Lactobacilli-containing vaginal probiotics to cureor prevent bacterial or fungal vaginal dysbiosis:a systematic review and recommendations forfuture trial designsJHHM van de Wijgert,a,b MC Verwijsa
a Institute of Infection and Global Health, University of Liverpool, Liverpool, UK b Julius Center for Health Sciences and Primary Care,
University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands
Correspondence: Professor JHHM van de Wijgert, 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.
Email: [email protected]
Accepted 17 June 2019. Published Online 8 August 2019.
Background Vaginal probiotics claiming to cure and/or prevent
bacterial and/or fungal vaginal dysbiosis are available on the
market but, until recently, did not have to be approved as drugs
for human use.
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)
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.
Keywords Bacterial vaginosis, lactobacilli, live biotherapeutic
products, probiotics, vaginal microbiota, vulvovaginal candidiasis.
Tweetable abstract Lactobacilli-containing vaginal probiotics hold
promise for bacterial vaginosis cure and prevention, but not for
vulvovaginal candidiasis.
Please cite this paper as: van de Wijgert JHHM , Verwijs MC. Lactobacilli-containing vaginal probiotics to cure or prevent bacterial or fungal vaginal
dysbiosis: a systematic review and recommendations for future trial designs. BJOG 2019; https://doi.org/10.1111/1471-0528.15870
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 Opti-
mal 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, Lactobacillus
iners, Lactobacillus gasseri, Lactobacillus jensenii, and Lacto-
bacillus 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
Prospero registration: CRD42017075717.
1ª 2019 Royal College of Obstetricians and Gynaecologists
DOI: 10.1111/1471-0528.15870
www.bjog.orgSystematic review
lactobacilli and produces several other antimicrobial com-
pounds.3
Bacterial vaginosis 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 Enterobacteri-
aceae) is much less common, but clinically relevant.2
Almost all cases of fungal vaginal dysbiosis are caused by
Candida species, predominantly Candida albicans.2 In con-
trast 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 increased VMB knowledge has led to a renewed
interest in using exogenous lactobacilli (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 antifun-
gal 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 suffi-
ciently by antibiotics.4 In addition, 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 can-
not expand and biofilms cannot be established, increased
local production or release of lactic acid and other antimi-
crobial compounds, modulation of local cervicovaginal
mucosal immune responses, and/or inhibition of C. albi-
cans hyphae formation.3,8,9
We performed a systematic review to examine the
impact of vaginally applied lactobacilli-containing probi-
otics on the VMB in clinical studies. We also offer recom-
mendations for future trial designs based on this review
and our own experiences.10
Methods
The systematic review focused on BV and VVC cure and/or
recurrence as defined by conventional diagnostic methods,
as well as VMB composition and vaginal detection of pro-
biotic strains as assessed by molecular methods. It was con-
ducted according to the PRISMA 200911 guidelines and
registered on PROSPERO (CRD42017075717).12 Patients
and 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), and also included a trial completed by our
group that had been fully analysed, and that had been pre-
sented at an international conference, but was not yet pub-
lished, on that date.10 No filters were used except for
‘English language’. Duplicates were manually removed.
Two researchers independently screened titles and abstracts
(with JvdW as tiebreaker) and additional articles were iden-
tified by screening the reference lists of selected articles, rel-
evant reviews, and expert opinion pieces. The
corresponding author of a conference abstract was con-
tacted but this did not yield sufficient information for
inclusion. We included studies of any vaginally applied
probiotic in sexually active women regardless of menopau-
sal, 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, bacte-
rial culture, other forms of Gram stain scoring (Lactobacil-
lus 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 System-
atic Reviews of Interventions.17 We also determined an
overall risk of bias per study. Articles for which all poten-
tial biases (selection, performance, detection/ascertainment,
attrition, reporting, and ‘other’ bias, with the latter includ-
ing confounding) were assessed as low risk were judged as
‘overall low risk of bias’, those with a maximum of two
high or unclear risks 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 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-square testing
(cross-sectional comparisons between groups) or McNe-
mar’s testing (pre-post within-arm comparisons) using
STATA 13 (StataCorp, College Station, TX, USA). The full
data extraction database can be requested from the corre-
sponding author by email.
