595
Effects of Contraceptive Method on the Vaginal Microbial Flora:
A Prospective Evaluation
Kalpana Gupta,1 Sharon L. Hillier,2
Thomas M. Hooton,1 Pacita L. Roberts,1
and Walter E. Stamm1
1
Department of Medicine, Division of Allergy and Infectious Diseases,
University of Washington, Seattle; 2Department of Obstetrics,
Gynecology, and Reproductive Sciences, University of Pittsburgh,
Magee-Women’s Research Institute, Pittsburgh, Pennsylvania
A prospective evaluation of 331 university women who were initiating use of oral contraceptive pills (OCPs), a cervical cap, diaphragm-spermicide, or other spermicidal methods was
done to assess the effects of commonly used contraceptive methods on the vaginal flora.
Vaginal introital cultures were performed at baseline and then weekly for 1 month. The
prevalence of Escherichia coli vaginal colonization and of abnormal vaginal Gram stain scores
(Nugent criteria) increased significantly among women using a cervical cap or diaphragmspermicide but not among women using OCPs. Women with E. coli colonization were significantly more likely to have an abnormal Nugent score and an absence of lactobacilli. In a
multivariate model, use of spermicidal contraception in the preceding week was associated
with an abnormal Nugent score and with colonization with E. coli, Enterococcus species, and
anaerobic gram-negative rods. Thus, spermicidal methods of contraception are associated
with alterations of the vaginal microflora that consequently may predispose women to genitourinary infections.
The normal vaginal ecosystem is increasingly recognized as
an important host defense mechanism against exogenous
urogenital infection. Alteration of the normally Lactobacillusdominant flora has been associated with an increased risk of
bacterial vaginosis (BV), gonorrhea, infection with human immunodeficiency virus, and Escherichia coli vaginal colonization,
the critical step preceding urinary tract infection (UTI) [1–5].
The vaginal flora can be disrupted by a variety of factors,
including sexual intercourse, antimicrobial use, and douching
[1, 6, 7]. The effects of specific contraceptive methods on the
vaginal flora, particularly those involving spermicide use, are
not clear. Whereas some studies have shown a significant decrease in the prevalence of vaginal lactobacilli among diaphragm-spermicide users [6, 8] or among spermicide users alone
[9–11], others have found no such effect [12, 13]. The effects
of other birth control methods, such as oral contraceptive pills
(OCPs) and the cervical cap, on the vaginal microbial ecology
are even less well delineated. To assess the effects of these contraceptives and of sexual activity on the vaginal microflora, we
Received 12 July 1999; revised 12 October 1999; electronically published
14 February 2000.
Presented in part: annual meeting of the Infectious Diseases Society for
Obstetrics and Gynecology, Barrier Creek, Colorado, 1996.
Written informed consent was obtained from all participants, and human
experimentation guidelines of the University of Washington Human Subjects
Review Committee were followed.
Grant support: National Institutes of Health (DK 47549 and DK 53369).
Reprints or correspondence: Dr. Kalpana Gupta, 1959 NE Pacific St.,
University of Washington, HSB, BB1221, Box 356523, Seattle, WA 98195
([email protected]).
The Journal of Infectious Diseases 2000; 181:595–601
q 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18102-0024$02.00
prospectively studied a cohort of 331 healthy, sexually active
university women who were initiating a new method of contraception.
Methods
Study population. Women included in the present study were
participants in a larger 2-site prospective cohort study of risk factors for UTI in women [14]. Only women enrolled at 1 of the sites,
the student health service at the University of Washington, were
included in this aspect of the study. Complete details of the overall
study design have been published [14]. Briefly, women between
18–40 years of age who were initiating a new method of birth
control, were not pregnant, had <1 UTI in the previous year, had
no chronic illnesses, and had no known anatomic or functional
abnormalities of the urinary tract were eligible to participate.
