Prevalence of Bacterial Vaginosis and Vaginal Flora Changes in Peri- and Postmenopausal Women

Our aim was to evaluate the prevalence of bacterial vaginosisand decrease in lactobacillus colonization in women 40 yearsold or older in relation to menopausal status by evaluationof Gram-stained smears. A total of 1,486 smears from ItalianCaucasian women aged 40 to 79 years were examined. Women wereclassified as follows: fertile (regular cycles) (n = 328), perimenopausal(irregular cycles) (n = 237), and postmenopausal (n = 921),including 331 women on estroprogestinic hormone replacementtherapy (HRT). The prevalences of bacterial vaginosis (assessedas a Nugent score of $>=$ 7) in fertile (9.8%) and perimenopausal(11.0%) women were not statistically different, whereas theprevalence was significantly lower overall in postmenopausalwomen (6.0%) (P = 0.02). Specifically, 6.3% of postmenopausalwomen without HRT and 5.4% of postmenopausal women with HRTwere positive for bacterial vaginosis. The Nugent score systemwas not adequate for evaluating the normal and intermediatevaginal flora in women over the age of 40 years. High numbersof peri- and postmenopausal women had no lactobacilli and nobacterial-vaginosis-associated microorganisms. This nonpathologicalabsence of lactobacilli in women with a Nugent score of 4 wasscored as 4_*, and this group was considered separately fromthe intermediate flora group. A score of 4_* was obtained for2.1% of fertile women, 11.4% of perimenopausal women, 44.1%of postmenopausal women without HRT, and 6.9% of postmenopausalwomen with HRT. The physiological reduction in lactobacilluscolonization of the vagina in postmenopausal women does notcause an increase in bacterial-vaginosis prevalence. Reversionof lactobacillus flora to premenopausal levels due to HRT doesnot increase the prevalence of bacterial vaginosis in postmenopausalwomen.

INTRODUCTION

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Introduction

Bacterial vaginosis (BV) is a polymicrobial disorder characterizedby an increase in the vaginal pH over 4.5, a reduction in orabsence of lactobacillus colonization, and overgrowth of severalfacultatively and obligately anaerobic bacteria (10). BV isassociated with adverse pregnancy outcomes, upper genital tractinfections such as pelvic inflammatory disease, endometritis,post-gynecologic-surgery infections, cervicitis, urinary tractinfections, cervical intraepithelial neoplasia, and increasedrisk of sexual acquisition of human immunodeficiency virus infection(7, 13, 16, 17, 19, 24, 25, 29-31). BV is usually diagnosedby the clinical Amsel's criteria (1) or by the Nugent method(26) of Gram stain interpretation. Both these methods, however,were developed to analyze the vaginal flora of pregnant women,and then their use was extended to evaluation of the vaginaflora of fertile women. To our knowledge, no clear indicationson how to diagnose vaginal flora alterations in peri- and postmenopausalwomen have been reported. In fact, in postmenopausal women BVcannot be clinically diagnosed by Amsel's criteria (1) becauseone of these criteria, the vaginal pH value, is constitutivelyelevated (3, 23); moreover, the scarcity of vaginal dischargemakes it difficult to judge the kind of secretion. The Nugentmethod is based on the assumption that normal women have fullvaginal colonization by lactobacilli (26); this is valid forpregnant and fertile women but not for postmenopausal women(3, 15, 23).

Very few studies have evaluated the prevalence of BV in postmenopausalwomen (3, 15), and no study has assessed the prevalence of BVin perimenopausal women. Moreover, no data are available onthe effect of hormone replacement therapy (HRT) on BV prevalence.

The present study is a cohort study to determine the changesin vaginal flora and the prevalence of BV by standardized evaluationof Gram-stained smears for women 40 years old or older as afunction of reproductive condition: fertile, perimenopausal,or postmenopausal with or without HRT.