2 ª 2019 Royal College of Obstetricians and Gynaecologists
Wijgert, Verwijs
Results
The search identified 2949 unique articles but only 34 stud-
ies 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 tech-
niques (Table S1).10,26,27,30,39,42–50 The selected studies eval-
uated 22 different vaginal probiotics (Table S2). The most
studied were Gynoflor (L. acidophilus KS400 plus 30 mi-
crogram oestriol; three studies with 359 partici-
pants),32,33,39,47 Lactin-V (L. crispatus CTV-05; four studies
with 132 participants),30,31,46 and L. reuteri RC-14 with
L. rhamnosus GR-1 (four studies with 47 partici-
pants)23,26,42,44. 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).
BV and molecular VMB composition outcomesOf the 13 studies with BV and/or molecular VMB compo-
sition as outcome, five were judged 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 con-
trols,10,21,23,25 and one with a vaginal pH-lowering tablet
control24 (two trials included both placebo/‘no interven-
tion’ 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 improve-
ment (2/9 after initial antibiotic treatment and 7/9 without
antibiotic use), nine on BV recurrence (6/9 and 3/9,
• Full-text articles by snowball-ling of reference lists (n = 28)
• Unpublished study by the authors (n = 1)
Records identified through databases search string
(n = 5416)
Records after duplicates removed,screened based on title/abstract
(n = 2949)
Full-text articles assessed foreligibility(n = 105)
Potentially relevant articles identified
(n = 76)
Articles excluded based on title/abstract (n = 2873)
• Oral probiotics (n = 44)• Useful for introduction
and/or discussion (n = 34)• Irrelevant (n = 2795)
Excluded articles (n = 71)• Amsel criteria only (n = 15)• Irrelevant outcome (n = 10)• Not in English (n = 9)• No full-text available (n = 9)• No probiotics used (n = 8)• Non-human study (n = 6)• Oral probiotics (n = 5)• Data uninterpretable (n = 3)• Yoghurt used (n = 2)• Conference/poster abstract
(n = 2)• Data not released by authors
(n = 1)• Safety data only (n = 1)
Articles from which data were extracted (n = 34, including 34
efficacy results)• One article reported results of
two separate trials• In two cases, two separate
articles reported results of the same trial
• One article included results for two vaginal probiotics
Figure 1. PRISMA flow diagram.
3ª 2019 Royal College of Obstetricians and Gynaecologists
Systematic review of vaginal probiotics
Table 1. Summary of BV/VMB study results
References Probiotic Study design Results
Articles with a medium overall risk of bias
Larsson et al.18 EcoVag (L. gasseri EB01-DSM
14869 + L. rhamnosus Lbp
PB01-DSM 14870)
RCT: placebo control 10 days of EcoVag after clindamycin did not improve BV cure
(by Ison-Hay; 32/50 cured) compared with placebo (37/50
cured) but significantly reduced the cumulative incidence of
BV/intermediate microbiota within 4 menstrual cycles when
used for 10 days after each menses (13/37 versus 21/39
incident cases, respectively)
Petricevic et al.19 Gynophilus (L. casei
rhamnosus Lcr35)
RCT: no-intervention
control
Seven days of Gynophilus after clindamycin resulted in
significantly improved BV cure (by Nugent 0–3) compared
with women receiving clindamycin only (69/83 versus 31/88,
respectively) assessed 5 weeks after cessation of the
intervention period
Mastro-marino
et al.20Florisia (L. brevis
CD2 + L. salivarius subsp.
