Download Influence of Menstruation on the Microbiota of Healthy Women`s

Jpn. J. Infect. Dis., 64, 76-80, 2011
Short Communication
Influence of Menstruation on the Microbiota of
Healthy Women's Labia Minora as Analyzed Using
a 16S rRNA Gene-Based Clone Library Method
Tsukasa Shiraishi*, Kazumasa Fukuda1, Nobuo Morotomi1, Yuri Imamura,
Junko Mishima, Shigeo Imai, Kiyoshi Miyazawa, and Hatsumi Taniguchi1
Life Science Laboratory, Unicharm Corporation, Kagawa 769-1602; and
of Microbiology, School of Medicine, University of Occupational and
Environmental Health Japan, Fukuoka 807-8555, Japan
1Department
(Received October 15, 2010. Accepted December 17, 2010)
SUMMARY: The aim of this study was to determine the influence of menstruation on the bacterial
population of healthy Japanese women's vulvas, especially the labia minora. Labia minora swabs were
obtained from 10 premenopausal, nonpregnant Japanese women at premenstruation and on day 2 of
menstruation. Vaginal swabs were also obtained from 3 out of the 10 women. No significant difference
was found in the average bacterial cell count between the menstruation and premenstruation samples.
Molecular analysis using a 16S rRNA gene-based clone library method detected 22 genera from the labia
minora swabs (total 20), with the genus Lactobacillus being predominant at both premenstruation and
during menstruation in 7 out of the 10 women. Of the other 3 women, 2 showed various kinds of bacterial species, including oral and fecal bacteria, with Atopobium vaginae and Gardnerella vaginalis
predominating in the remaining woman's vulva in both conditions. In total, 6 out of 10 cases (60z)
showed significantly different microbiota of the labia minora between the two conditions. These results
imply that menstruation may promote a distortion of the bacterial flora around the vulva, although it
causes no significant increase of the bacterial count.
due to their location (entrance of the vagina). The labia
majora also exhibit a much higher microbial typediversity (6), thus impying that the microbial flora of
the human vulva may be a significant affecting force
due to the fact that the vulva is exposed to both internal
(vaginal fluid and menstruation) and external (urine and
fecal material) influences. Furthermore, the microbial
organisms present on the vulva may trigger various vaginal diseases, such as vulvovaginitis (7), bartholinitis (8),
and urinary tract infection (9).
Recently, in an attempt to understand the comprehensive microbial community of the vulva and the vagina,
molecular methods using the 16S rRNA gene sequence
were used to analyze the whole bacterial population
structure (6,10–14). These studies found that a wider
range of populations inhabited the labia majora (including bacterial species known to be commensals of the
skin) than the labia minora (6). Since the labia minora
are located close to the vagina, menstruation may have a
significant effect on their bacterial community. A complete understanding of the composition and ecology of
the microbial community of the labia minora, which
may play a role as the ``gatekeeper'' of the vagina, may
therefore be important for understanding the etiology
of infectious diseases of the vagina and the vulva.
There are two central themes to this study, both of
which are focused on the labia minora. The first of these
involves a characterization of the structure of the
microbial communities found on the labia minora of 10
healthy Japanese women using a 16S rRNA gene-based
clone library method, whereas the second involves the
clarification of the possible effects of menstruation on
Microbial organisms that have become adapted to the
peculiar environment provided by various anatomical
locations on the human body are known to affect the
biological and/or physical condition of the host (1). Indeed, the vagina is an exceptional organ in this respect
because of its constant acidic conditions (2). A variety
of bacteria are found in the vaginas of healthy women,
with those from the genus Lactobacillus tending to
predominate. In fact, the lactobacilli found in the
normal microbial flora of the vagina are known to play
an important role in preventing infections. These
microorganisms produce large amounts of lactic acid
during growth in the vagina, thereby maintaining its
acidity. Furthermore, these bacteria inhibit the growth
of many pathogenic organisms by producing hydrogen
peroxide (H2O2) (3). Several attempts to use viable
lactobacilli as a probiotic treatment have been reported
to be sucessful (4), thus suggesting that Lactobacillus
introduced into the vagina remained there for a
sufficient time period posttreatment (5). These findings
suggest that Lactobacillus may well be the most typical
and important bacterial group found in the human
vagina.