Women were evaluated at enrollment and then weekly for 1 month
after initiation of their new contraceptive method. Vaginal introital
cultures and detailed interviews were obtained at each of these
visits. In addition, women maintained a daily diary, which was
provided at enrollment to record occurrences of sexual intercourse,
contraceptive use, antimicrobial use, menses, and genitourinary
symptoms.
Laboratory methods. Vaginal cultures were collected by inserting a sterile cotton swab ∼1 inch into the vagina and were
transported to the laboratory in Amies transport medium. Semiquantitative cultures for aerobic gram-negative rods and cocci, anaerobic gram-negative rods, and lactobacilli were performed by
using standard laboratory methods [15, 16]. Results were categorized semiquantitatively as 0 (no growth), 11 (colonies in the first
streak zone), 21 (colonies in the second streak zone), 31 (colonies
in the third streak zone), and 41 (colonies in the fourth streak
zone). Vaginal smears were prepared by rolling a second vaginal
596
Gupta et al.
swab across a microscope slide at the patient’s bedside and allowing
it to air-dry. The smears were then Gram stained, and the flora
were scored as normal (Nugent scores 0–3), intermediate (Nugent
scores 4–6), or consistent with BV (Nugent scores 7–10), by use of
criteria and methods described elsewhere [16, 17].
Statistical methods. Changes in vaginal flora were assessed by
comparing the baseline visit culture results to the week 1 and week
4 culture results by use of McNemar’s test. Only women who completed the baseline visit and 4 weekly follow-up visits were included
in this analysis. Women starting their birth control method before
the baseline culture, reporting no episodes of vaginal intercourse
during the study period, or using an alternative method of birth
control in addition to their primary method were excluded. Condom use in conjunction with OCP use was allowed if the frequency
of spermicide use was less than once per week.
A second analysis examining the temporal relationship between
vaginal microbial flora and vaginal intercourse and contraceptive
use was performed by using logistic regression analysis. For the
present analysis, each visit was analyzed separately and was correlated with sexual activity and contraceptive use in the week before
the visit as ascertained from the patients’ diaries. Visits for which
diary information in the preceding week was not available were
excluded from this analysis. The analyses were adjusted for multiple
observations per woman using the method of generalized estimating equations [18].
Results
Of the 348 university women enrolled in the larger study, 331
(95%) met criteria for inclusion in the present analysis. A total
of 1644 visits that yielded vaginal culture data were available
from these women. The majority of the women were white
(79%), were never married (85%), were secretors of blood group
antigens (75%), and had no previous history of UTIs (72%).
The mean age was 23 years, and all women were sexually active
or initiating sexual activity. More detailed specific characteristics of the study group have been reported [14].
Changes in vaginal flora after initiating a new contraceptive
method. All 5 visits were completed by 213 women, resulting
in 1065 visits with vaginal cultures for inclusion in this analysis.
The new contraceptive methods used by these women were
OCPs (103 women), diaphragm-spermicide (75 women), and
cervical cap (35 women). The proportions of women colonized
with specific organisms at baseline and at 1 week and 4 weeks
after initiating the new contraceptive methods are shown in
figure 1. The prevalence of each organism at baseline was similar among women in the 3 groups, except for Enterococcus
species, which were present in 16% and 17% of women in the
OCP and diaphragm-spermicide groups, respectively, and in
only 3% of women in the cervical cap group. Most organisms
were present in <20% of women at baseline, except for lactobacilli, which were present in 85%–94% of women. The prevalence of women with lactobacilli did not change significantly
1 week and 4 weeks after initiating any of the 3 contraceptive
methods. In contrast, the prevalence of women with E. coli
JID 2000;181 (February)
increased significantly in the cervical cap group, from 17% at
baseline to 40% at weeks 1 and 4 (P = .03 and P = .03, respectively), and in the diaphragm-spermicide group, from 15%
at baseline to 48% at week 1 and to 32% at week 4 (P = .001
and P = .005, respectively). There was a small decrement in the
prevalence of E. coli in the OCP group, from 22% at baseline
to 12% at week 4 (P = .04). Women without vaginal E. coli at
baseline were more likely to be colonized with E. coli at any
follow-up visit if they were in the diaphragm-spermicide (60%)
and cervical cap groups (49%) than if they were in the OCP
group (27%). Conversely, women with vaginal E. coli at baseline
were more likely to subsequently become negative in the OCP
group (11%) than in the diaphragm-spermicide (5%) or cervical
cap groups (0%).
