Randomized, double-blind, placebo

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European Journal of Obstetrics & Gynecology and Reproductive Biology 141 (2008) 54–57
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European Journal of Obstetrics & Gynecology and
Reproductive Biology
journal homepage: www.elsevier.com/locate/ejogrb
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Randomized, double-blind, placebo-controlled study of oral lactobacilli to
improve the vaginal flora of postmenopausal women
Ljubomir Petricevic a, Frank M. Unger b, Helmut Viernstein b, Herbert Kiss a,*
a
b
Department of Obstetrics and Gynaecology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria
Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 14 January 2008
Received in revised form 29 April 2008
Accepted 26 June 2008
Objective: The purpose of this randomized, double-blind, placebo-controlled study was to evaluate the
influence of the orally administered probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus
reuteri RC-14 on the quality of the vaginal flora in postmenopausal women.
Study design: Postmenopausal women with Nugent scores between 4 and 6 in initial vaginal swab, were
randomized into two groups. Women in the intervention group received probiotic capsules containing
2.5 109 CFU (colony forming units) each of lyophilized L. rhamnosus GR-1 and L. reuteri RC-14 and
women in the control group received an oral placebo once daily, in both groups for 14 days. Final vaginal
swabs were taken 1 day after the last administration of the medication. The primary efficacy variable was
a change in the Nugent score between baseline and the end of the study of at least two grades in each
individual patient.
Results: Seventy two women were recruited in the study, 35 assigned to the intervention group and 37 to
the control group. Twenty-one of the 35 subjects (60%) in the intervention group and 6 of the 37 subjects
(16%) in the control group showed a reduction in the Nugent score by at least two grades. The difference in
the number of patients with improvement was highly significant (p = 0.0001). The median difference in
Nugent scores between baseline and the end of the study was 3 in the intervention group and 0 in the
control group (p = 0.0001).
Conclusion: Our results provide evidence for an alternative modality to restore the normal vaginal flora
using specific probiotic strains administered orally.
ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Lactobacilli
Oral administration
Vaginal flora
1. Introduction
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The healthy vaginal flora is dominated by lactobacilli, which
plays an important role in protecting against genital infections. In
women of child-bearing age, a reduction or absence of lactobacilli
can upset the microbial balance of the vaginal flora, lead to an
overgrowth of Gram-negative bacteria, and ultimately result in
bacterial vaginosis [1]. Standardized diagnosis of bacterial
vaginosis is based either on the clinical criteria described by
Amsel et al. [2] or on the more objective criteria reported by
Nugent et al. [3].
In premenopausal women, estrogen encourages the vaginal
colonization with lactobacilli, which metabolize glycogen to
produce lactic acid and maintain a low vaginal pH that inhibits
the growth of many pathogens. The genitourinary atrophy
* Corresponding author. Tel.: +43 1 40400 2822; fax: +43 1 40400 2861.
E-mail address: [email protected] (H. Kiss).
0301-2115/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ejogrb.2008.06.003
following menopause is associated with a decline in estrogen
secretion, leading to the depletion of lactobacilli, a rise in pH, and
increased vaginal colonization with enterobacteria. Clinically, this
manifests as an increased prevalence of recurrent urinary tract
infections [4,5].
Data on the role of lactobacilli in the treatment or prevention of
urogenital infection are scarce. In 2005, Ozkinay et al. reported that
restoration of the vaginal flora can be significantly enhanced by the
administration of live lactobacilli in combination with low-dose
oestriol [6]. Uehara et al. found that local application of lactobacilli
seems a promising treatment for the prevention of recurrent
urinary tract infections [7].
There is increasing evidence to suggest a strong association
between the gastrointestinal and vaginal ecosystems. Thus,
Antonio et al. found that lactobacilli colonizing the rectum may
be a reservoir for vaginal lactobacilli, contributing to the
maintenance of the vaginal microflora and decreasing the risk of
bacterial vaginosis [8]. Morelli et al. showed that lactobacilli can
also be delivered to the vaginal environment by oral ingestion [9].
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L. Petricevic et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 141 (2008) 54–57
Fig. 1. The consort diagram randomized, double-blind, placebo-controlled study of oral lactobacilli to improve the vaginal flora of postmenopausal women.