The microbial types of the labia majora are significantly different from those found inside the vagina
*Corresponding author: Mailing address: Laboratory of
Microbial Physiology, Research Faculty of Agriculture,
Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo
060-8589, Japan. Tel: ＋81-11-706-4115, Fax: ＋81-11706-2501, E-mail: shiraishi1020＠gmail.com
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the microbial communities present on the labia minora.
This study was performed in accordance with
Japanese ethical guidelines regarding epidemiologic
studies.
Labia minora swabs were obtained from 10
premenopausal, nonpregnant Japanese women between
the ages of 31 and 43 years. Written informed consent
was obtained from all participants in this study. The
conditions set for the sample donors were as follows:
(i) regular menstrual cycle, (ii) regular sexual intercourse
during the examination period, (iii) no symptoms or
signs of vaginal and/or urinary tract infection (as determined by a physician), (iv) no antimicrobial agent use
for 6 weeks prior to sampling, (v) no body piercing of
the vulva, thigh, or buttock area.
Subjects were excluded if they self-reported: (vi) pregnancy, (vii) immunocompromised status, (viii) potential
carrier of a sexually transmitted disease, (ix) AIDS,
and/or (x) hepatitis. In addition, the women were
checked for infectious diseases (including Nugent score
examination) at each sampling visit by a physician from
the Kagawa Inoshita Hospital.
To maintain similar conditions in the vaginal areas of
the subjects, the subjects were asked to use the same
soap, wear the same underwear, and use the same menstrual tools.
Samples were collected at Kagawa Inoshita Hospital.
Scrape samples were collected from the labia minora
using a sterile cotton swab 2 days after menstruation
began. The premenstrual samples were collected 1 week
prior to expected menstruation. Samples of vaginal
fluid were taken from the mid-vagina using sterile cotton swabs. All samples were placed in a transport vial
C.
and stored at 49
Bacterial cell counting, total DNA extraction, and
16S rRNA gene clone library analysis were performed as
described previously (15,16). Briefly, the swabs were
vigorously shaken in 2.0 ml of Tris-HCl buffer (100 mM
Tris-HCl, 50 mM EDTA-2Na, [pH 8.0]) to dislodge the
cells. After treatment with ethidium bromide (EtBr) solution (100 mg/ml), bacteria-shaped objects in the swab
suspension were counted under an Olympus BX50
microscope (Olympus Optical, Tokyo, Japan) and the
number of bacteria per milliliter of solution calculated.
The results of bacterial cell counting (epifluorescence
staining method) are shown in Fig. 1. Around 105–107
cells/ml were detected in each bacterial cell suspension
prepared from the labia minora premenstruation (Fig.
1A). However, although the average number of bacterial flora cells was 1.2 × 107 cells/ml both premenstruation and during menstruation (the P-value calculated
with a paired Student's t test was 0.97), the cell numbers
at menstruation showed a significant diversity (standard
deviation of 0.7 × 107 for premenstruation and 2.2 ×
107 during menstruation) (Fig. 1B). Before starting this
study, we hypothesized that there would be an increase
in both bacterial numbers and their diversity due to the
extra nutrients provided by the menstrual blood and the
high humidity of the location. However, somewhat surprisingly, a significant increase in the bacterial cell
count was not observed during menstruation. These
results therefore suggest that menstruation might be one
of the factors that alter the bacterial cell density of the
labia minora in healthy women.
Fig. 1. Bacterial cell counts in the sample solutions prepared
from labia minora swabs at premenstruation and during
menstruation of 10 healthy Japanese women. The bacterial cell
counts of each sample were shown in A and the variation of cell
counts was represented in B. White bars and black bars in A indicate the bacterial cell numbers at premenstruation and during
menstruation, respectively. The horizontal bars in B indicate
the average numbers of bacterial cells. The P value obtained
using paired Student's t test is shown.
The total DNA of the swab solutions was extracted
using sodium dodecyl sulfate (SDS, final concentration
3.0z), approximately 0.3 g of glass beads, and a Micro
Smash MS-100 apparatus (Tomy Seiko, Tokyo, Japan).