salicinius
FV2 + L. plantarum FV9)
RCT: placebo control Seven days of Florisia (without any antibiotic) was significantly
more efficacious in curing BV (15/18 Nugent 0–3, 3/18
Nugent 4–6) than placebo (2/16 Nugent 4–6; 14/16
persistent Nugent 7–10) and was associated with significantly
lower BV (Nugent 7–10) cumulative incidence in the 2 weeks
after cessation of Florisia (7/18 and 13/16, respectively)
Bradhaw et al.21 Gynoflor (L. acidophilus
KS400 + 0.03 mg estriol)
RCT: placebo and
clindamycin cream
controls
Twelve days of Gynoflor after oral metronidazole treatment
for BV (Nugent 7–10) resulted in a borderline (P = 0.13)
lower BV recurrence (9/133) at Month 1 compared with
12 days of placebo (13/135) but higher compared with
7 days of vaginal clindamycin (5/140). At Month 6, BV
recurrence was comparable between the three arms (37/133,
36/135, and 42/140, respectively)
van de Wijgert
et al.10Ecologic Femi+ (B. bifidum
W28 + L. acidophilus
W70 + L. helveticus
W74 + L. brevis
W63 + L. plantarum
W21 + L. salivarius W24)
RCT: no-intervention and
oral metronidazole
controls
Intermittent Ecologic Femi+ use after oral metronidazole BV
treatment resulted in significantly lower BV (Nugent 7–10)
recurrence than no-intervention (incidence 3.58/PY versus
10.18/PY, respectively), and significantly reduced BV-
anaerobes concentration by sequencing, during the 2 months
of use, but the effect disappeared by 4 months after
cessation of use
Van de Wijgert
et al.10Gynophilus LP (L. rhamnosus
Lcr35 regenerans)
RCT: no-intervention and
oral metronidazole
controls
Intermittent Gynophilus LP use after oral metronidazole BV
treatment resulted in a non-significant lower BV (Nugent 7–
10) recurrence than controls (incidence 5.36/PY), and non-
significantly reduced BV-anaerobe concentration by
sequencing, during the 2 months of use, but the effect
disappeared by 4 months after cessation of use
Articles with a high overall risk of bias
Eriksson et al.22 L. gasseri + L. casei var
rhamnosus + L. fermentum
(impregnated tampons)
RCT: placebo control Use of lactobacilli-impregnated tampons for 5 or more days
during the first and second menses after clindamycin
treatment was not efficacious in reducing BV recurrence (by
Nugent 4–10) by the end of the two menstrual cycles (41/91)
compared with placebo tampons (34/96)
Anukam et al.23 RC-14/GR-1 (L. fermentum
RC-14 + L. rhamnosus GR-
1)
RCT: metronidazole gel
control
5 days of GR-1 + RC-14 capsules (without any antibiotic) was
significantly more efficacious in curing BV (12/20 Nugent 0–
3) than 5 days of metronidazole gel (6/20) when assessed
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)
Hemalatha et al.24 Florisia (L. brevis
CD2 + L. salivarius subsp.
