieces. The corresponding author of a conference abstract was contacted but

this did not yield sufficient information for inclusion. We included studies of any vaginally-

applied probiotic performed in sexually active women regardless of menopausal, pregnancy,

or vaginal dysbiosis status. We excluded two studies that assessed yoghurt because the

bacteria present in the yoghurt were not specified. All study designs and probiotic use

schedules (with or without additional oral or vaginal antibiotic or antifungal treatment) were

allowed. BV had to be diagnosed by Nugent15 or Ison-Hay16 Gram stain scoring or the

bacterial VMB comprehensibly assessed by molecular methods. Studies using Amsel criteria,

bacterial culture, other forms of Gram stain scoring (Lactobacillus counts only), rapid tests,

or participant-reported symptoms and/or clinician-observed signs only, or with unclear

diagnostic procedures, were excluded. VVC had to be diagnosed by wet mount, culture, or

Candida polymerase chain reaction (PCR). Studies using participant-reported symptoms

and/or clinician-observed signs only or with unclear diagnostic procedures were excluded.

Both authors extracted data from all selected articles and discussed their findings. Each

article was assessed for risk of bias as described in the Cochrane Handbook for Systematic

Reviews of Interventions.17 We also determined an overall risk of bias per study. Articles for

which all potential biases (selection, performance, detection/ ascertainment, attrition,

reporting, and ‘other’ bias, with the latter including confounding) were assessed as low risk

were judged as ‘overall low risk of bias’, those with a maximum of two high or unclear risks

This article is protected by copyright. All rights reserved.

 of bias as ‘overall medium risk of bias’, and those with three or more high or unclear risks of

bias as ‘overall high risk of bias’. Articles were not excluded based on risk of bias
Accepted Article
assessments. Risk of bias plots were made using RevMan 5.3.5 (The Cochrane Collaboration,

Copenhagen, Denmark). If no statistical testing was reported, but absolute numbers of cases

per exposure group were reported, we performed Chi-squared testing (cross-sectional

comparisons between groups) or McNemar’s testing (pre-post within-arm comparisons) using

Stata 13 (StataCorp, College Station, USA). The full data extraction database can be

requested from the corresponding author by email.

Results

The search identified 2,949 unique articles but only 34 studies were eligible (flow diagram in

Figure 1).10,18–50 Thirteen studies reported 14 results on BV and/or molecular VMB

composition (Table 1),10,18–29 12 studies reported 12 results on VVC (Table 2),29–41 and 14

studies reported 15 results on vaginal probiotic strain detection using molecular techniques

(Table S1).10,26,27,30,39,42–50 The selected studies evaluated 22 different vaginal probiotics

(Table S2). Gynoflor (L. acidophilus KS400 plus 30 μg oestriol; three studies with 359

participants),32,33,39,47 Lactin-V (L. crispatus CTV-05; four studies with 132

participants),30,31,46,56–58 and L. reuteri RC-14 with L. rhamnosus GR-1 (four studies with 47

participants)23,26,42,44 were most studied. The majority of probiotics contained 108-1010

colony-forming units per dose, and all contained lactobacilli as an active ingredient.

Additional active ingredients included oestriol (Gynoflor), Streptococcus thermophilus

(Lactagyn, Femilac), Bifidobacterium bifidum (Ecologic Femi+), Pediococcus acidilactici

(Ellen capsules), and acidifying agents (Kramegin, Florisia, ActiCand 30, and one unnamed

probiotic).

This article is protected by copyright. All rights reserved.

 BV and molecular VMB composition outcomes

Of the 13 studies with BV and/or molecular VMB composition as outcome, five were judged
Accepted Article
to have medium, and eight high, overall risk of bias (Figure 2). Most studies (11/13) were

randomised controlled trials, including six with placebo controls,18,20–22,26,28 two with ‘no

intervention’ controls,10,19 four with metronidazole or clindamycin controls,10,21,23,25 and one

with a vaginal pH lowering tablet control24 (two trials included both placebo/‘no intervention’

and antibiotic controls10,21) (Table 1). Five clinical trials were judged medium risk10,18–21 and

the other six high risk.22–29 The two remaining studies (both judged high risk) were pre-post

intervention studies in women receiving a vaginal probiotic without an antibiotic.27,29

Nine of the 14 main results reported in the 13 studies were on BV cure or post-treatment

Nugent score improvement (2/9 after initial antibiotic treatment and 7/9 without antibiotic

use), nine on BV recurrence (6/9 and 3/9, respectively), and four on BV cure and recurrence

(Table 1). Most studies showed beneficial effects of vaginal probiotic interventions for both

BV cure and recurrence, and the distribution of results did not differ significantly between

studies judged to be of medium or high risk. Of the seven randomised controlled trials that

evaluated BV cure, four showed a significant increase,19,20,23,25 one a non-significant

increase,26 and two no increase in BV cure fraction compared to controls when assessed

immediately to five weeks after probiotic use cessation.18,24 One of the two studies with

negative BV cure results went on to show significantly reduced BV recurrence after

continued use during the next four menstrual cycles,18 and the other one used a pH-lowering

tablet as the control product (the cure fractions were less than 50% in both groups).24 The two

pre-post intervention studies enrolled women with a range of baseline Nugent scores and

showed significantly higher proportions of women with Nugent 0-3 immediately after

probiotic use cessation, which lasted up to 21-28 days post-treatment.27,29 Of the nine BV

This article is protected by copyright. All rights reserved.

 recurrence results in eight randomised controlled trials, six were significant

reductions,10,18,20,23,25,28 two non-significant reductions,10,21 and one no reduction.22 The one

Accepted Article
study that showed no reduction in BV recurrence included women who used the vaginal

probiotic during menses only.22 All except one of the other studies avoided probiotic use

during menses, and in the one study that did not, women used probiotics in between and

during menses.10 The two medium risk randomised controlled trials that compared vaginal

probiotic use after initial antibiotic treatment to placebo or no-intervention controls as well as

prolonged antibiotic use controls both showed that prolonged antibiotic use was more

efficacious in reducing BV recurrence than probiotic use.10,21

VVC outcomes

Of the 12 studies with VVC as outcome, one was judged to have medium, and 11 high,

overall risk of bias (Figure 2). Six of 12 studies were pre-post intervention

studies,29,31,33,35,37,38,41 two were non-randomised cohort studies with parallel comparison

groups (one with a placebo group36 and one comparing fluconazole plus probiotic to

fluconazole only39), and four were randomised controlled trials (two with a placebo

group,30,32 one with a ‘no intervention’ group,40 and one comparing itraconazole plus

probiotic to itraconazole only34) (Table 2). The one medium risk study was a randomised

placebo-controlled trial.30

Four of the 12 probiotic studies with a VVC outcome reported on VVC cure (all without any

antifungal use), eight on VVC recurrence or incidence (2/8 with concurrent antifungal use,

2/8 after antifungal use, 2/8 after antibiotic treatment for another indication and no antifungal

use, and 2/8 without any antifungal or antibiotic use), and two on both (Table 2, Table S3).