MATERIALS AND METHODS

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Materials and Methods

Study population. Nonpregnant women aged 40 to 79 years were consecutively recruitedduring routine gynecologic examinations (Papanicolaou [Pap]smear tests) in three clinics (located in Udine, Bologna, andTrieste) in northern Italy from April 1998 to April 2001. Womenwere enrolled after giving informed consent according to localEthics Committee guidelines, and clinical research was conductedin accordance with guidelines for human experimentation issuedby the authors' institutions. As a routine practice, at thetime of scheduling of the Pap test, patients were requestedto refrain from sexual intercourse and from any vaginal treatmentfor 3 days prior to their checkups. All women were Caucasian;92% had $>=$ 8 years of education, and 67% had $>=$ 13 years of education.All women included were eligible for the Pap test (no bleedingor major vaginal inflammatory signs) and had no malignanciesor severe medical illnesses. None had overt yeast vaginitis,and none was positive for Neisseria gonorrhoeae infection. Womenwith positive Trichomonas vaginalis results (on a wet smearand/or Pap smear exam) were excluded. A Chlamydia trachomatistest by ligase chain reaction (LCx; Abbott Diagnostics, Rome,Italy) was performed on the first 500 women enrolled. Becauseonly one woman was positive (0.2% C. trachomatis prevalence),further tests were not carried out. Further exclusion criteriawere partial or total hysterectomy, menopause induced by drugtreatments, HRT terminated less than 6 months earlier, HRT takenfor less than 3 months, vaginal estrogenic treatment, progestinictreatment in perimenopause, and tamoxifen or analogous antiestrogendrug therapy. When interviewed at the times of their visits,women included in the study said that they had not had sexualintercourse or engaged in any vaginal practice (such as douchingor using vaginal suppositories) in the past 3 days and had notused antibiotics in the past 2 weeks. Among 1,557 eligible womenover the age of 40, 71 were excluded for reasons such as anincomplete questionnaire, positive cervical intraepithelialneoplasia discovered as a result of the Pap test performed atthe time of the visit, or an inadequate vaginal secretion smear,leaving a study base of 1,486 participants for analysis.

The mean age ± standard deviation of the 1,486 womenenrolled was 53.4 ± 7.0 years.

Groups of women. Postmenopausal condition was defined as the absence of naturalmenses for at least 12 months.

From the study base of 1,486 women, four different groups ofparticipants were identified. The first group consisted of 328fertile women who had regular menses. The mean age of this groupwas 45.3 ± 3.7 years (range, 40 to 55 years);

The second group consisted of 237 perimenopausal women who hadirregular menses, or absence of menstruation for less than 12months (8). The mean age was 50.1 ± 3.0 years (range,40 to 58 years);

The third group consisted of 590 postmenopausal women who hadnot received HRT in the past 6 months (95% had never receivedHRT). The mean age was 58.1 ± 6.0 years (range, 42 to79 years); the mean age of onset of menopausal status was 50.2± 3.6 years (range, 33 to 59 years); the average numberof postmenopausal years was 7.9 ± 6.1 (range, 1 to 34years);

The fourth group consisted of 331 postmenopausal women currentlyreceiving oral or transdermal estroprogestinic replacement therapy.The mean age was 55.4 ± 4.4 years (range, 42 to 69 years);the mean onset of menopausal status was 49.3 ± 4.1 years(range, 32 to 58 years); the average number of postmenopausalyears was 6.1 ± 3.5 (range, 1 to 26 years). The averagetime of HRT was 3.9 ± 2.6 years. One hundred ninety-eightof these women were receiving transdermal estrogenic treatment,and 133 were on oral treatment; 9 of those on oral treatmentwere receiving tibolone. Progestinic treatment was transdermalin 25 cases, vaginal in 2 cases, and oral in the remaining cases.Two hundred sixty-eight protocols were sequential; the restwere combined estroprogestinic protocols.

Overall, 921 women were postmenopausal, with a physiologicaltermination of menstruation. Their mean age was 57.1 ±5.6 years (range, 42 to 79 years); the mean age of onset ofmenopausal status was 49.9 ± 3.8 years (range, 32 to59 years); the average number of postmenopausal years was 7.3± 5.4 (range, 1 to 34 years).