salicinius + L. plantarum)
RCT: pH-lowering tablet
control
8 days of Florisia (without any antibiotic) was not efficacious
in curing BV (by Nugent; 36/75 cured) compared with pH-
lowering tablet (29/73 cured)
4 ª 2019 Royal College of Obstetricians and Gynaecologists
Wijgert, Verwijs
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 with controls when assessed immediately
to 5 weeks after cessation of probiotic use.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 <50% in both groups).24 The two pre-post
intervention studies enrolled women with a range of base-
line Nugent scores and showed significantly higher propor-
tions of women with Nugent 0–3 immediately after
cessation of probiotic use, which lasted up to 21–28 days
post-treatment.27,29 Of the nine BV 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 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
Table 1. (Continued)
References Probiotic Study design Results
Ling et al.25 L. delbrueckii subsp. lactis
DM8909
RCT: metronidazole
control
7 days of unnamed L. delbrueckii-containing probiotic
(without any antibiotic) was equally effective as vaginal
metronidazole gel in curing BV (assessed 5 days after
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
Bisanz et al.26 RC-14/GR-1 (L. reuteri RC-
14 + L. rhamnosus GR-1)
Randomised controlled
cross-over trial: placebo
control
In women with Nugent 4–6, 3 days of GR-1/RC-14 use
(without any antibiotic) did not result in more women with
Nugent score improvement compared with placebo use (2/
10 and 0/10 improved 1 day after use, and 2/9 and 1/8
improved 8 days after use). However, the relative
abundance of Lactobacillus was significantly increased, and
the relative abundance of Atopobium significantly
decreased, 1 day after use
Verdenelli et al.27 SYNBIO gin (L. rhamnosus
IMC 501 + L. paracasei IMC
502)
Pre-/post-intervention
study
In women with Nugent 0–6, 7 days of SYNBIO gin (without
any antibiotic) resulted in a significantly higher proportion of
women with Nugent 0–3 immmediately after therapy (28/35
versus 21/35 at baseline), and this persisted for 21 days after
cessation of therapy (28/35 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)
Bohbot et al.28 Physioflor (L. crispatus IP
174178)
RCT: placebo control Physioflor for 14 days immediately after metronidazole
therapy, plus 14 days in three subsequent menstrual cycles,
resulted in significantly lower BV cumulative incidence
(Nugent 7–10), and time to BV recurrence, compared with
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)
Rapisarda et al.29 L. acidophilus LA14 Pre-/post-intervention
study
In women with Nugent 4–10, 14 days of L. acidophilus LA14
use (without any antibiotic) resulted in 46/60 women cured
(Nugent 0–3) at the end of therapy, and this persisted for
28 days after product cessation (50/60)
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 stains) and/or molecular VMB composition
endpoints: 14 results in 13 studies.
5ª 2019 Royal College of Obstetricians and Gynaecologists
Systematic review of vaginal probiotics
Table 2. Summary of VVC study results
References Probiotic Study design Results
Articles with a medium overall risk of bias
Czaja et al.30 L. crispatus CTV-05 (not
Lactin-V)
RCT: placebo control In women with a history of recurrent UTI but without
current dysuria, 5 days of L. crispatus CTV-05 use resulted
in a similar VVC cumulative incidence (4/15) compared with
placebo use (2/15) up to 25 days after cessation of use
Articles with a high overall risk of bias€Ozmen et al.31 Gynoflor (L. acidophilus
KS400 + 0.03 mg estriol)
Pre-/post-intervention
study in three
separate, non-
randomised groups
12 days of Gynoflor with or without metronidazole,
compared with metronidazole alone (to treat BV), resulted
in significantly lower cumulative VVC incidence (3/96 and
2/97, versus 14/114, respectively) in the 10–13 days after
cessation of use
Pirotta et al.32 Femilac (L. rhamnosus +
L. delbrueckki +
L. acidophilus +
S. thermophilus)
2 9 2 factorial design
RCT: placebo control
10 days of Femilac during (6 days) and after (4 days) non-
metronidazole antibiotic use, compared with placebo
controls, was not effective in reducing VVC incidence
within 18 days after cessation of Femilac use (17/59 versus