The four studies that reported on VVC cure were pre-post intervention studies in women who

This article is protected by copyright. All rights reserved.

 received probiotic treatment in the absence of antifungal treatment.29,33,35,41 They reported

cure fractions ranging from 57-100% after therapy durations ranging from six days to two
Accepted Article
months, but VVC recurrence beyond one month after treatment cessation was not assessed.

The four studies that assessed VVC recurrence after combined probiotic/antifungal treatment

showed mixed results: two studies that compared antifungal plus probiotic use with

antifungal use alone showed no difference in VVC recurrence between the groups,34,39 and

two studies that compared probiotic after antifungal use with either placebo after antifungal

use36 or antifungal use alone40 showed significantly lower VVC recurrence. One of the two

studies that assessed VVC incidence after antibiotic use for a non-VVC indication showed

reduced VVC incidence31 and the other one did not.32 Finally, the two studies in women who

used a vaginal probiotic in the absence of antifungal or antibiotic therapy showed similar

VVC incidence in probiotic and placebo users,30 or no difference in proportions of women

with VVC before and after probiotic use.37,38

Vaginal detection outcomes

Fourteen studies assessed vaginal detection of probiotic strains (with one study reporting

results for two probiotics) using a variety of molecular techniques on culture isolates (10/14)

or on DNA extracted from vaginal swabs (4/14; Table S1). Probiotic strains were detected in

56-100% of probiotic users, or 20-39% of collected swabs, during or directly after the

intervention period. The timing between last probiotic insertion and vaginal sample collection

for detection measurement was never reported, and was the most clear in a study by Dausset

et al that evaluated two, three, or five times per week dosing and assessed probiotic strain

concentrations by quantitative PCR every day.50 This study showed rapid probiotic strain

concentration increases and decreases after each administration. The 11 studies that assessed

probiotic strain detection more than a week after cessation of use showed that detection

This article is protected by copyright. All rights reserved.

 always decreased within weeks; mean probiotic strain survival in the vagina cannot be

determined more precisely because most studies included only one post-use assessment
Accepted Article
ranging from 14 days to six months after cessation.10,26,27,39,42,44–46,49 None of the three studies

that assessed the association between vaginal detection and self-reported adherence found

such an association,10,39,47 but one study showed higher probiotic strain concentrations in

women who ever had any strain detected during the intervention period compared to women

who did not.10 This sequencing study also highlighted the high inter-individual variability in

vaginal probiotic strain detection and concentration, as well as overall VMB composition.

Two L. crispatus CTV-05 studies reported that the probiotic strain was less often detected in

women who had had sexual intercourse during the product use period (with or without

condoms),45,48 and four studies found that detection was less likely in women who had native

lactobacilli prior to probiotic use (L. crispatus in the case of three L. crispatus CTV-05

studies and any lactobacilli in the case of an EcoVag study).39,43,45,48

Quality assessment

We judged overall risk of bias to be high for 26 studies, medium for eight studies, and low for

no studies (Figure 2). The majority of efficacy studies were likely underpowered and the

majority of descriptive vaginal detection studies had low precision, with 9/14 studies

assessing fewer than 20 women per probiotic. None of the studies compared responders to

non-responders in a comprehensive manner, partly due to sample size limitations. Only 12

studies reported adherence data, and only two of those included adherence measures other

than self-report.10,47 The exact timing between last probiotic insertion and vaginal sample

collection for outcome assessment was never reported. Most BV/molecular VMB studies

(11/13), but only 4/12 VVC studies, were randomised controlled trials and participants and

clinicians in 16/24 trials were not blinded. Outcome ascertainment rigour and quantification

This article is protected by copyright. All rights reserved.

 improved over time with the increased availability of molecular technologies (Figure 2).

However, microbiological inclusion criteria often differed from microbiological outcome

Accepted Article
assessments, and it was usually unclear whether these assessments were blinded. Insufficient

consideration of potential confounding factors, such as hormonal contraception use,

pregnancy, menses, vaginal practices, sexual behaviour, any antimicrobial use that was not

part of the study design, and presence of sexually transmitted infections, was also common.

Discussion

Main findings and interpretations

The systematic review results suggest that lactobacilli-containing vaginally applied probiotics

hold promise for BV cure and prevention, but much less so for VVC cure and prevention.

Four of the six medium risk randomised controlled trials with BV endpoints showed

significant benefits, and the other two showed non-significant benefits. No major safety

concerns were reported in any of the studies. In addition, we found consistent evidence that

vaginal detection of probiotic strains does not last long beyond the dosing period, suggesting

that none of the evaluated probiotic strains colonise the vagina. However, these findings are

not definitive because heterogeneity was high and the quality of most studies suboptimal.

Furthermore, publication bias is likely to be high, although we did not formerly assess this

due to high heterogeneity.