Vaginal sample collection. Samples were collected from the posterior fornix or lateralvaginal wall of nonbleeding women with an Ayre's spatula usinga nonlubricated speculum to perform a smear on a glass slide.The smear was then stained according to the Gram procedure.Clue cells and vaginal flora were evaluated on the Gram-stainedsmear.

Gram-stained-smear evaluation. The vaginal flora was determined by evaluation of five differentfields under oil immersion (magnification, x1,000). For thefirst analysis we adopted the Nugent score method (26). Threedifferent morphotypes are quantitatively evaluated: lactobacilli,Gardnerella-like species (including Gardnerella vaginalis, Bacteroidesspp., Prevotella spp., and Porphyromonas spp.), and Mobiluncusspp. Lactobacilli and Gardnerella-like morphotypes are rankedby the average number in five fields as follows: 0, <1, 1to 4, 5 to 30, and >30. These correspond to 4 to 0 points,respectively, for the lactobacillus morphotype and to 0 to 4points, respectively, for the Gardnerella-like morphotype. TheMobiluncus morphotype (curved rods) is ranked as 0, <1 to4, and $>=$ 5, corresponding to 0, 1, and 2 points, respectively.The sum of the points assigned to each of the three morphotypesis the final Nugent score of the patient, which is classifiedas normal (a Nugent score from 0 to 3), intermediate alteredflora (a Nugent score from 4 to 6), or full BV (a Nugent scorefrom 7 to 10).

In a more-refined analysis, the absence of lactobacilli, ofGardnerella-like bacteria, and of Mobiluncus spp., which isvery frequent in peri- or postmenopausal women, was consideredapart from the group with intermediate flora (which denotesa partially pathological change in the vaginal flora). For mostof these women only epithelial host cells are visible in thesmear; occasionally gram-positive cocci and/or enterobacteriaceaeare detectable. In fact, the decrease in lactobacilli is a commonlyobserved phenomenon in healthy postmenopausal women. Since sucha physiological condition characterizes a subgroup of womenwith a Nugent score of 4, we refer to it below as score 4_*.None of the women with score 4_* had clue cells.

Women with Nugent scores from 4 to 10 who were positive forclue cells were considered to have abnormal anaerobic vaginalflora (6, 32).

Full lactobacillus colonization was defined as more than 30lactobacilli observed in all the five (magnification, x1,000)fields examined.

All the Gram smear evaluations were performed by two independentinvestigators, with more than 90% agreement; discrepant readingswere reexamined, and a third investigator was consulted in caseof persistent disagreement.

Statistical analysis. Univariate comparisons of proportions were carried out by usingthe Pearson ${chi}$ ² test. Any P value of <0.05 was considered statisticallysignificant. The SPSS (Statistical Package for Social Sciences)software package was used for data analysis.

RESULTS

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Results

Gram-stained smear scores are shown in Table 1, subdivided asNugent scores 0 to 3, 4 to 6, and 7 to 10 (normal, intermediate,and full BV flora, according to Nugent et al. [26], respectively)and abnormal anaerobic flora (a Nugent score of $>=$ 4 and the presenceof clue cells); values are reported for all women as well asfor specific subgroups of women defined as fertile, perimenopausal,postmenopausal, and postmenopausal with or without HRT (seeMaterials and Methods). The overall prevalence of full BV was7.6%, and that of abnormal anaerobic vaginal flora was 9.9%.

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TABLE 1. Prevalence of normal, intermediate, and full BV vaginal flora^a and of abnormal anaerobic flora^b in specific subgroups women over the age of 40 years

Fertile and perimenopausal women showed no statistically significantdifferences in the prevalences of BV and abnormal anaerobicflora (9.8 versus 11.0% [P = 0.639] for BV; 12.5 versus 13.1%[P = 0.838] for abnormal anaerobic flora). For all postmenopausalwomen together, the prevalences of BV (6.0%) and abnormal vaginalflora (8.1%) were lower than those in fertile women (P = 0.021and P = 0.020, respectively) and perimenopausal women (P = 0.007and P = 0.019, respectively). The subgroup of postmenopausalwomen with HRT had slightly lower prevalences of BV (5.4%) andabnormal anaerobic flora (7.6%) than postmenopausal women withoutHRT (6.3 and 8.5%, respectively), but the differences were notstatistically significant (P = 0.609 for full BV; P = 0.624for abnormal anaerobic flora).