9/54, respectively)
Di Pierro et al.33 Kramegin (L. acidophilus +
Krameria triandra plant
extract + 15 mg lactic acid)
Pre-/post-intervention
study
10 days of Kramegin (without antifungal) cured 75/75
women with acute VVC, and 20/30 women with recurrent
VVC (both significant compared with baseline). Cure was
assessed 7 days after last administration
Witt et al.34 L. gasseri RCT: probiotic +
itraconazole versus
itraconazole only
In women with acute VVC and a history of recurrent VVC,
after induction with itraconazole twice weekly for 1 month,
itraconazole (once per month) with L. gasseri (6
consecutive days per month) for 6 months resulted in a
similar VVC recurrence rate as itraconazole (once per
month) only (15/45 versus 12/31, respectively). Results
were also similar 6 months after product cessation (6/25
versus 5/23, respectively)
Vicariotto et al.35 ActiCand (L. fermentum
LF10 + L. acidophilus LA02)
Pre-/post-intervention
study
In women with acute VVC and a history of recurrent VVC,
ActiCand (without antifungal; 7 consecutive nights,
followed by 3 nights per week for 3 weeks, 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 Seta et al.36 Gyno-Canesflor (L. plantarum
P17630)
Non-randomised
prospective cohort
study
In women with acute VVC but no history of recurrent VVC,
34 days of Gyno-Canesflor after clotrimazole treatment,
compared with placebo use, resulted in a non-significant
(Fisher’s P = 0.095) lower cumulative VVC incidence (1/40
versus 5/40, respectively)
Donders et al.37 &
Donders et al.38Gynoflor (L. acidophilus
KS400 + 0.03 mg estriol)
Pre-/post-intervention
study
An 84-day single-arm Gynoflor treatment scheme (once
daily for 4 weeks followed by thrice weekly for 8 weeks)
for atrophic vaginitis in postmenopausal women with
breast cancer temporarily increased asymptomatic VVC
prevalence after 2 weeks of use (from 2/14 to 7/16) but
returned to baseline values after 1 (3/16), 2 (3/16), and 3
(3/13) months of use
Pendharkar- Trial II
et al.39EcoVag (L. gasseri DSM
14869 + L. rhamnosus DSM
14870)
Non-randomised
prospective cohort
study
In women with recurrent VVC, fluconazole (7 days in cycle
1 followed by once weekly in cycles 2 + 3, and biweekly in
cycles 4–6) with EcoVag (10 days in cycle 2, and once
weekly in cycles 2–6) did not significantly improve the VVC
cure proportion 6 months after treatment cessation
compared with the fluconazole regimen without EcoVag
(8/9 versus 7/10, respectively) but almost all women in
both groups were cured
6 ª 2019 Royal College of Obstetricians and Gynaecologists
Wijgert, Verwijs
in the one study that did not, women used probiotics inbe-
tween and during menses.10 The two medium risk ran-
domised controlled trials that compared vaginal probiotic
use after initial antibiotic treatment with placebo or no-in-
tervention controls as well as prolonged antibiotic use con-
trols both showed that prolonged antibiotic use was more
efficacious in reducing BV recurrence than was probiotic
use.10,21
VVC outcomesOf the 12 studies with VVC as outcome, one was judged
to have medium and 11 high overall risk of bias (Fig-
ure 2). Six of 12 studies were pre-post intervention stud-
ies,29,31,33,35,37,38,41 two were non-randomised cohort
studies with parallel comparison groups (one with a pla-
cebo group36 and one comparing fluconazole plus probi-
otic with 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 itracona-
zole plus probiotic to itraconazole only34) (Table 2). The
one medium risk study was a randomised placebo-con-
trolled 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). The four studies that reported on VVC
cure were pre-post intervention studies in women who
received probiotic treatment in the absence of antifungal
treatment.29,33,35,41 They reported cure fractions ranging
from 57 to 100% after therapy durations ranging from
6 days to 2 months, but VVC recurrence beyond 1 month
after treatment cessation was not assessed. The four studies
that assessed VVC recurrence after combined probiotic/an-
tifungal 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 inci-
dence 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 pro-
biotic 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
Table 2. (Continued)