We opted to evaluate all vaginally applied probiotics for bacterial and fungal vaginal

dysbiosis indications because no single product-indication combination had been sufficiently

evaluated to allow for a meaningful synthesis of results. While all identified probiotics

contained lactobacilli, it is not yet clear which Lactobacillus strains are the most promising, if

any. The probiotics included in this review therefore varied in terms of Lactobacillus strains,

This article is protected by copyright. All rights reserved.

 other active ingredients, excipients, application methods, and doses. We could not identify

any one product that seemed to outperform the others in any of the clinical indications or
Accepted Article
vaginal detection. Most of the probiotic strains originated from the gut or from traditional

fermented foods, and have been marketed for decades by non-pharmaceutical companies,

even before clinical evidence became available.6 In recent years, the regulatory landscape for

vaginal probiotics has changed considerably. When health claims are made, the US Food and

Drug Administration has required human drug approval since 2016, and the European

Medicines Authority will follow suit per 1 May 2020. As far as we know, only one of the

products included in this review is being developed as a drug for human use: Lactin-V. It

contains a L. crispatus strain (CTV-05) that was isolated from a healthy woman.43 While the

results of the five vaginal detection and safety trials conducted with L. crispatus CTV-05 to

date warranted continued development,30,43,45,47,48,51 we did not find early indications that this

strain is likely to outperform other Lactobacillus strains. A Lactin-V efficacy trial for BV

recurrence is currently ongoing.52

The mixed results of trials with VVC indications are not surprising to us given that molecular

studies have shown that Candida species often co-exist with lactobacilli in the VMB, and

epidemiological studies have shown negative associations between BV and VVC, including

increased VVC incidence after BV treatment.5 In fact, some BV intervention trials assessed

VVC incidence as a safety endpoint.10,53 While the use of exogenous lactobacilli to cure or

prevent BV is much more in line with the molecular and epidemiological evidence to date, it

is not clear what the best dosing strategies would be: as a main treatment in the absence of

antibiotic treatment, as an adjuvant treatment to antibiotic treatment, as a maintenance

therapy to prevent incident or recurrent BV, or a combination of these. This review could not

provide definitive answers to these questions because none of the studies directly compared

This article is protected by copyright. All rights reserved.

 different treatment strategies with the same probiotic. However, the adjuvant and

maintenance therapy studies were of higher quality, and more convincing, than the studies
Accepted Article
that used a vaginal probiotic as a stand-alone BV treatment. Only two studies directly

compared antibiotic plus probiotic use with prolonged antibiotic use and they both found that

the latter was more efficacious in reducing BV recurrence. This would not be a reason to

discontinue vaginal probiotic development because most would agree that prolonged or

frequent antibiotic use is not desirable given risks of side effects and antimicrobial resistance.

None of the articles explained why specific doses (about 108 colony-forming units per

insertion for most products) and dosing frequency and duration were chosen. We suspect that

many of these depended on marketing approvals dating from before the American and

European drug regulatory approval restrictions (as was the case in our study),10 and are not

necessarily based on clinical evidence. While probiotic strains may not have to be present in

high concentrations to exert beneficial effects, the vaginal detection findings of this review

beg the question whether doses and/or dosing frequency/duration should be increased. Some

studies suggested that baseline VMB composition and sexual behaviour during probiotic use

may influence efficacy,10,39,43,45,48 suggesting that stratified, or even personalised, VMB

modulation may be required in the most difficult cases of persistent or recurrent BV.

Strengths and limitations

The quality of most studies was suboptimal. Some of the traditional epidemiological biases

(such as insufficient statistical power, and lack of randomisation, blinding, appropriate

controls, and adjustment for confounding) could have been avoided, but others are unique to

vaginal probiotic trials and are methodologically challenging. The latter tended to improve

over time, with the increasing availability of molecular methods. An important limitation of

This article is protected by copyright. All rights reserved.

 all studies was that the time duration between last probiotic insertion and vaginal sample

collection for endpoint assessment and adherence were not known with sufficient precision,
Accepted Article
and were not taken into account in analyses. This is particularly problematic in the context of

BV endpoints by Nugent or Ison-Hay scoring, because those scores heavily depend on

counting bacteria with a Lactobacillus morphotype (Gram-positive rod) under a microscope.

The scoring cannot differentiate between probiotic lactobacilli and autologous lactobacilli,

and is prone to inflated counts if the sample is taken soon after insertion. Only the two most

recent studies managed to partially address this problem. Dausset et al used daily sampling in

the context of intermittent dosing, as well as quantitative PCR to differentiate between

probiotic and autologous Lactobacillus strains.10,50 Our study employed 16S rRNA gene

quantification and sequencing, which enabled us to not only quantify probiotic and

autologous Lactobacillus strains, but also all BV-associated anaerobes combined.10 Reduction

of the overall BV-anaerobes concentration over time, in addition to the increased lactobacilli

concentration over time, provided convincing evidence for the beneficial effects of

intermittent probiotic use. Our study also showed that host responses to vaginal probiotic

treatment are highly variable, which calls the appropriateness of cross-sectional and pre-post

assessments, as well as the use of group means/medians, into question.

Moving forward, we recommend that future trials incorporate quantitative molecular methods

that differentiate between probiotic and autologous Lactobacillus strains; assess efficacy and

safety endpoints regularly before, during, and after cessation of probiotic use; standardise and

optimise the time duration between insertion and sample collection; improve statistical power

to allow for direct comparisons between responders and non-responders; take all known

confounders and adherence into account; and avoid generic epidemiological biases as much

as possible (Table 3). Ideally, future trials would also quantify BV-anaerobes, and assess the

This article is protected by copyright. All rights reserved.

 impact on biofilm formation, Candida species, and bacterial pathobionts other than BV-

anaerobes.
Accepted Article
Conclusion

Vaginal probiotics hold promise for BV cure and prevention. We expect that the availability

of vaginal probiotics for these indications will decline in 2020 because of regulatory changes.

We therefore urge the field to invest in clinical evidence-based product development, and to

conduct future clinical trials more rigorously.

Acknowledgments

The authors thank medical student Connie Rees for assistance with the search and data

extraction.

Disclosures of Interests

The authors conducted a clinical trial with Ecologic Femi+ (Winclove Probiotics,

Amsterdam, Netherlands) and Gynophilus LP (Biose, Aurillac, France). This trial was funded

by the UK Medical Research Council and the University of Liverpool, but the two companies

donated their products for use in the trial free of charge. The authors have no financial or

intellectual investments in these products, and do not report any other competing interests.

Completed disclosure of interest forms are available to view online as supporting

information.

Contribution to Authorship

JvdW conceived and planned the systematic reviewed, and JvdW and MCV performed the

review and analyses, and wrote the manuscript, together.

This article is protected by copyright. All rights reserved.

 Details of Ethics Approval
Accepted Article
Not applicable.

Funding

None.