In a randomized, placebo-controlled study of healthy women given
daily oral capsules of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14, Reid et al. found a significant increase in
vaginal lactobacilli for treated subjects versus controls [10]. These
results suggest that selected intestinal lactobacilli also colonize the
vagina, suggesting that the intestinal tract can be used as a delivery
system for specific lactobacilli. We undertook this prospective,
randomized, double-blind, placebo-controlled study to determine
whether orally administered L. rhamnosus GR-1 and Lactobacillus
reuteri RC-14 exert a measurable effect on the vaginal flora.
2. Materials and methods
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This study was performed with the approval of the Ethics
Committee of the Medical University of Vienna. Between March
2005 and May 2006, we enrolled consecutive postmenopausal
women aged 55–65 years without vaginal bleeding or abnormal
vaginal discharge from among outpatients of the Department of
Obstetrics and Gynaecology. To determine the effect of lactobacilli
in the absence of estrogen, patients receiving hormone replacement therapy (HRT) were not included in the study.
From each potential participant, an initial vaginal smear was
taken and transferred to a microscopy slide. Smears were Gramstained and evaluated using the Nugent scoring system [3] at an
experienced central laboratory. The Nugent scoring system is a
objective, semi-quantitative evaluation of vaginal secretion. This
score (0–10) was described as a weighted combination of the
following morphotypes: lactobacilli, Gardnerella vaginalis or
bacteroides (small Gram-variable rods or Gram-negative rods),
and curved Gram-variable rods. The criteria for bacterial vaginosis
is a score of 7 or higher; a score of 4–6 is considered intermediate
and a score of 0–3 is considered normal. To detect even minor
treatment effects, scoring was performed using the entire 10-grade
Nugent scale rather than a simplified system grouping several
grades together. A flow diagram according to consort depicting
information about the number of participants at the different stage
of the trial is shown in Fig. 1.
Following evaluation of the initial swabs, only women with
Nugent scores between 4 and 6 were invited to participate in the
study after giving written informed consent. Between 1 and 2 days
after the initial swab, patients were randomized to one of two
study groups using a computer-generated randomization list.
Women in the intervention group received probiotic capsules
containing 2.5 109 CFU (colony forming units) each of lyophilized L. rhamnosus GR-1 and L. reuteri RC-14 as well as sodium
alginate to protect the bacteria against stomach acid once daily.
Women in the control group were given an oral lactose placebo
once daily.
The study duration was 14 days. Compliance was assessed by
interview and by having patients return the capsule packs. Final
vaginal swabs for Nugent scoring were taken on the day following
the last administration of the study medication. Participants,
primary investigator as well as Gram-stain readers were blinded to
the treatment assignment. The primary efficacy variable was a
change in the Nugent score between baseline and the end of the
study of at least two grades in each individual patient. As
secondary efficacy variable, we assessed whether or not treatment
improved the Nugent score, regardless of the extent of change.
2.1. Statistics
The proportion of subjects with score changes between
baseline and the end of the study as defined by the primary
and secondary endpoints was compared between treatment
groups using a chi-square test. A p-value < 0.05 (two-sided) was
considered statistically significant. Mean Nugent scores were
compared descriptively using the Mann–Whitney U-test. Assuming a score change of at least two grades in more than 50% of
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L. Petricevic et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 141 (2008) 54–57
patients in the intervention group and not more than 20% in the
control group, a sample size of 30 subjects per group was
considered sufficient to show a statistically significant superiority
with a power of at least 80%. SPSS version 11.0 was used for
statistical analyses.