Postmenopausal women's smears analyzed according to the methodof Nugent showed clearly disproportionate figures for the normaland intermediate flora groups. Among postmenopausal women withoutHRT, only 46.3% (P < 0.001 with respect to fertile women)had normal flora, and as many as 47.5% (P < 0.001 with respectto fertile women) had intermediate flora, i.e., a partiallypathological vaginal condition (Table 1).

In a more-refined analysis, the prevalence of score 4_* (absenceof lactobacilli, no Gardnerella-like species, and no Mobiluncusspp., i.e., disappearance of lactobacillus colonization thatcannot be ascribed to the presence of BV-associated bacteria)(Fig. 1) was evaluated (Table 2). Fertile women had the lowestprevalence of score 4_* (2.1%). A 5.4-fold-higher percentageof perimenopausal women than of fertile women had score 4_* (P< 0.001). For all postmenopausal women together, the prevalenceof score 4_* was nearly 3-fold that among perimenopausal women(P < 0.001) and nearly 15-fold that among fertile women (P< 0.001). The prevalence of score 4_* was 21-fold elevatedin postmenopausal women who were not hormonally treated overthat among women with regular menses (P < 0.001). The subgroupsof postmenopausal women with and without HRT were markedly differentin prevalence of physiological absence of lactobacilli (score4_*): 6.9% versus 44.1% (P < 0.001). The prevalence of score4_* in women receiving HRT was close to that found in all premenopausalwomen over the age of 40 (the sum of women with regular andirregular menses) (6.9 versus 6.0%; P = 0.582).

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FIG. 1. Gram-stained vaginal smear from a postmenopausal woman with a score of 4_* (magnification, x400). Epithelial cells are visualized; bacteria are absent.

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TABLE 2. Prevalence of women with score 4*^a and redefined intermediate and normal flora groups in specific subgroups of women over the age of 40 years

Since a decrease in lactobacillus levels in postmenopausal womenis generally considered a normal physiologic change (3, 15,23), women over the age of 40 with a score of 4_* appear to bemore properly ranked in the normal than in the intermediatevaginal flora group. Accordingly, the number of women with ascore of 4_* was subtracted from the number of women in the groupwith Nugent scores of 4 to 6, to obtain a redefined intermediateflora group, and was added to the number of women in the groupwith Nugent scores of 0 to 3 to create a redefined normal group(see Table 2). When score 4_* was subtracted, the prevalenceof intermediate flora in the fertile subgroup was not statisticallydifferent from those in all the other subgroups (Table 2). Inaddition, when score 4_* was added, there were no statisticallysignificant differences in the prevalence of normal flora betweenthe fertile group, the perimenopausal group, and the group ofpostmenopausal women without HRT (Table 2).

Figure 2 shows the profile of full lactobacillus colonization(see Materials and Methods) and of the physiological absenceof vaginal lactobacilli (score 4_*) among the subgroups of womenover the age of 40. The fertile women had a very high prevalenceof full vaginal lactobacillus colonization (68.3%) in parallelwith the lowest prevalence of score 4_*. Full lactobacillus colonizationdecreases from fertile to perimenopausal to postmenopausal womenwithout HRT. Postmenopausal women without HRT had the lowestpercentage of full lactobacillus colonization (34.1%) and thehighest prevalence of score 4_* (44.1%). Women with HRT showeda level of full lactobacillus colonization almost identicalto that found in fertile women (68.9 versus 68.3%; P = 0.871).Overall, HRT causes lactobacillus colonization to revert tolevels found in premenopausal women without provoking the restorationof conditions causing BV, as evidenced by the fact that BV prevalencein postmenopausal women with HRT is nearly half that found inall premenopausal women (5.4 versus 10.3%; P = 0.012).

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FIG. 2. Profile of full lactobacillus colonization (stippled bars) and of score 4_* (physiological absence of lactobacilli) (dark shaded bars) in subgroups of women over the age of 40 years.