References
1. van de Wijgert JHHM, Borgdorff H, Verhelst R, Crucitti T, Francis S, Verstraelen H, et al. The
vaginal microbiota: what have we learned after a decade of molecular characterization? PLOS
ONE. 2014 Aug 22;9(8):e105998.
2. van de Wijgert JHHM, Jespers V. The global health impact of vaginal dysbiosis. Research in
Microbiology. 2017 Nov 1;168(9):859–64.
3. Tachedjian G, Aldunate M, Bradshaw CS, Cone RA. The role of lactic acid production by
probiotic Lactobacillus species in vaginal health. Research in Microbiology. 2017 Nov
1;168(9):782–92.
4. Muzny CA, Schwebke JR. Biofilms: an underappreciated mechanism of treatment failure and
recurrence in vaginal infections. Clin Infect Dis. 2015 Aug 15;61(4):601–6.
5. van de Wijgert JHHM. The vaginal microbiome and sexually transmitted infections are
interlinked: consequences for treatment and prevention. PLOS Medicine. 2017 Dec
27;14(12):e1002478.
6. O’Toole PW, Marchesi JR, Hill C. Next-generation probiotics: the spectrum from probiotics to
live biotherapeutics. Nature Microbiology. 2017 May;2(5):17057.
7. Bradshaw CS, Brotman RM. Making inroads into improving treatment of bacterial vaginosis –
striving for long-term cure. BMC Infectious Diseases. 2015 Jul 29;15:292.
8. Petrova MI, Imholz NCE, Verhoeven TLA, Balzarini J, Van Damme EJM, Schols D, et al.
Lectin-like molecules of Lactobacillus rhamnosus GG inhibit pathogenic Escherichia coli and
Salmonella biofilm formation. PLoS One 2016 Aug;11(8): e0161337.
9. Allonsius CN, Vandenheuvel D, Oerlemans EFM, Petrova MI, Donders GGG, Cos P, et al.
Inhibition of Candida albicans morphogenesis by chitinase from Lactobacillus rhamnosus GG.
Scientific Reports. 2019 Feb 27;9(1):2900.
10. van de Wijgert JHHM, Verwijs MC, Agaba SK, Bronowski C, Mwambarangwe L, Uwineza M,
et al. Intermittent use of vaginal probiotics or oral metronidazole to prevent bacterial vaginosis
recurrence: safety and preliminary efficacy by Nugent scoring and 16S rRNA gene sequencing.
Submitted for publication.
11. Moher D, Liberati A, Tetzlaff J, Altman DG, Group TP. Preferred reporting items for systematic
reviews and meta-analyses: the PRISMA statement. PLOS Medicine. 2009 Jul
21;6(7):e1000097.
12. Prospero - International prospective register of systematic reviews [Internet]. [cited 2018 Jul
30]. Available from:
https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=75717
13. PubMed - NCBI [Internet]. [cited 2018 Jul 17]. Available from:
https://www.ncbi.nlm.nih.gov/pubmed/
14. Embase | Elsevier [Internet]. [cited 2018 Jul 17]. Available from:
https://www.elsevier.com/solutions/embase-biomedical-research

This article is protected by copyright. All rights reserved.

 15. Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by
a standardized method of gram stain interpretation. J Clin Microbiol. 1991 Feb 1;29(2):297–
301.
16. Ison C, Hay P. Validation of a simplified grading of Gram stained vaginal smears for use in
Accepted Article
genitourinary medicine clinics. Sex Transm Infect. 2002 Dec;78(6):413–5.
17. Cochrane handbook for systematic reviews of interventions version 5.1.0 [Internet]. The
Cochrane Collaboration; 2011 [cited 2018 Jul 11]. Available from: http://handbook-5-
1.cochrane.org/
18. Larsson P-G, Stray-Pedersen B, Ryttig KR, Larsen S. Human lactobacilli as supplementation of
clindamycin to patients with bacterial vaginosis reduce the recurrence rate; a 6-month, double-
blind, randomized, placebo-controlled study. BMC Women’s Health [Internet]. 2008 Dec [cited
2018 May 23];8(1). Available from:
http://bmcwomenshealth.biomedcentral.com/articles/10.1186/1472-6874-8-3
19. Petricevic L, Witt A. The role of Lactobacillus casei rhamnosus Lcr35 in restoring the normal
vaginal flora after antibiotic treatment of bacterial vaginosis. BJOG. 2008 Oct;115(11):1369–74.
20. Mastromarino P, Macchia S, Meggiorini L, Trinchieri V, Mosca L, Perluigi M, et al.
Effectiveness of Lactobacillus-containing vaginal tablets in the treatment of symptomatic
bacterial vaginosis. Clinical Microbiology and Infection. 2009 Jan;15(1):67–74.
21. Bradshaw CS, Pirotta M, De Guingand D, Hocking JS, Morton AN, Garland SM, et al. Efficacy
of oral metronidazole with vaginal clindamycin or vaginal probiotic for bacterial vaginosis:
randomised placebo-controlled double-blind trial. Abdel-Aleem H, editor. PLoS ONE. 2012 Apr
3;7(4):e34540.
22. Eriksson K, Carlsson B, Forsum U, Larsson P-G. A double-blind treatment study of bacterial
vaginosis with normal vaginal lactobacilli after an open treatment with vaginal clindamycin
ovules. Acta Dermato-Venereologica. 2005 Jan 1;85(1):42–6.
23. Anukam KC, Osazuwa E, Osemene GI, Ehigiagbe F, Bruce AW, Reid G. Clinical study
comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat
symptomatic bacterial vaginosis. Microbes and Infection. 2006 Oct;8(12–13):2772–6.
24. Hemalatha R, Mastromarino P, Ramalaxmi BA, Balakrishna NV, Sesikeran B. Effectiveness of
vaginal tablets containing lactobacilli versus pH tablets on vaginal health and inflammatory
cytokines: a randomized, double-blind study. European Journal of Clinical Microbiology &
Infectious Diseases. 2012 Nov;31(11):3097–105.
25. Ling Z, Liu X, Chen W, Luo Y, Yuan L, Xia Y, et al. The restoration of the vaginal microbiota
after treatment for bacterial vaginosis with metronidazole or probiotics. Microbial Ecology.
2013 Apr;65(3):773–80.
26. Bisanz JE, Seney S, McMillan A, Vongsa R, Koenig D, Wong L, et al. A systems biology
approach investigating the effect of probiotics on the vaginal microbiome and host responses in
a double blind, placebo-controlled clinical trial of post-menopausal women. PLoS ONE. 2014
Aug 15;9(8):e104511.
27. Verdenelli MC, Cecchini C, Coman MM, Silvi S, Orpianesi C, Coata G, et al. Impact of
probiotic SYNBIO® administered by vaginal suppositories in promoting vaginal health of
apparently healthy women. Current Microbiology. 2016 Oct;73(4):483–90.
28. Bohbot JM, Daraï E, Bretelle F, Brami G, Daniel C, Cardot JM. Efficacy and safety of vaginally
administered lyophilized Lactobacillus crispatus IP 174178 in the prevention of bacterial
vaginosis recurrence. Journal of Gynecology Obstetrics and Human Reproduction. 2018
Feb;47(2):81–6.
29. Rapisarda AMC, Caldaci L, Valenti G, Brescia R, Sapia F, Sarpietro G, et al. Efficacy of
vaginal preparation containing Lactobacillus acidophilus, lactic acid and deodorized garlic
extract in treatment and prevention of symptomatic bacterial vaginitis: result from a single-arm
pilot study. Italian Journal of Gynaecology & Obstetrics. 2018;30(1):21–31.
30. Czaja CA, Stapleton AE, Yarova-Yarovaya Y, Stamm WE. Phase I trial of a Lactobacillus
crispatus vaginal suppository for prevention of recurrent urinary tract infection in women.
Infectious Diseases in Obstetrics and Gynecology. 2007:1–8.
31. Özmen S, Turhan NO, Seckin NC. Gardnerella-associated vaginitis: comparison of three
treatment modalities. Turk J Med Sci. 1998;28:171–3.

