Influence of Menstruation on the Microbiota of HealthyWomen'sLabiaMinoraasAnalyzedUsing a 16S rrna Gene-Based Clone Library Method

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1 Jpn. J. Infect. Dis., 64, 76-80, 2011 Short Communication Influence of Menstruation on the Microbiota of HealthyWomen'sLabiaMinoraasAnalyzedUsing a 16S rrna Gene-Based Clone Library Method Tsukasa Shiraishi*, Kazumasa Fukuda 1, Nobuo Morotomi 1,YuriImamura, Junko Mishima, Shigeo Imai, Kiyoshi Miyazawa, and Hatsumi Taniguchi 1 Life Science Laboratory, Unicharm Corporation, Kagawa ; and 1 Department of Microbiology, School of Medicine, University of Occupational and Environmental Health Japan, Fukuoka , Japan (Received October 15, 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. 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 (H 2 O 2 ) (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 , Japan. Tel: , Fax: , shiraishi1020@gmail.com 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 76

2 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 andstoredat49c. 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 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 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 for premenstruation and 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?- CCTACGGGAGGCAGCAG-3? and 907R, 5?- 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 ( and the DNA Data Bank of Japan ( 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. 77

3 Table 1. Bacterial composition of the clone libraries of labia minora and vaginal fluid samples from 10 healthy Japanese women Reference type strain (accsession no.) Labia minora swab 1) (z clones) Vaginal fluid swab 2) (z clones) No.1 No.2 No.3 No.4 No.5 No.6 No.7 No.8 No.9 No.10 No.5 No.7 No.10 B A B A B A B A B A B A B A B A B A B A BF AF BF AF BF AF 78 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) Atopobium vaginae (Y17195) Bacteroides coagulans (DQ497990) 1.1 Porphyromonas somerae (AY968205) 1.1 Prevotella bivia (L16475) P. buccalis (L16476) 1.1 Gemella haemolysans (L14326) 1.1 Staphylococcus epidermidis (D83363) Facklamia hominis (Y10772) 1.1 Lactobacillus crispatus (Y17362) L. fermentum (M58819) 3.3 L. fornicalis (Y18654) L. gasseri (M58820) L. iners (Y16329) L. kitasatonis (AB107638) 2.1 Streptococcus difficilis (AB112407) 7.2 S. intermedius (AF104671) S. parasanguinis (AF003933) 30.4 S. pseudopneumoniae (AY485599) 4.3 Dialister micraerophilus (AF473837) 1.2 D. propionicifaciens (AY850119) Selenomonas noxia (AF287799) 1.1 Anaerococcus prevotii (D14139) Micromonas micos (AF542231) 1.1 Peptoniphilus harei (Y07839) P. ivorii (Y07840) P. lacrimalis (D14141) Peptostreptococcus stomatis (DQ160208) 1.2 Ureaplasma parvum (AF073456) Fusobacterium canifelinum (AY162220) Leptotrichia hofstadii (AY029803) 1.1 Vibrio gigantis (AJ582810) 1.1 Unclassified Total clones ) : B, labia minora swab at pre-menstruation; A, labia minora swab during menstruation. 2) : BF, vaginal fluid swab at pre-menstruation; AF, vaginal fluid swab during menstruation.

4 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 ( z) insample No. 4 and No. 7 at both premenstruation and during menstruation, whereas L. iners clones were found to be predominant ( z) 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) and10.5z (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 menstruationin6(no.1,2,3,5,6,and8)ofthe10 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, andgardnerella, 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) thanincaucasianwomen (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

5 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. Acknowledgments The authors acknowledge Masao Nagahara (Kagawa Inoshita Hospital) for assistance in sample collecting and medical examinations. Conflict of interest None to declare. REFERENCES 1. Costello, E.K., Lauber, C.L., Hamady, M., et al. (2009): Bacterial community variation in human body habitats across space and time. Science, 326, Farage, M. and Maibach, H. (2006): Lifetime changes in the vulva and vagina. Arch. Gynecol. Obstet., 273, Mijac, V.D., Dukic, S.V., Opavski, N.Z., et al. (2006): Hydrogen peroxide producing lactobacilli in women with vaginal infections. Eur. J. Obstet. Gynecol. Reprod. Biol., 129, Reid, G., Charbonneau, D., Erb, J., et al. (2003): Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol. Med. Microbiol., 35, Burton, J.P., Cadieux, P.A. and Reid, G. (2003): Improved understanding of the bacterial vaginal microbiota of women before and after probiotic instillation. Appl. Environ. Microbiol., 69, Brown, C.J., Wong, M., Davis, C.C., et al. (2007): Preliminary characterization of the normal microbiota of the human vulva using cultivation-independent methods. J. Med. Microbiol., 56, Sobel, J.D., Funaro, D. and Kaplan, E.L. (2007): Recurrent group A streptococcal vulvovaginitis in adult women: family epidemiology. Clin. Infect. Dis., 44, Sing, A., Roggenkamp, A., Kress, K., et al. (1998): Bartholinitis due to Streptococcus pneumoniae: case report and review. Clin. Infect. Dis., 27, Reid, G. and Bruce, A.W. (2003): Urogenital infections in women: can probiotics help? Postgrad. Med. J., 79, Zhou, X., Brown, C.J., Abdo, Z., et al. (2007): Differences in the composition of vaginal microbial communities found in healthy Caucasian and black women. ISME J., 1, Zhou, X., Westman, R., Hickey, R., et al. (2009): Vaginal microbiota of women with frequent vulvovaginal candidiasis. Infect. Immun., 77, Oakley, B.B., Fiedler, T.L., Marrazzo, J.M., et al. (2008): Diversity of human vaginal bacterial communities and associations with clinically defined bacterial vaginosis. Appl. Environ. Microbiol., 74, Yamamoto, T., Zhou, X., Williams, C.J., et al. (2009): Bacterial populations in the vaginas of healthy adolescent women. J. Pediatr. Adolesc. Gynecol., 22, Zhou, X., Bent, S.J., Schneider, M.G., et al. (2004): Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods. Microbiology, 150, Akiyama, T., Miyamoto, H., Fukuda, K., et al. (2010): Development of a novel PCR method to comprehensively analyze salivary bacterial flora and its application to patients with odontogenic infections. Oral. Surg. Oral. Med. Oral. Pathol. Oral. Radiol. Endod., 109, Kawanami, T., Fukuda, K., Yatera, K., et al. (2010): A higher significance of anaerobes: the clone library analysis of bacterial pleurisy. Chest, Aug 5 (Epub ahead of print). 17. Verhelst, R., Verstraelen, H., Claeys, G., et al. (2005): Comparison between Gram stain and culture for the characterization of vaginal microflora: definition of a distinct grade that resembles grade I microflora and revised categorization of grade I microflora. BMC Microbiol., 5, Bradshaw, C.S., Tabrizi, S.N., Fairley, C.K., et al. (2006): The association of Atopobium vaginae and Gardnerella vaginalis with bacterial vaginosis and recurrence after oral metronidazole therapy. J. Infect. Dis., 194, Matu, M.N., Orinda, G.O., Njagi, E.N., et al. (2010): In vitro inhibitory activity of human vaginal lactobacilli against pathogenic bacteria associated with bacterial vaginosis in Kenyan women. Anaerobe, 16, Verstraelen, H., Verhelst, R., Claeys, G., et al. (2009): Longitudinal analysis of the vaginal microflora in pregnancy suggests that L. crispatus promotes the stability of the normal vaginal microflora and that L. gasseri and/or L. iners are more conducive to the occurrence of abnormal vaginal microflora. BMC Microbiol., 9,