References Probiotic Study design Results
Kovachev et al.40 Lactagyn (L. acidophilus,
L. rhamnosus,
S. thermophilus,
L. delbrueckii subsp.
bulgaricus)
RCT: no-intervention
control
In women with acute VVC, antifungal treatment
(fluconazole and fenticonazole) followed by 10-day
Lactagyn therapy resulted in lower VVC recurrence (10/
209) at the final follow-up visit 25–30 days after product
cessation compared with 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
Rapisarda et al.29 L. acidophilus LA14 Pre-/post-intervention
study
In women with Nugent 4–10 and 21/60 with acute VVC,
14 days of L. acidophilus LA14 without antifungals or
antibiotics resulted in a significant reduction of VVC cases
to 9/60, 1 day after cessation of use, and 6/60, 4 weeks
after cessation of use. Mean Candida culture counts also
decreased significantly
Murina et al.41 Estromineral Probiogel
(L. fermentum
LF10 + L. plantarum LP02)
Pre-/post-intervention
study
Estromineral Probiogel (without antifungal) cured 51/82
women with acute VVC and 27/27 in women with
recurrent VVC 20–30 days after therapy initiation (both
significant compared with baseline), but therapy longer
than the intended 6 days was required in 57.3% and
63.0% women, respectively, due to persistence of
symptoms
BV, bacterial vaginosis; UTI, urinary tract infection; VMB, vaginal microbiome; VVC, vulvovaginal candidiasis.
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.
7ª 2019 Royal College of Obstetricians and Gynaecologists
Systematic review of vaginal probiotics
A B
Figure 2. Risk of bias assessments. ‘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.
8 ª 2019 Royal College of Obstetricians and Gynaecologists
Wijgert, Verwijs
Vaginal detection outcomesFourteen 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 clearest
in a study by Dausset et al.50 that evaluated two, three, or
five times per week dosing and assessed probiotic strain
concentrations by quantitative PCR every day. 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 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 ranging from
14 days to 6 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 probi-
otic 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 high-
lighted the high interindividual variability in vaginal probi-
otic 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 stud-
ies found that detection was less likely in women who had
autologous 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 assessmentWe judged overall risk of bias to be high for 26 studies,
medium for eight studies, and low for no studies (Fig-
ure 2). The majority of efficacy studies were likely under-
powered 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 com-
pared responders with 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 vagi-
nal 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
improved over time with the increased availability of
molecular technologies (Figure 2). However, microbiologi-
cal inclusion criteria often differed from microbiological
outcome assessments, and it was usually unclear whether
these assessments were blinded. Insufficient consideration
of potential confounding factors, such as hormonal contra-
ception use, pregnancy, menses, vaginal practices, sexual
behaviour, any antimicrobial use that was not part of the
study design, and presence of sexually transmitted infec-
tions, was also common.
Discussion
Main findings and interpretationsThe systematic review results suggest that lactobacilli-con-
taining 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 con-
trolled 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 probi-
otic strains colonised the vagina. However, these findings
are not definitive because heterogeneity was high and the
quality of most studies suboptimal. Furthermore, publica-
tion bias is likely to be high, although we did not formally
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 suffi-
ciently evaluated to allow for a meaningful synthesis of
results. Although all identified probiotics contained lacto-
bacilli, 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,
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 indi-
cations or vaginal detection. Most of the probiotic strains
originated from the gut or from traditional fermented
foods, and have been marketed for decades by non-phar-
maceutical 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 from 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:
9ª 2019 Royal College of Obstetricians and Gynaecologists
Systematic review of vaginal probiotics
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 develop-
ment,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 cur-
rently ongoing.52
The mixed results of trials with VVC indications did not
surprise us given that molecular studies have shown that
Candida species often co-exist with lactobacilli in the VMB,
and epidemiological studies have shown negative associa-
tions between BV and VVC, including increased VVC inci-
dence 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 epidemio-
logical 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 different treatment
strategies with the same probiotic. However, the adjuvant
and maintenance therapy studies were of higher quality,
and more convincing, than the studies that used a vaginal
probiotic as a stand-alone BV treatment. Only two studies
directly compared antibiotic plus probiotic use with pro-
longed antibiotic use, and they both found that the latter
Table 3. Recommendations for vaginal probiotic efficacy evaluation
Topic Recommendations
Study design,
populations,
sample size
Randomised placebo-controlled blinded trials are the preferred study design. Avoid pre-post studies without any parallel control
groups
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 interindividual
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 relation 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
BV, bacterial vaginosis; VMB, vaginal microbiota; VVC, vulvovaginal candidiasis.