This article is protected by copyright. All rights reserved.

 32. Pirotta M, Gunn J, Chondros P, Grover S, O’Malley P, Hurley S, et al. Effect of lactobacillus in
preventing post-antibiotic vulvovaginal candidiasis: a randomised controlled trial. BMJ. 2004
Sep 4;329(7465):548.
33. Di Pierro F, Catacchio V, Candidi C, Zerbinati N, Alfonso R. Rhatany-based preparation in
Accepted Article
vulvovaginitis and vaginosis. Gazz Med Ital. 2009;168:338–46.
34. Witt A, Kaufmann U, Bitschnau M, Tempfer C, Özbal A, Haytouglu E, et al. Monthly
itraconazole versus classic homeopathy for the treatment of recurrent vulvovaginal candidiasis:
a randomised trial: Monthly itraconazole versus classic homeopathy for treatment of RVVC.
BJOG: An International Journal of Obstetrics & Gynaecology. 2009 Oct;116(11):1499–505.
35. Vicariotto F, Del Piano M, Mogna L, Mogna G. Effectiveness of the association of 2 probiotic
strains formulated in a slow release vaginal product, in women affected by vulvovaginal
candidiasis: a pilot study. Journal of Clinical Gastroenterology. 2012 Oct;46:S73–80.
36. De Seta F, Parazzini F, De Leo R, Banco R, Maso GP, De Santo D, et al. Lactobacillus
plantarum P17630 for preventing Candida vaginitis recurrence: a retrospective comparative
study. European Journal of Obstetrics & Gynecology and Reproductive Biology. 2014
Nov;182:136–9.
37. Donders G, Neven P, Moegele M, Lintermans A, Bellen G, Prasauskas V, et al. Ultra-low-dose
estriol and Lactobacillus acidophilus vaginal tablets (Gynoflor®) for vaginal atrophy in
postmenopausal breast cancer patients on aromatase inhibitors: pharmacokinetic, safety, and
efficacy phase I clinical study. Breast Cancer Research and Treatment. 2014 Jun;145(2):371–9.
38. Donders G, Bellen G, Neven P, Grob P, Prasauskas V, Buchholz S, et al. Effect of ultra-low-
dose estriol and lactobacilli vaginal tablets (Gynoflor®) on inflammatory and infectious markers
of the vaginal ecosystem in postmenopausal women with breast cancer on aromatase inhibitors.
European Journal of Clinical Microbiology & Infectious Diseases. 2015 Oct;34(10):2023–8.
39. Pendharkar S, Brandsborg E, Hammarström L, Marcotte H, Larsson P-G. Vaginal colonisation
by probiotic lactobacilli and clinical outcome in women conventionally treated for bacterial
vaginosis and yeast infection. BMC Infectious Diseases. 2015 Dec;15(1).
40. Kovachev SM, Vatcheva-Dobrevska RS. Local probiotic therapy for vaginal Candida albicans
infections. Probiotics & Antimicro Prot. 2015 Mar 1;7(1):38–44.
41. Murina F, Vicariotto F, Di Francesco S. Thymol, eugenol and lactobacilli in a medical device
for the treatment of bacterial vaginosis and vulvovaginal candidiasis. New Microbiol.
2018;41(3):220–4.
42. Gardiner GE, Heinemann C, Bruce AW, Beuerman D, Reid G. Persistence of Lactobacillus
fermentum RC-14 and Lactobacillus rhamnosus GR-1 but not L. rhamnosus GG in the human
vagina as demonstrated by randomly amplified polymorphic DNA. Clinical and Vaccine
Immunology. 2002 Jan 1;9(1):92–6.
43. Antonio MAD, Hillier SL. DNA fingerprinting of Lactobacillus crispatus strain CTV-05 by
repetitive element sequence-based PCR analysis in a pilot study of vaginal colonization. Journal
of Clinical Microbiology. 2003 May 1;41(5):1881–7.
44. Burton JP, Cadieux PA, Reid G. Improved understanding of the bacterial vaginal microbiota of
women before and after probiotic instillation. Applied and Environmental Microbiology. 2003
Jan 1;69(1):97–101.
45. Antonio MAD, Meyn LA, Murray PJ, Busse B, Hillier SL. Vaginal colonization by probiotic
Lactobacillus crispatus CTV‐ 05 is decreased by sexual activity and endogenous lactobacilli.
The Journal of Infectious Diseases. 2009 May 15;199(10):1506–13.
46. Ehrström S, Daroczy K, Rylander E, Samuelsson C, Johannesson U, Anzén B, et al. Lactic acid
bacteria colonization and clinical outcome after probiotic supplementation in conventionally
treated bacterial vaginosis and vulvovaginal candidiasis. Microbes and Infection. 2010
Sep;12(10):691–9.
47. Hemmerling A, Harrison W, Schroeder A, Park J, Korn A, Shiboski S, et al. Phase 2a study
assessing colonization efficiency, safety, and acceptability of Lactobacillus crispatus CTV-05 in
women with bacterial vaginosis: Sexually Transmitted Diseases. 2010 Dec;37(12):745–50.
48. Ngugi BM, Hemmerling A, Bukusi EA, Kikuvi G, Gikunju J, Shiboski S, et al. Effects of
bacterial vaginosis-associated bacteria and sexual intercourse on vaginal colonization with the