Like E. coli, the prevalence of women with vaginal Enterococcus species also increased significantly at weeks 1 and 4
among the cervical cap group (P = .008 and P = .014, respectively) and at week 1 among the diaphragm-spermicide group
(P = .001). There were only minor changes in enterococcal colonization in the OCP group. The prevalence of women with
Candida species decreased slightly from 16% at baseline to 5%
at week 4 (P ! .01) among those in the OCP group. None of
the other organisms isolated, including Staphylococcus aureus,
anaerobic gram-negative rods, group B streptococci, and Gardnerella vaginalis, changed significantly among the 3 groups of
women over the 1-month observation period. Increases in the
prevalence of anaerobic gram-negative rods that approached
statistical significance were seen at the week 1 follow-up visit
in the cervical cap (14%–29%) and diaphragm-spermicide
(20%–32%) groups but not in the OCP group.
By using semiquantitative cultures, we further determined
whether initiation of these contraceptive methods altered the
quantities of specific vaginal microbes (data not shown). The
majority of women in each contraceptive group had either 21
or 31 lactobacilli throughout the study period, with little evident quantitative change after initiation of their contraceptive
method. In contrast, although the majority of women in each
contraceptive group had an absence of E. coli throughout the
study period, there were significant increases in the percentages
of women with 21, 31, and 41 E. coli among the diaphragmspermicide and cervical cap groups over the study period. These
changes were not observed in the OCP group.
Changes in Nugent score after initiating a new contraceptive
method. Nugent scores were evaluated at each visit. At baseline, a score consistent with normal flora (1–3) was present in
86% (cervical cap group), 67% (diaphragm-spermicide group),
and 65% (OCP group) of the women (figure 2). At week 1, a
significant reduction in the prevalence of women with Nugent
scores of 1–3 was observed in the cervical cap group (54%,
P = .002) and in the diaphragm-spermicide group (44%, P =
.001), although little change occurred in the OCP group (62%,
P = .55). At week 4, the percentage of women with normal
scores remained significantly reduced, as compared with base-
JID 2000;181 (February)
Contraceptive Method and Vaginal Flora
597
Figure 1. Changes in the prevalence of vaginal microflora stratified by contraceptive method. Bars represent the percentage of women with a
vaginal culture positive for 111 of each microorganism at baseline (black bar); week 1 (light gray bar); and week 4 (dark gray bar). Bacteroides
includes organisms belonging to the genera Bacteroides, Prevotella, and Porphyromonas. Statistically significant differences between baseline and
follow-up weeks denoted by * (P ! .05) and † (P ! .01). CNS, coagulase-negative staphylococci; GBS, Group B streptococci.
line rates in the cervical cap group (53%, P = .001), although
smaller nonsignificant differences were seen in the diaphragmspermicide (57%) and the OCP (56%) groups, as compared with
baseline scores. A majority of these score changes represented
shifts from normal flora to intermediate flora rather than to
BV.
Relationship between Nugent score and vaginal colonization
with E. coli and enterococci. The association between Nugent
scores and vaginal colonization with E. coli and enterococci
were also examined. As the prevalence of women with abnormal
flora as measured by Nugent score increased, the prevalence
and semiquantitative counts of E. coli and enterococcal vaginal
colonization also increased (figure 3). A score consistent with
intermediate or BV flora was present among 62% of women
with 41 E. coli but only 31% of women with no E. coli (P !