After treatment of the DNA with phenol-chloroformisoamyl alcohol (25:24:1, vol/vol), the solution was
concentrated and replaced by about 30 ml of TE buffer
using a Montage PCR Centrifugal Filter Device (Millipore, Bedford, Mass., USA). For clone library construction using the extracted DNA as template, the partial 16S rRNA gene fragments (approximately 580 bp)
were amplified by PCR using AmpliTaq Gold DNA
polymerase (Applied Biosystems, Foster City, Calif.,
USA) and a pair of universal primers (341F, 5?and
907R,
5?CCTACGGGAGGCAGCAG-3?
CCGTCAATTCMTTTRAGTTT-3?). PCR inhibition
as a result of blood composition was not observed in
this study. The resulting PCR products were cloned using a TOPO TA Cloning kit (Invitrogen, Carlsbad,
Calif., USA) according to the manufacturer's instructions.
The nucleotide sequences were determined for 96 randomly chosen clones for each sample. Highly accurate
sequences were compared to an in-house database containing only 16S rRNA gene sequences of type strains
(5,878 species) obtained from the Ribosome Database
Project II (http://rdp.cme.msu.edu/) and the DNA
Data Bank of Japan (http://www.ddbj.nig.ac.jp/) using the BLAST algorithm. Those clones with 97z or
higher sequence similarity to the reference type strain
were presumed to be members of the same species,
whereas those whose sequences were º97z similar
were defined as unclassified bacteria.
A total of 2,347 clone sequences were determined,
compared to the reference sequences of type strains, and
the most similar type strain selected for each clone
(Table 1). A total of 2,113 of 2,347 sequences determined (approximately 90z) showed 97z or higher
sequence similarity to the reference type strains.
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Table 1. Bacterial composition of the clone libraries of labia minora and vaginal fluid samples from 10 healthy Japanese women
Labia minora swab1) (z clones)
Reference type strain
(accsession no.)
No. 1
B
A
No. 2
B
A
No. 3
B
A
No. 4
B
A
No. 5
B
A
No. 6
B
A
Vaginal fluid swab2) (z clones)
No. 7
B
A
No. 8
B
A
No. 9
B
A
No. 10
B
A
No. 5
BF
AF
No. 7
BF
AF
No. 10
BF
AF
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Actinomyces gerencseriae (X80414)
1.1
Corynebacterium accolens (X80500)
1.1
C. afermentans (X82054)
1.1
C. kroppenstedtii (Y10077)
1.2
C. matruchotii (X82065)
3.3
Bifidobacterium breve (M58731)
84.4
B. pseudocatenulatum (D86187)
6.7
B. scardovii (AJ307005)
3.3
Gardnerella vaginalis (M58744)
28.4 61.6
2.3
1.1
19.3 19.3
Atopobium vaginae (Y17195)
58.0 27.9
70.5 7.2
Bacteroides coagulans (DQ497990)
1.1
Porphyromonas somerae (AY968205)
1.1
Prevotella bivia (L16475)
2.2 1.0
1.1
P. buccalis (L16476)
1.1
Gemella haemolysans (L14326)
1.1
Staphylococcus epidermidis (D83363)
2.2
2.4
Facklamia hominis (Y10772)
1.1
Lactobacillus crispatus (Y17362)
26.0 92.6 100 100
88.8 10.5 97.9 93.7
94.5 98.9
L. fermentum (M58819)
3.3
L. fornicalis (Y18654)
9.4 2.1
1.1
L. gasseri (M58820)
13.2
5.7
6.8
L. iners (Y16329)
64.6 2.1
10.1 81.4
98.8 80.7 98.9 98.8 94.6 97.7
96.7 95.8
L. kitasatonis (AB107638)
2.1
Streptococcus difficilis (AB112407)
7.2
S. intermedius (AF104671)
3.3
2.4
2.4
S. parasanguinis (AF003933)
30.4
S. pseudopneumoniae (AY485599)
4.3
Dialister micraerophilus (AF473837)
1.2
D. propionicifaciens (AY850119)
6.6 1.0
2.2
Selenomonas noxia (AF287799)
1.1
Anaerococcus prevotii (D14139)
15.4 58.3
1.2
32.5
Micromonas micos (AF542231)
1.1
Peptoniphilus harei (Y07839)
2.2 4.2
6.5
1.2
3.6
P. ivorii (Y07840)
4.4
1.1
P. lacrimalis (D14141)
1.1
2.2
Peptostreptococcus stomatis
(DQ160208)
1.2
Ureaplasma parvum (AF073456)
1.1
1.2
Fusobacterium canifelinum (AY162220)
1.1
1.2
Leptotrichia hofstadii (AY029803)
1.1
Vibrio gigantis (AJ582810)
1.1
Unclassified
44.0 35.4 7.8 35.9
8.0 10.5
5.8 2.1 4.2 1.2 3.6 1.1
5.4 2.3 3.4 31.3 5.5 1.1 3.3 4.2
Total clones
1):
2):
91
96
90
92
96
94
91
94 88
B, labia minora swab at pre-menstruation; A, labia minora swab during menstruation.