This article is protected by copyright. All rights reserved.

 probiotic Lactobacillus crispatus CTV-05. Sexually Transmitted Diseases. 2011
Nov;38(11):1020–7.
49. Tomusiak A, Strus M, Heczko P, Adamski P, Stefański G, Mikołajczyk-Cichońska A, et al.
Efficacy and safety of a vaginal medicinal product containing three strains of probiotic bacteria:
Accepted Article
a multicenter, randomized, double-blind, and placebo-controlled trial. Drug Design,
Development and Therapy. 2015 Sep;9:5345–54.
50. Dausset C, Patrier S, Gajer P, Thoral C, Lenglet Y, Cardot J-M, et al. Comparative phase I
randomized open-label pilot clinical trial of Gynophilus® (Lcr regenerans®) immediate release
capsules versus slow release muco-adhesive tablets. Eur J Clin Microbiol Infect Dis.
2018;37(10):1869–80.
51. Stapleton AE, Au-Yeung M, Hooton TM, Fredricks DN, Roberts PL, Czaja CA, et al.
Randomized, placebo-controlled Phase 2 trial of a Lactobacillus crispatus probiotic given
intravaginally for prevention of recurrent urinary tract infection. Clinical Infectious Diseases.
2011 May 15;52(10):1212–7.
52. LACTIN-V study for recurrent bacterial vaginosis - ClinicalTrials.gov [Internet]. [cited 2019
Apr 23]. Available from: https://clinicaltrials.gov/ct2/show/NCT02766023
53. McClelland RS, Richardson BA, Hassan WM, Chohan V, Lavreys L, Mandaliya K, et al.
Improvement of vaginal health for Kenyan women at risk for acquisition of Human
Immunodeficiency Virus type 1: results of a randomized trial. J Infect Dis. 2008 May
15;197(10):1361–8.

Tables, Figures, and Supplementary Materials Caption List

Table 1: Summary of BV/VMB study results.

Table 2: Summary of VVC study results.

Table 3: Recommendations for vaginal probiotic efficacy evaluation

Figure 1: PRISMA flow diagram

Figure 2: Risk of bias assessments

Appendix S1: Search terms and filters

Table S1: Summary of vaginal probiotic strain detection results

Table S2: Product characteristics

This article is protected by copyright. All rights reserved.

 Table 1: Summary of BV/VMB study results.
BV bacterial vaginosis, PY person-years at risk, RCT randomised controlled trial, VMB vaginal
microbiota.
Main results of studies with BV cure and/or recurrence (by Nugent or Ison-Hay scoring of Gram
Accepted Article
stains) and/or molecular VMB composition endpoints: 14 results in 13 studies.
Reference Probiotic Study design Results
Articles with a medium overall risk of bias
Larsson18 EcoVag (L. gasseri RCT: placebo 10 days of EcoVag after clindamycin did not improve BV cure (by Ison-Hay; 32/50 cured)
EB01-DSM 14869 + L. control compared to placebo (37/50 cured) but significantly reduced the cumulative incidence of
rhamnosus Lbp BV/intermediate microbiota within 4 menstrual cycles when used for 10 days after each
PB01-DSM 14870) menses (13/37 versus 21/39 incident cases, respectively).
Petricevic19 Gynophilus (L. casei RCT: no- 7 days of Gynophilus after clindamycin resulted in significantly improved BV cure (by
rhamnosus Lcr35) intervention Nugent 0-3) compared to women receiving clindamycin only (69/83 versus 31/88,
control respectively) assessed 5 weeks after cessation of the intervention period.
Mastro- Florisia (L. brevis CD2 RCT: placebo 7 days of Florisia (without any antibiotic) was significantly more efficacious in curing BV
marino20 + L. salivarius subsp. control (15/18 Nugent 0-3, 3/18 Nugent 4-6) than placebo (2/16 Nugent 4-6; 14/16 persistent
salicinius FV2 + L. Nugent 7-10) and was associated with significantly lower BV (Nugent 7-10) cumulative
plantarum FV9) incidence in the 2 weeks after Florisia cessation (7/18 and 13/16, respectively).
Bradhaw21 Gynoflor (L. RCT: placebo 12 days of Gynoflor after oral metronidazole treatment for BV (Nugent 7-10) resulted in a
acidophilus KS400 + and borderline (p=0.13) lower BV recurrence (9/133) at Month 1 compared to 12 days of
0.03mg oestriol) clindamycin placebo (13/135) but higher compared to 7 days of vaginal clindamycin (5/140). At
cream controls Month 6, BV recurrence was comparable between the three arms (37/133, 36/135, and
42/140, respectively).
van de Ecologic Femi+ (B. RCT: no- Intermittent Ecologic Femi+ use after oral metronidazole BV treatment resulted in
Wijgert10 bifidum W28 + L. intervention significantly lower BV (Nugent 7-10) recurrence than no-intervention (incidence 3.58/PY
acidophilus W70 + L. and oral versus 10.18/PY, respectively), and significantly reduced BV-anaerobes concentration by
helveticus W74 + L. metronidazole sequencing, during the 2 months of use, but the effect disappeared by 4 months after
brevis W63 + L. controls cessation of use.
plantarum W21 + L.
salivarius W24)
Van de Gynophilus LP (L. RCT: no- Intermittent Gynophilus LP use after oral metronidazole BV treatment resulted in a non-
Wijgert10 rhamnosus Lcr35 intervention significant lower BV (Nugent 7-10) recurrence than controls (incidence 5.36/PY), and
regenerans) and oral non-significantly reduced BV-anaerobes concentration by sequencing, during the 2
metronidazole months of use, but the effect disappeared by 4 months after cessation of use.
controls
Articles with a high overall risk of bias
Eriksson22 L. gasseri + L. casei RCT: placebo Use of lactobacilli-impregnated tampons for 5 or more days during the first and second
var rhamnosus + L. control menses after clindamycin treatment was not efficacious in reducing BV recurrence (by
fermentum Nugent 4-10) by the end of the two menstrual cycles (41/91) compared to placebo
(impregnated tampons (34/96).
tampons)
Anukam23 RC-14/GR-1 (L. RCT: 5 days of GR-1+RC-14 capsules (without any antibiotic) was significantly more efficacious
fermentum RC-14 + L. metronidazole in curing BV (12/20 Nugent 0-3) than 5 days of metronidazole gel (6/20) when assessed
rhamnosus GR-1) gel control directly after the intervention, and also in reducing BV recurrence (Nugent 7-10) up to 25
days post-intervention (2/17 versus 9/18, respectively).
Hemalatha24 Florisia (L. brevis CD2 RCT: pH 8 days of Florisia (without any antibiotic) was not efficacious in curing BV (by Nugent;
+ L. salivarius subsp. lowering tablet 36/75 cured) compared to pH-lowering tablet (29/73 cured).
salicinius + L. control
plantarum)
Ling25 L. delbrueckii subsp. RCT: 7 days of unnamed L. delbrueckii-containing probiotic (without any antibiotic) was
lactis DM8909 metronidazole equally effective as vaginal metronidazole gel in curing BV (assessed 5 days after
control treatment completion; 22/25 and 25/30 cured, respectively) and was associated with a
significantly lower recurrence 30 days after treatment completion than metronidazole
gel (0/25 versus 5/30 recurrences). The molecular VMB data also suggest benefits of
using the probiotic.
Bisanz26 RC-14/GR-1 (L. Randomised In women with Nugent 4-6, 3 days of GR-1/RC-14 use (without any antibiotic) did not
reuteri RC-14 + L. controlled result in more women with Nugent score improvement compared to placebo use (2/10
rhamnosus GR-1) cross-over trial: and 0/10 improved one day after use, and 2/9 and 1/8 improved 8 days after use).
placebo control However, the Lactobacillus relative abundance was significantly increased, and
Atopobium relative abundance significantly decreased, one day after use.
Verdenelli27 SYNBIO gin (L. Pre-/post- In women with Nugent 0-6, 7 days of SYNBIO gin (without any antibiotic) resulted in a
rhamnosus IMC 501 intervention significantly higher proportion of women with Nugent 0-3 directly after therapy (28/35
+ L. paracasei IMC study versus 21/35 at baseline), and this persisted for 21 days after cessation of therapy (28/35
502) still had Nugent 0-3; insufficient data for McNemar testing). SYNBIO gin use also resulted
in a significant increase in Lactobacillus concentration (p<0.01).
Bohbot28 Physioflor (L. RCT: placebo Physioflor for 14 days immediately after metronidazole therapy, plus 14 days in three
crispatus IP 174178) control subsequent menstrual cycles, resulted in significantly lower BV cumulative incidence
(Nugent 7-10), and time to BV recurrence, compared to placebo (8/39 and 16/39 had at
least one recurrence, respectively). However, the effects disappeared 84 days after
product cessation (p=0.922, absolute numbers not given).
Rapisarda29 L. acidophilus LA14 Pre-/post- In women with Nugent 4-10, 14 days of L. acidophilus LA14 use (without any antibiotic)
intervention resulted in 46/60 women cured (Nugent 0-3) at the end of therapy, and this persisted for
study 28 days after product cessation (50/60).