DISCUSSION

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Discussion

The present study differs from previously published studieson the vaginal flora of postmenopausal women in the larger numberof subjects and in the direct comparison among fertile women,perimenopausal women, and postmenopausal women with and withoutHRT, all of whom belong to a homogeneous ethnic group. We examinedwomen presenting for routine Pap smears in order to specificallyassess the prevalence of BV in the general population, sincethese data are missing in the literature and very recently thenecessity for such information has been highlighted (24). Themean age at menopause in the present investigation (49.9 years)is close to that recently reported for the general Italian population(50.9 years) (20).

We examined the vaginal flora by the objective and reproducibleevaluation of Gram-stained smears (26), avoiding subjectiveclinical evaluations (1), because vaginal pH in women closeto or in menopausal status is not exclusively a marker of anaerobiccolonization (3). To determine the prevalence of BV, we appliedthe strict definition given by Nugent et al. (26), comprisingscores from 7 to 10, but we also considered the enlarged groupwith abnormal anaerobic vaginal flora, comprising women withNugent scores of $>=$ 4 and with Gram-stained smears positive forclue cells, as this kind of classification has a sensitivitycloser to that of the clinical method (6, 32).

A number of trends emerged from the present study. We foundthat in the group of all premenopausal women over the age of40, the prevalence of BV was 10.3% and that of abnormal anaerobicflora was 12.7%. The presence of regular (fertile women) orirregular (perimenopausal women) menses did not statisticallyaffect the percentages of full BV and abnormal anaerobic flora.In the group of all postmenopausal women, the prevalence ofBV was 6.0% and that of abnormal anaerobic flora was 8.1%, bothstatistically lower than those found in premenopausal women.Estroprogestinic treatment of postmenopausal women had no statisticallysignificant effect on either full BV (prevalence, 5.4%) or abnormalanaerobic flora (prevalence, 7.6%). To the best of our knowledge,no previous study has evaluated the effects of HRT on the prevalenceof BV or abnormal anaerobic flora.

The absence of lactobacilli without detectable BV-associatedmicroorganisms is a rather rare event in women over the ageof 40 who still have regular menses (2.1%), whereas this conditionincreases markedly in perimenopausal women (11.4%) and is presentin as many as 44.1% of postmenopausal women without HRT. Wedefined this condition as score 4_* because it constitutes asubset of women with a Nugent score of 4. It appears that thisdistinction has to be made especially for peri- and postmenopausalwomen, because the absence of lactobacillus colonization inthese women appears to be a normal physiologic condition ratherthan an intermediate abnormal flora. In fact, in healthy fertilewomen, full vaginal colonization by lactobacilli is properlyconsidered the normal physiologic condition. However, the mereabsence of lactobacillus colonization (without any evidenceof Gardnerella-like or Mobiluncus microorganisms) should bedistinguished from other conditions resulting in a Nugent scoreof 4, which derive from a partial decrease in lactobacilluscolonization and a limited increase in growth of BV-associatedbacteria. This kind of mixed flora, in our opinion, should bemore properly defined as intermediate abnormal flora. On thisbasis, we propose a new scoring system, a refinement of theNugent score method, that distinguishes score 4_* among womenhaving a Nugent score of 4. We consider it advisable that ourmethod be adopted for all women regardless of hormonal status.

The large increase in the prevalence of score 4_* among postmenopausalwomen without HRT is paralleled by a large decrease in the percentageof women with full lactobacillus colonization (34.1% in postmenopausalwomen versus 68.3% in fertile women and 57.0% in perimenopausalwomen). The overall pronounced decrease in lactobacillus levelsthat we found in postmenopausal women is in line with valuesobtained in previous studies carried out on more limited numbersof subjects (3, 15, 23).