.001). Similarly, such scores were present among 64% of women
with 41 enterococci and only 35% of women with no enterococci (P = .01).
Figure 2. Changes in Nugent score stratified by contraceptive method. Bars represent the percentage of women within each contraceptive
group with a Nugent score of 1–3 (dark gray bar); 4–6 (light gray bar); and 7–10 (black bar) at each visit. *P ! .05 for comparison of baseline
versus follow-up week.
JID 2000;181 (February)
Contraceptive Method and Vaginal Flora
599
users of the cervical cap and trended toward a decrease among
diaphragm-spermicide users. The results of a second multivariate model that combined all 3 spermicidal contraceptive methods into 1 independent variable are also presented in table 1.
In this analysis, the risk of having an abnormal Nugent score
or colonization with E. coli, Enterococcus species, or anaerobic
gram-negative rods was increased among spermicide users,
whereas the risk of Candida species colonization was decreased.
Discussion
Figure 3. Association between abnormal Nugent score and vaginal
colonization with Escherichia coli and enterococci. Significant increase
was observed in the percentage of women who had an abnormal Nugent score (defined as a score >4) with increasing semiquantitative
amounts of E. coli and enterococci (P < .01).
Relationship between vaginal colonization with E. coli and lactobacilli. There was an inverse association between vaginal
lactobacilli and E. coli colonization. As shown in figure 4, as
the quantity of lactobacilli per vaginal culture increased from
0 to 41, the prevalence of E. coli vaginal colonization decreased
from 34% to 18%. Furthermore, after controlling for intercourse, antimicrobial use, and birth control method in the preceding week, the risk of E. coli colonization at any visit was
2-fold greater among women without lactobacilli, as compared
with women with lactobacilli (P = .01).
Multivariate model assessing the risk of vaginal colonization
with normal and abnormal flora by contraceptive method. To
determine the relative risk of vaginal colonization with each
organism among women using a diaphragm-spermicide, cervical cap, or other spermicidal method in the preceding 7 days,
a multivariate model with these exposures as covariates was
constructed (table 1). Sexual intercourse and antimicrobial use
in the preceding 7 days were also included in the same model.
After controlling for sexual activity and antimicrobial use, the
relative risk of being colonized with lactobacilli, coagulase-negative staphylococci, group B streptococci, or G. vaginalis among
diaphragm-spermicide, cervical cap, and spermicide users was
not statistically significant. Conversely, there was a significantly
increased risk of E. coli vaginal colonization of ∼2-fold among
spermicide users, 3-fold among cervical cap users, and 4-fold
among diaphragm-spermicide users. Intercourse also increased
the risk of E. coli vaginal colonization by 3.5-fold. As with E.
coli, the risk of vaginal colonization with Enterococcus species
was also significantly increased in association with intercourse
or diaphragm-spermicide use in the previous week but was not
significantly elevated among users of the cervical cap or spermicide alone. The risk of vaginal colonization with anaerobic
gram-negative rods and the risk of having an abnormal Nugent
score were both significantly increased among users of a diaphragm-spermicide in the preceding week, whereas the risk of
Candida species colonization was significantly decreased among
The effects of contraceptive choice on the vaginal microbial
flora have important implications for women who suffer from
infections associated with disruptions in the vaginal ecology,
such as BV and UTIs. The present prospective study of women
who were initiating a new method of contraception (and, in
some cases, sexual activity) showed that OCPs have little effect
on the vaginal microbial flora, whereas diaphragm-spermicide
use is associated with vaginal colonization with E. coli, Enterococcus species, and anaerobic gram-negative rods, as well as
with an elevated Nugent score. Cervical cap use and other
spermicide use were also each associated with similar alterations
in flora, particularly E. coli colonization. Conversely, neither
intercourse alone, nor use of any of the contraceptive methods,
significantly affected the overall prevalence of vaginal lactobacilli.