3. Results
Nugent
score
Intervention group (N = 35)
Control group (N = 37)
Initial
swab n (%)
Final
swab n (%)
Initial
swab n (%)
Final
swab n (%)
1
2
3
4
5
6
7
8
–
–
–
31
2
1
–
1
18
2
2
10
1
1
0
1
–
–
–
33
0
3
–
1
3
1
2
26
0
1
1
3
(88.6)
(5.7)
(2.9)
(2.9)
(51.4)
(5.7)
(5.7)
(28.6)
(2.9)
(2.9)
(0.0)
(2.9)
(89.2)
(0.0)
(8.1)
(2.7)
(8.1)
(2.7)
(5.4)
(70.3)
(0.0)
(2.7)
(2.7)
(8.1)
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Between March 2005 and May 2006, a total of 200 postmenopausal women of Caucasian origin were screened for
enrolment in the study. Of these, 72 women were included in
the study. Overall, two women with Nugent score 8 were
erroneously included, one in the intervention group and one in
the control group. This protocol violation did not have an influence
on either the statistical analysis or the result. The mean age of the
study participants was 57.6 (S.D. 7.8) years, mean weight was
70.9 kg (S.D. 12.0), and mean height was 164.2 cm (S.D. 6.6), with
no significant differences between study groups. Likewise, there
were no between-group differences in terms of smoking habits,
alcohol consumption, or the number of previous pregnancies or
deliveries. All patients were postmenopausal and were not taking
any hormone replacement therapy.
Outcome data were available from 35 women in the intervention group and 37 women in the control group. The distributions of
initial and final Nugent scores in the intervention and control
groups are shown in Table 1. Mean Nugent scores of initial and final
swabs were 4.23 (S.D. 0.77) and 2.49 (S.D. 1.82) in the intervention
group and 4.27 (S.D. 0.84) and 4.11 (S.D. 1.61) in the control group.
The median difference in Nugent scores between initial and final
swabs was 3 in the intervention group and 0 in the control group.
This difference between groups was highly significant (p = 0.0001).
Table 2 shows the shifts in Nugent scores between initial and
final swabs in individual patients. Thus, Nugent scores remained
unchanged in 9 women in the intervention group and in 25 women
in the control group. Twenty-one of the 35 women (60%) in the
intervention group and 6 of the 37 women (16%) in the control
group showed a reduction of the Nugent score by at least two
grades. The between-group difference in the number of patients
with improvement was highly significant (p = 0.0001).
At the end of the study, Nugent scores had decreased by even
three grades in 54.3% (n = 19) of patients in the intervention group
and in 10.8% (n = 4) of patients in the control group. Again, the
between-group difference was highly significant (p = 0.0005). One
woman in the intervention group and four women in the control
group had bacterial vaginosis as demonstrated by Nugent scores of
either 7 or 8. Whereas the vaginal flora of the two women
erroneously included into the study had remained unchanged,
Table 1
Distribution of Nugent scores of initial and final swabs in the intervention and
control groups
three patients in the control group, who had an intermediate
vaginal flora at inclusion, had progressed to bacterial vaginosis by
the end of the study.
4. Discussion
The results of this randomized, double-blind, placebo-controlled study suggest that oral application of lactobacilli once daily
for 2 weeks results in a substantial improvement in the vaginal
flora of postmenopausal women with Nugent scores between 4
and 6 as demonstrated by a score reduction by at least two grades.
Lactobacilli constitute most of the healthy vaginal flora [1]. A
loss of lactobacilli frequently leads to bacterial vaginosis or
recurrent genitourinary infection. Local substitution has been
shown to significantly enhance the restoration of the vaginal flora,
leading to a significant reduction in the recurrence of urinary tract
infections [6,7].
In 2003, Reid et al. used species-specific PCR-amplification to
demonstrate that L. rhamnosus and L. fermentum can be delivered
to the vagina when administered orally and found a significant
increase in vaginal lactobacilli in treated subjects versus controls
[10]. One year later, Morelli et al. also showed, using genetic
identification, that the lactobacilli L. rhamnosus and L. fermentum
can be delivered to the vaginal environment even when taken
orally [9]. Both studies were performed in healthy premenopausal
women. Also, a recent study by Antoni et al. found that rectal and
vaginal co-colonization with lactobacilli was associated with the
lowest prevalence of bacterial vaginosis [8]. The authors concluded
that Lactobacillus sp. in the rectum may contribute to the
maintenance of the vaginal microflora. Thus, certain strains of
lactobacilli appear to inhabit the human gut as well as the
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Table 2
Difference in Nugent scores between initial and final swabs in individual patients in the intervention and control groups
L. Petricevic et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 141 (2008) 54–57
Oral probiotic supplements, which so far have been used only to
restore a healthy gastrointestinal flora, are now beginning to gain
ground in gynaecology. The results of our study suggest that
nutritional supplementation by orally administered capsules
containing 2.5 109 CFU each of L. rhamnosus GR-1 and L. reuteri
RC-14 may be a useful aid in raising floral quality and improving
urogenital health. Recent publications have shown that the quality
of the vaginal flora can be significantly enhanced by local
administration of lactobacilli [6,7]. The results of our study
suggest an alternative modality to restore the normal vaginal
flora using specific probiotic strains of lactobacilli administered
orally, warranting confirmation in a large-scale follow-up study
including PCR specific analyses.