In our study HRT restores the percentage of full lactobacilluscolonization recorded for fertile women. Among postmenopausalwomen on estroprogestinic treatment, the prevalence of score4_* (6.9%) is sixfold reduced from that for postmenopausal womenwithout HRT and is threefold higher than that for fertile women;however, it is close to that found in the group of all premenopausalwomen (the sum of the fertile and perimenopausal subgroups)over the age of 40 (6.0%). The positive influence of HRT onlactobacilli is a known phenomenon, although we were unableto find quantitative data in the published literature (12, 21,22). The growth of lactobacilli observed in women receivingestroprogestinic treatment is very likely the consequence ofthe estrogen-induced increase in glycogen content in vaginalepithelial cells, since glucose derived from the metabolismof glycogen is believed to constitute the main nutritional factorfor lactobacilli (which convert it to lactic acid, loweringthe vaginal pH) (2, 11, 14). On the other hand, the availabilityof glucose potentially could also promote the growth of othermicroorganisms (18). In our study HRT did not increase the prevalenceof full BV or of abnormal anaerobic flora. Such a finding highlightsthe fact that some irreversible changes occur in the postmenopausalvaginal environment. Among possible factors that are not significantlyaffected by estroprogestinic treatments, but that could be involvedin the persistence of BV-associated bacteria in the vagina,are the availability of receptors for bacteria on host cells,variations in the biofilm lining the vaginal epithelium, thinningof the vaginal epithelium, changes in mucus abundance, viscosity,and glycoprotein content, availability of nutritional factors(other than glucose) such as metal ions, especially iron, andchanges in the innate and/or adaptive immune response (4-6,9, 27, 28). The paradox of the postmenopausal condition is thatin spite of the impressive decrease in lactobacillus colonization,increase in vaginal pH, increase in levels of gram-positivecocci, and increase in coliform colonization (15), the prevalenceof BV is significantly lower than those in perimenopausal andfertile women.

To our knowledge, no detailed data on vaginal flora in perimenopausalwomen have been reported previously. In our study, perimenopausalwomen show a grade of lactobacilli which is intermediate betweenthat of women who still have regular cycles and that of postmenopausalwomen without HRT. Perimenopausal women have almost the sameBV prevalence as fertile women, which is roughly twice thatof postmenopausal women, indicating that those factors preventingBV-associated microorganism colonization in postmenopausal womenhave not yet developed. From our findings it follows that, indealing with the vaginal flora of women over the age of 40,perimenopausal women should be considered as a distinct groupof subjects.

Among the limitations of the present investigation is the lackof information on body mass, sexual habits (frequency of sexualintercourse, number of partners over the life span, age of sexualdebut), and hygienic, alimentary, and smoking habits. No serumsamples were available on which to perform measurements of follicule-stimulatinghormone, luteinizing hormone, and estradiol levels.

In summary, our data reveal that postmenopausal status involvesmajor vaginal changes that are not confined to the glycogencontent of epithelial cells, which can be modulated by HRT.Other, still unclarified subtle differences occur in the vaginasof postmenopausal women, and their future identification couldcontribute to understanding of the general mechanisms of colonizationby BV-associated microorganisms. Our data show that the absenceof lactobacilli by itself is not related to an elevated prevalenceof BV, as observed in peri- and postmenopausal women. This findingsuggests that the decrease in lactobacillus colonization ismore likely a consequence than a leading cause of the anaerobicalteration of the vaginal ecology.

ACKNOWLEDGMENTS

This work was supported by Cofin 1998 and Cofin 2000 grantsfrom the "Ministero Istruzione, Università, Ricerca"of Italy, a year 2000 Regione Friuli Venezia Giulia grant, theUniversity of Udine, the University of Trieste, the Universityof Bologna, and the IRCCS Burlo Garofolo Hospital, Trieste,Italy.

We thank Sandra Flatow, New Haven, Conn., for reading the manuscript,Nunziata Verdolina, Azienda Servizi Sanitari n. 4, Udine, Italy,for nursing assistance, and Marina Cudin, Dipartimento di Scienzee Tecnologie Biomediche, Udine, Italy, for laboratory technicalhelp.

FOOTNOTES

* Corresponding author. Mailing address: Dipartimento di Scienze e Tecnologie Biomediche, Facoltà di Medicina e Chirurgia, Piazzale Kolbe 4, 33100 Udine, Italy. Phone: 39 0432 494312. Fax: 39 0432 494301. E-mail: scauci{at}mail.dstb.uniud.it.

REFERENCES

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