BF, vaginal fluid swab at pre-menstruation; AF, vaginal fluid swab during menstruation.
86
89
86
95
95
85
83
92
85
93
88
88
83
91
90
91
95
The genus Lactobacillus was found to predominate in
7 out of 10 women at both premenstruation and during
menstruation. This genus is known to be the predominant microbe in the vagina under normal conditions. Indeed, it is commonly used in the field of obstetrics and gynecology as one of the indicators for the
diagnosis of vaginal infectious diseases. In a previous
study, the microbial flora from the vulva of 4 healthy
Caucasian women was analyzed using a cultivation-independent method (6). This study found that the most
abundant phylotypes in the labia minora of 3 out of
these 4 women were most similar to either Lactobacillus
crispatus or L. iners (6). Likewise, the results of our
study also suggest that L. crispatus or L. iners are the
predominant bacteria on the labia minora of healthy
Japanese women at premenstruation. Clones similar to
L. crispatus were predominant (97.9–100.0z) in sample
No. 4 and No. 7 at both premenstruation and during
menstruation, whereas L. iners clones were found to be
predominant (94.6–98.9z) under both conditions in
No. 8, No. 9, and No. 10. Clones of the same two
Lactobacillus spp. (L. iners and L. crispatus) were detected in No. 3 and No. 6, both of whom underwent
predominant species inversion between the premenstruation and menstruation periods. The compositions of
clones similar to L. iners and L. crispatus were 64.6 and
26.0z, respectively, in No. 3B (premenstruation),
whereas the composition of L. iners clones decreased to
2.1z, and that of L. crispatus clones increased to
92.6z in No. 3A (during menstruation). The detection
ratios of these species for No. 6 were 10.1z (L. iners)
and 88.8z (L. crispatus) at premenstruation (No. 6B)
and 81.4z (L. iners) and 10.5z (L. crispatus) during
menstruation (No. 6A). These findings suggest that
although Lactobacillus spp. remain predominant,
menstruation may nevertheless cause an alteration of
the bacterial flora. It should be noted that significant
differences were observed between premenstruation and
menstruation in 6 (No. 1, 2, 3, 5, 6, and 8) of the 10
women subjects. The characteristics of the bacterial
flora from the labia minora of healthy Japanese women
were used to create two groups: non-predominance of
Lactobacillus spp. (Group A, No. 1, 2, and 5), and
predominance of Lactobacillus spp. (Group B, No. 3, 4,
6, 7, 8, 9, and 10). We were unable to decide whether the
former type was normal or not. The latter type was subdivided into three groups on the basis of stable
predominance of L. crispatus (No. 4 and 7), and L.
iners (No. 8, 9, and 10), and the reversion of the
predominant species during menstruation (No. 3 and 6).
Interestingly, L. fornicalis was also detected along with
L. crispatus and L. iners for No. 3 and 6, thus suggesting that L. fornicalis may also cause alterations of the
microbial flora.