This article is protected by copyright. All rights reserved.

 Table 2: Summary of VVC study results.

BV bacterial vaginosis, UTI urinary tract infection, VMB vaginal microbiome, VVC vulvovaginal
candidiasis.
Accepted Article
Main results of the studies with VVC cure and/or incidence (by culture or wet mount) endpoints.
None of the studies used Candida PCR: 12 results in 12 studies.
Reference Probiotic Study design Results
Articles with a medium overall risk of bias
Czaja30 L. crispatus CTV-05 (not RCT: placebo In women with a history of recurrent UTI but without current dysuria, 5
Lactin-V) control days of L. crispatus CTV-05 use resulted in a similar VVC cumulative
incidence (4/15) compared to placebo use (2/15) up to 25 days after
cessation of use.
Articles with a high overall risk of bias
Özmen31 Gynoflor (L. acidophilus KS400 Pre-/post- 12 days of Gynoflor with or without metronidazole, compared to
+ 0.03mg oestriol) intervention metronidazole alone (to treat BV), resulted in significantly lower cumulative
study in three VVC incidence (3/96 and 2/97, versus 14/114, respectively) in the 10-13
separate, non- days after cessation of use.
randomised
groups
Pirotta32 Femilac (L. rhamnosus + L. 2x2 factorial 10 days of Femilac during (6 days) and after (4 days) non-metronidazole
delbrueckki + L. acidophilus + design RCT: antibiotic use, compared to placebo controls, was not effective in reducing
S. thermophilus) placebo control VVC incidence within 18 days after cessation of Femilac use (17/59 versus
9/54, respectively).
Di Pierro33 Kramegin (L. acidophilus + Pre-/post- 10 days of Kramegin (without antifungal) cured 75/75 women with acute
Krameria triandra plant intervention VVC, and 20/30 women with recurrent VVC (both significant compared to
extract + 15mg lactic acid) study baseline). Cure was assessed 7 days after last administration.
Witt34 L. gasseri RCT: probiotic In women with acute VVC and a history of recurrent VVC, after induction
+ itraconazole with itraconazole twice weekly for one month, itraconazole (once per
versus month) with L. gasseri (6 consecutive days per month) during 6 months
itraconazole resulted in a similar VVC recurrence rate as itraconazole (once per month)
only only (15/45 versus 12/31, respectively). Results were also similar 6 months
after product cessation (6/25 versus 5/23, respectively).
Vicariotto35 ActiCand (L. fermentum LF10 Pre-/post- In women with acute VVC and a history of recurrent VVC, ActiCand (without
+ L. acidophilus LA02) intervention antifungal; 7 consecutive nights, followed by 3 nights per week for 3 weeks,
study and one night per week for 4 weeks) resulted in a significant reduction in
VVC cases to 7/30 women after cessation of therapy at Day 56.
De Seta36 Gyno-Canesflor (L. plantarum Non- In women with acute VVC but no history of recurrent VVC, 34 days of Gyno-
P17630) randomised Canesflor after clotrimazole treatment, compared to placebo use, resulted
prospective in a (non-significant, Fisher's p=0.095) lower cumulative VVC incidence
cohort study (1/40 versus 5/40, respectively).
Donders37 & Gynoflor (L. acidophilus KS400 Pre-/post- A 84-day single-arm Gynoflor treatment scheme (once daily for 4 weeks
Donders38 + 0.03mg oestriol) intervention followed by thrice weekly for 8 weeks) for atrophic vaginitis in
study postmenopausal women with breast cancer temporarily increased
asymptomatic VVC prevalence after two weeks of use (from 2/14 to 7/16)
but returned to baseline values after one (3/16), two (3/16), and three
(3/13) months of use.
Pendharkar EcoVag (L. gasseri DSM 14869 Non- In women with recurrent VVC, fluconazole (7 days in cycle 1 followed by
- Trial II39 + L. rhamnosus DSM 14870) randomised once weekly in cycles 2+3, and biweekly in cycles 4-6) with EcoVag (10 days
prospective in cycle 2, and once weekly in cycles 2-6) did not significantly improve the
cohort study VVC cure fraction 6 months after treatment cessation compared to the
fluconazole regimen without EcoVag (8/9 versus 7/10, respectively) but
almost all women in both groups were cured.
Kovachev40 Lactagyn (L. acidophilus, L. RCT: no- In women with acute VVC, antifungal treatment (fluconazole and
rhamnosus, S. thermophilus, intervention fenticonazole) followed by 10-day Lactagyn therapy resulted in lower VVC
L. delbrueckii subsp. control recurrence (10/209) at the final follow-up visit 25-30 days after product
bulgaricus) cessation than antifungal treatment alone (76/207). The authors report this
as two non-significant pre/post results, but these are significant when the
appropriate test (McNemar) is used, and when the two groups are
compared directly.
Rapisarda29 L. acidophilus LA14 Pre-/post- In women with Nugent 4-10 and 21/60 with acute VVC, 14 days of L.
intervention acidophilus LA14 without antifungals or antibiotics resulted in a significant
study reduction of VVC cases to 9/60 one day after cessation of use, and 6/60 4
weeks after cessation of use. Mean Candida culture counts also decreased
significantly.
Murina41 Estromineral Probiogel (L. Pre-/post- Estromineral Probiogel (without antifungal) cured 51/82 women with acute
fermentum LF10 + L. intervention VVC and 27/27 in women with recurrent VVC between 20-30 days after
plantarum LP02) study therapy initiation (both significant compared to baseline), but therapy
longer than the intended 6 days was required in 57.3% and 63.0% women,
respectively, due to persistence of symptoms.