Acknowledgments
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urogenital tract and may be able to pass from the gut to the vagina,
the most likely route being through the perineum.
In premenopausal women, estrogen encourages the vaginal
colonization with lactobacilli. To test whether oral lactobacilli
exert a measurable effect on the vaginal flora even in the absence of
estrogen, we performed a study in postmenopausal women not
taking any hormone replacement therapy to eliminate the
influence of estrogen. L. rhamnosus GR-1 and L. reuteri RC-14 were
selected because they had previously been shown to be capable of
colonizing the vagina [9–11] and because they are available in a
consistent capsule formation.
In our study, oral probiotics lead to the significant improvements in the postmenopausal vaginal flora as demonstrated on the
basis of Nugent scores. The ‘normal’ vaginal flora described by
Spiegel [1] corresponds to a Nugent et al. [3] score between 0 and 3,
whereas what Spiegel describes as an ‘intermediate’ vaginal flora
corresponds to a Nugent score between 4 and 6. In our study, oral
administration of lactobacilli in the absence of estrogen resulted in
a shift from an intermediate to a normal vaginal flora in 60% of
women in the intervention group, compared with only 16% of
patients in the control group, a difference that was highly
significant.
An interesting finding of our study is that three patients in the
control group had progressed to bacterial vaginosis by the end of
the study, compared to no patient in the intervention group. Even
though this difference was not significant, it certainly is of clinical
relevance. Another aspect of clinical interest is that the vaginal
flora of one patient in the intervention who had had a Nugent score
of 8 at inclusion failed to improve despite oral lactobacilli,
suggesting that oral substitution with lactobacilli, even though
protective against bacterial vaginosis, may lack therapeutic
efficacy.
One limitation of our study is the small sample size. While the
size of the study population was tested and found acceptable in an
appropriate power analysis, it is obviously desirable to corroborate
and substantiate the present results in a multicenter setting.
However, the subsequently calculated power of 98% supports this
proof of concept design. Another limitation is that assessment of
the vaginal microflora was based on Gram-stain only. However, the
Nugent scoring system is generally considered an adequate and
objective method for the evaluation of the vaginal flora. Also, the
oral lactobacilli we used have previously been shown to be
identifiable in the vagina using PCR [9,10].
It has long been known that oral application of lactobacilli can
exert beneficial effects on the gut flora [12] and contribute to the
treatment of intestinal complaints, such as enteritis and various
disorders causing diarrhoea [13,14]. Conceivably, the beneficial
effects of lactobacilli may not be limited to the intestine, but may
also extend to the vaginal area. It is unclear to what extent an
imbalance of the gut flora can be detrimental to the survival of
lactobacilli. However, the stimulating influence of lactobacilli on
the synthesis of IgA antibodies in the context of the gut-associated
lymphoid system is well established [15,16]. IgA antibodies may
travel to the vaginal epithelia and exert both anti-infective and
anti-inflammatory effects [15,16]. Whether through this or other
mechanisms, it may be speculated that a healthy gut flora
contributes to the protection against female genitourinary tract
infections and that an imbalance of the intestinal flora favours
suppression of lactobacilli with consecutive overgrowth of anaerobes in the vagina.
57
The authors thank Dr. Günther Nirnberger, Bioconsult GmbH,
Perchtoldsdorf, Austria for the statistical design and data evaluation in connection with the present study. Chr. Hansen A/S,
Hørsholm, Denmark, are gratefully acknowledged for providing
the probiotic and placebo capsules.
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