Our data also suggest that these alterations in vaginal flora
do not occur independently of one another. Even after controlling for intercourse, spermicide use, and antimicrobial use
in the previous week, the inverse association between lactobacilli and vaginal E. coli colonization was highly statistically
significant. Although we did not specifically assay hydrogen
peroxide (H2O2) production by lactobacilli strains in the present
study, it is likely that a majority of lactobacilli isolated did
produce H2O2, because these cultures were taken from healthy
premenopausal women, a population in which the prevalence
of H2O2-producing lactobacilli approaches 95% [19]. An inverse
Figure 4. Association between vaginal lactobacilli and Escherichia
coli colonization. A significant decrease was observed in the percentage
of women who had 111 vaginal E. coli colonization with increasing
semiquantitative amounts of lactobacilli (P < .01).
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Gupta et al.
Table 1.
JID 2000;181 (February)
Adjusted risk of vaginal colonization by contraceptive method.
a
b
Adjusted odds ratio (95% CI) for colonization by exposure in the previous week
Microbial organism
Intercourse
Lactobacilli
Escherichia coli
Enterococcus species
Candida species
Anaerobic gram-negative rods
Abnormal Nugent score
0.8
3.5
1.8
1.3
0.9
1.3
(0.5–1.2)
(2–6)
(1.1–2.8)
(0.7–2.4)
(0.6–1.3)
(0.9–1.7)
Diaphragm or
spermicide
1 (0.6–1.8)
3.8 (2.5–5.6)
2.5 (1.7–3.8)
0.5 (0.2–1.1)
1.7 (1.2–2.3)
1.5 (1.1–2.1)
Cervical cap
1.5
2.7
1.4
0.3
1.5
1.1
(0.7–3.4)
(1.6–4.4)
(0.8–2.6)
(0.1–0.8)
(0.9–2.3)
(0.7–1.8)
Other spermicidal
method
1.4 (0.8–2.5)
1.7 (1.1–2.6)
1.6 (1–2.7)
0.9 (0.4–1.7)
1.1 (0.7–1.7)
1 (0.7–1.5)
Any spermicidal
c
method
1.3
2.8
1.8
0.5
1.5
1.3
(0.8–2.1)
(2–4)
(1.3–2.7)
(0.3–0.9)
(1.1–2.1)
(1–1.7)
a
The same multivariate model was applied to each organism. Group B streptococci, coagulase-negative staphylococci,
and Gardnerella vaginalis were not significantly associated with any of the exposures.
b
Defined as the presence of the organism in a semiquantitative concentration of 111.
c
All 3 spermicidal contraceptive methods were combined into 1 variable in this second model.
relationship between H2O2-producing lactobacilli and vaginal
E. coli has been reported in women with a history of recurrent
UTI [5]. Our data suggest that this phenomenon may also be
important in the pathogenesis of acute E. coli cystitis.
In addition, women who had an abnormal Nugent score,
suggesting an overgrowth of anaerobic flora, were also more
likely to have E. coli and enterococcal colonization. The prevalence and semiquantitative amount of these aerobic bacteria
increased as the prevalence of abnormal Nugent scores increased, suggesting a linear relationship between these perturbations in vaginal ecology. Recent appreciation that the loss of
H2O2-producing lactobacilli may play a role in the pathogenesis
of both BV [1] and E. coli [5] vaginal colonization provides a
plausible common pathway for these events, and further evidence that these events are not entirely independent [1, 5]. Thus,
exposures that lead to a loss of H2O2-producing lactobacilli
likely predispose a woman to vaginal colonization with both
anaerobic and aerobic bacteria. The extent and type of microbial alteration, and the resulting clinical syndrome, such as BV
or UTI, likely depends on a combination of additional factors,
such as intercourse frequency, spermicide use, douching, a new
sex partner, or new sexually acquired pathogens. Additional
host factors, such as secretor status or estrogen state, may also
be important. The association between BV and E. coli vaginal
colonization has been reported among women with acute UTI
symptoms who use diaphragm-spermicide [20] but was not
found among asymptomatic pregnant women in labor [16]. The
latter study did find an increased prevalence of enterococcal
colonization among women with BV flora, suggesting that aerobic bacteria are associated with the anaerobic overgrowth
characterizing BV.