This article is protected by copyright. All rights reserved.

 Table 3: Recommendations for vaginal probiotic efficacy evaluation.
Accepted Article
BV bacterial vaginosis, VMB vaginal microbiota, VVC vulvovaginal candidiasis.

Topic Recommendations

Study design, - Randomised placebo-controlled blinded trials are the preferred study design. Avoid pre-post studies without
populations, any parallel control groups.
sample size - Define the target population and recruitment strategy a priori. Avoid convenience samples.
- Evaluate in women with and without urogenital symptoms, and in women at various levels of vaginal
dysbiosis risk.
- Conduct sample size calculations. Take into account that vaginal probiotic efficacy tends to have high inter-
individual variability and that efficacy analyses should be controlled for known confounders such as hormonal status,
menses, sexual behaviour, and presence of sexually transmitted infections. Allow for comparisons between responders
and non-responders.

Interventions - Consider dose per insertion, frequency of insertion, timing of insertion (also in relationship to timing of
sample collection for efficacy evaluation), and how menses will be taken into account.
- Provide clear use instructions, also in relation to vaginal hygiene practices and menses, and assess whether
the participant understood them. Assess the participant’s ability to insert the probiotic correctly by direct observation.
- Consider frequent sampling for outcome assessments. Participants could self-sample at home.
- Assess adherence throughout the study, ideally not just by self-report.

Controls - A parallel placebo control group is best, but if not possible, use no-intervention controls.
- In addition, consider a positive control group of women using oral or vaginal metronidazole/clindamycin for
BV or an antifungal for VVC.
- Blind the laboratory technicians who conduct the outcome assessments, especially when blinding of the
clinicians and participants is not possible.

Outcomes - Assess outcomes prior to, during, and after cessation of probiotic use in all women at all time points
regardless of symptomatology. Use the same outcome assessments to determine eligibility and/or cure of the initial
BV/VVC prior to initiation of probiotic use (if applicable), at the start of probiotic use, and throughout follow-up.
- Use (semi-)quantitative molecular methods to differentiate between probiotic lactobacilli, autologous
lactobacilli, BV anaerobes, Candida species, and other organisms of interest.
- In addition, use Nugent scoring and Amsel criteria to allow for comparisons with older studies and with
current clinical practice.
- Consider and record the timing between last probiotic insertion and sample collection for efficacy
assessment.
- Consider the vaginal microbiota holistically, including bacteria, yeasts, and sexually transmitted pathogens.
- Always assess and report safety outcomes (adverse events).

Results reporting - Report the name and manufacturer of the vaginal probiotic that was evaluated, as well as all active
ingredients, excipients, and dosing information. Explain the rationale for selecting the product and be transparent about
competing interests.
- Report clearly how the probiotic was meant to be used: as a main therapy to cure BV or VVC, as an adjuvant
therapy to an antimicrobial therapy, or as a maintenance therapy to prevent incidence or recurrence.
- Report intent-to-treat and per-protocol statistical analysis results.

This article is protected by copyright. All rights reserved.

 Figure 1: PRISMA flow diagram.
Accepted Article

This article is protected by copyright. All rights reserved.

 Figure 2: Risk of bias assessments.
Accepted Article
A. B.

‘Other sources of bias’ included whether confounders were considered (in the eligibility criteria or statistical analyses), intent-to-treat results were reported,
and statistical testing was appropriate. A Authors’ judgements about each potential bias for each study, ordered by publication year. Studies with high overall
risk of bias (seventh column) are indicated with a minus symbol and studies with medium overall risk of bias with a plus symbol. No studies were judged to
have a low overall risk of bias. B Summary of each bias across all 34 studies.

This article is protected by copyright. All rights reserved.