10 ª 2019 Royal College of Obstetricians and Gynaecologists
Wijgert, Verwijs
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 the 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 sus-
pect that many of these depended on marketing approvals
dating from before the American and European drug regu-
latory approval restrictions (as was the case in our study),10
and are not necessarily based on clinical evidence. Although
probiotic strains may not have to be present in high con-
centrations 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 sex-
ual behaviour during probiotic use may influence
efficacy,10,39,43,45,48 suggesting that stratified, or even per-
sonalised, VMB modulation may be required in the most
difficult cases of persistent or recurrent BV.
Strengths and limitationsThe quality of most studies was suboptimal. Some of the tra-
ditional epidemiological biases (such as insufficient statistical
power, and lack of randomisation, blinding, appropriate con-
trols, 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 molecu-
lar methods. An important limitation of all studies was that
the time duration between last probiotic insertion and vaginal
sample collection for endpoint assessment and adherence was
not known with sufficient precision, and was not taken into
account in analyses. This is particularly problematic in the
context of BV endpoints using Nugent or Ison-Hay scoring,
because those scores depend heavily on counting bacteria with
a Lactobacillus morphotype (Gram-positive rod) under a
microscope. The scoring cannot differentiate between probi-
otic 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 address this
problem partially. 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 quantifica-
tion and sequencing, which enabled us to quantify not only
probiotic and autologous Lactobacillus strains, but also all
BV-associated anaerobes combined.10 Reduction of the overall
BV-anaerobe concentration over time, in addition to the
increased lactobacilli concentration over time, provided con-
vincing evidence for the beneficial effects of intermittent pro-
biotic use. Our study also showed that host responses to
vaginal probiotic treatment are highly variable, which calls
into question the appropriateness of cross-sectional and pre-
post assessments, as well as the use of group means/medians.
Moving forward, we recommend the following in future
trials: incorporation of quantitative molecular methods that
differentiate between probiotic and autologous Lactobacillus
strains; regular assessment of efficacy and safety endpoints
before, during, and after cessation of probiotic use; stan-
dardisation and optimisation of time duration between
insertion and sample collection; improvement of statistical
power to allow for direct comparisons between responders
and non-responders; taking all known confounders and
adherence into account; and avoiding generic epidemiologi-
cal biases as much as possible (Table 3). Ideally, future tri-
als would also quantify BV-anaerobes, and assess the
impact on biofilm formation, Candida species, and bacte-
rial pathobionts other than BV-anaerobes.
Conclusion
Vaginal probiotics hold promise for BV cure and preven-
tion. We expect that the availability of vaginal probiotics
for these indications will decline in 2020 because of regula-
tory changes. We therefore urge the field to invest in clini-
cal evidence-based product development, and to conduct
future clinical trials more rigorously.
Disclosure of interestsThe authors conducted a clinical trial with Ecologic Femi+(Winclove Probiotics, Amsterdam, Netherlands) and Gyno-
philus 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 interests forms are available to view online as
supporting information.
Contribution to authorshipJvdW conceived and planned the systematic review, and
JvdW and MCV performed the review and analyses, and
wrote the manuscript together.
Details of ethics approvalNot applicable.
FundingNone.
AcknowledgementsThe authors thank medical student Connie Rees for assis-
tance with the search and data extraction.
11ª 2019 Royal College of Obstetricians and Gynaecologists
Systematic review of vaginal probiotics
Supporting Information
Additional supporting information may be found online in
the Supporting Information section at the end of the arti-
cle.
Table S1. Summary of vaginal probiotic strain detection
results.
Table S2. Product characteristics.
Appendix S1. Search terms and filters.&
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