The prospective nature of the present study and the use of
daily diaries allowed us to elucidate temporal relationships between various exposures and vaginal microbiology. Although
diaphragm-spermicide use has been associated with E. coli vaginal colonization [20–22], the individual contributions of intercourse, spermicide use, and contraceptive method have been
difficult to sort out. Our data show an incremental increase in
the effects on E. coli vaginal colonization of spermicide use
alone, cervical cap use, intercourse, and diaphragm-spermicide
use. A similar trend is seen with enterococcal colonization. Our
data also show a temporal relationship between diaphragmspermicide use and elevated Nugent scores. Most previous studies examining risk factors for BV have involved women attending sexually transmitted disease clinics, a population in
which sexual habits, frequency, and number of sex partners is
likely different from our study group of asymptomatic young
women attending a student health service. Unfortunately, exposures that have been previously associated with BV, such as
new sex partner, douching, and history of BV, were too infrequent to assess adequately among our study population.
We have previously proposed that spermicidal compounds
may predispose women to E. coli vaginal colonization and UTI
by virtue of their differential antimicrobial effects on lactobacilli
and E. coli [23]. Thus, most lactobacilli (especially H2O2 producers) are susceptible, but nearly all E. coli are highly resistant
to concentrations of nonoxynol-9 (N-9) that are likely present
during routine use [9]. In the present study, we did observe an
inverse relationship between E. coli vaginal colonization and
lactobacilli, but the overall prevalence of women with lactobacilli and the quantity of lactobacilli present (as ascertained
by our semiquantitative cultures) were not apparently altered
after spermicide exposure. It is possible that spermicide exposure exerts a significant but only transient effect on lactobacilli prevalence and concentrations. Thus, a spermicide-mediated antimicrobial effect reducing lactobacilli from 107 colony
forming units (cfu) per gram of vaginal fluid to 105 cfu per
gram of vaginal fluid (a 99% reduction) might not be detectable
24 h later because of regrowth of lactobacilli. This explanation
is supported by a recent study by Watts et al. [24], assessing
the effects of N-9 use in the absence of intercourse; the concentration of lactobacilli decreased significantly 4 h after exposure to N-9 but returned to baseline levels by 24 h after
exposure. The overall prevalence of lactobacilli was not significantly affected. Alternatively, because H2O2-producing lactobacilli are more sensitive to N-9 [25], perhaps N-9 exposure
induces a shift from strains that produce H2O2 to strains that
do not. However, a shift of this nature was not observed after
a single exposure to N-9 [24]. We did not measure H2O2 production by the strains in the present study and thus cannot
JID 2000;181 (February)
Contraceptive Method and Vaginal Flora
address this issue. Finally, both the abnormal Nugent scores
induced by spermicide exposure and the changes in prevalence
and concentration of anaerobic gram-negative rods that were
observed suggest that spermicides affect the anaerobic vaginal
flora in some manner. These effects could also be important in
predisposition to E. coli colonization and UTI.
In conclusion, barrier methods of contraception, such as the
cervical cap and diaphragm-spermicide, clearly alter the normal
vaginal ecosystem. Women who develop clinical syndromes associated with altered vaginal flora may choose to avoid these
methods of birth control in favor of OCPs, which appear to
have minimal effects on the vaginal microbial ecology. Elucidation of additional modifiable factors that disturb vaginal homeostasis is important for furthering our understanding of the
pathogenesis of altered vaginal ecology and for providing additional behavioral choices for women.
Acknowledgments
We gratefully acknowledge Carol Winter, ARNP, and Natalie
DeShaw, Research Study Coordinator, for patient enrollment and specimen collection.
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