A variety of bacterial compositions were detected in
the women from Group A (No. 1, 2, and 5). Unclassified bacteria were predominant (44z) in No. 1B (No. 1
premenstruation), although a further 11 species of bacteria were also detected. The unclassified 40 clones included 23 which were similar to Prevotella salivae (90z
similarity). Likewise, clones similar to Anaerococcus
prevotii and Lactobacillus gasseri were detected at 15.4
and 13.2z, respectively, of the 11 species of bacteria.
The number of species detected during menstruation
(No. 1A) decreased to 4, with clones similar to A.
prevotii being predominant (58.3z). Bifidobacterium
breve was predominant in No. 2B (84.4z), whereas 19
species of bacteria were detected in No. 2A. Atopobium
vaginae (58.0z) and Gardnerella vaginalis (28.4z)
were predominant in both samples from No. 5 (No. 5B,
premenstruation; No. 5A, menstruation), although the
compositional ratio of these clones was the inverse in
each case. These 3 women had been diagnosed as having
normal vaginal conditions using the Nugent score
method 1 week prior to this study. Despite this diagnosis, strains from the genus Prevotella, Anaerococcus,
Atopobium, and Gardnerella, all of which are known
bacterial vaginosis agents (17–19), were detected in No.
1 and 5. The reason for this discrepancy remains unclear, although it may depend on the interpretation of
the Nugent criteria and the stability of the vulvar bacterial flora.
A previous study involving the large-scale study of
Caucasian women's vaginal swabs suggested that L.
crispatus promotes the stability of the normal vaginal
microbial flora whereas L. gasseri and/or L. iners promote the occurrence of abnormal vaginal microflora to
some extent (20). In our study, clones similar to L. gasseri were detected in No. 1 and 5, although they appeared as a minor population. These results led us to
speculate that the 2 subjects may have been in a L. gasseri predominant state when the Nugent examination
was conducted, and that L. gasseri may cause destabilization of the microbial flora in the vagina and labia
minora.
The results of a vaginal fluid analysis from 3 subjects
(No. 5, 7, and 10) suggested that the bacterial flora
present on the labia minora at premenstruation were
similar to those in the vaginal fluid. Indeed, the bacterial flora in the vaginal fluid from No. 7 and 10 was very
similar to that present on the labia minora at both premenstruation and during menstruation. No. 5 also
presented a similar bacterial composition in both samples (No. 5B and 5BF), whereas No. 5AF displayed a
unique bacterial diversity involving an increase of A.
prevotii. These findings suggest that a vaginal community composed of bacteria other than lactobacilli
might become unstable during menstruation.
A previous study found that the vaginal flora of black
and Caucasian women are different, with nonpredominant lactobacilli accounting for a higher
proportion in black (33z) than in Caucasian women
(7z) (10). The data obtained in our study suggest that
the bacterial flora present on the labia minora of
healthy Japanese women mainly consisted of lactobacilli (in 7 of 10 cases) and that it was strongly affected by
vaginal fluid. The vaginal bacterial community of
healthy Japanese women may therefore be more similar
to that of black women than Caucasian women,
although a large-scale investigation will be required to
determine whether this is actually the case.
In summary, we have examined the influence of
menstruation on the bacterial flora present on the labia
minora of 10 healthy Japanese women. The bacterial
cell counts and bacterial flora were evaluated premenstruation and during menstruation. To the best of
our knowledge, an effect of menstruation on the bacterial flora of the labia minora and vaginal fluid has not
79
been reported previously. This study suggests that the L.
crispatus and L. iners populations remain relatively stable, even during menstruation, whereas other Lactobacillus spp. and bacteria from genera other than Lactobacillus may suffer alterations, such as a multiplication
of specific bacterial species, an increase of bacterial
variety, and an inversion of the predominant species.
Furthermore, our findings strongly suggest that the bacterial flora present on the labia minora is similar to that
in the vaginal fluid. These facts confirm the importance
of good personal hygiene during menstruation in order
to prevent bacterial vaginosis.
9.
10.
11.
12.
13.
Acknowledgments The authors acknowledge Masao Nagahara
14.
(Kagawa Inoshita Hospital) for assistance in sample collecting and
medical examinations.
Conflict of interest None to declare.
15.
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