Research ajog.

org

GYNECOLOGY
Colonization of the upper genital tract
by vaginal bacterial species in
nonpregnant women
Caroline M. Mitchell, MD, MPH; Anoria Haick, MS; Evangelyn Nkwopara, BA;
Rochelle Garcia, MD; Mara Rendi, MD, PhD; Kathy Agnew, BA;
David N. Fredricks, MD; David Eschenbach, MD

OBJECTIVE: The objective of the study was to evaluate the upper
genital tract (UGT) presence of vaginal bacterial species using sensitive
molecular methods capable of detecting fastidious bacterial vaginosis
(BV)eassociated bacteria.
STUDY DESIGN: Vaginal swabs were collected prior to hysterectomy.
The excised uterus was sterilely opened and swabs collected from the
endometrium and upper endocervix. DNA was tested in 11 quantitative
polymerase chain reaction (PCR) assays for 12 bacterial species:
Lactobacillus iners, L crispatus, L jensenii, Gardnerella vaginalis,
Atopobium vaginae, Megasphaera spp, Prevotella spp, Leptotrichia/
Sneathia, BVAB1, BVAB2, BVAB3, and a broad-range16S ribosomal
ribonucleic acid gene assay. Endometrial fluid was tested with Luminex
and an enzyme-linked immunosorbent assay for cytokines and
defensins and tissue for gene expression of defensins and cathelicidin.
RESULTS: We enrolled 58 women: mean aged 43
7 years, mostly
white (n ¼ 46; 79%) and BV negative (n ¼ 43; 74%). By species-
specific quantitative PCR, 55 (95%) had UGT colonization with at
least 1 species (n ¼ 52) or were positive by 16S PCR (n ¼ 3). The most
common species were L iners (45% UGT, 61% vagina), Prevotella spp
(33% UGT, 76% vagina) and L crispatus (33% UGT, 56% vagina).
Median quantities of bacteria in the UGT were lower than vaginal levels
by 2-4 log10 ribosomal ribonucleic acid gene copies per swab.
There were no differences in the endometrial inflammatory markers
between women with no bacteria, Lactobacillus only, or any BV-
associated species in the UGT.
CONCLUSION: Our data suggest that the endometrial cavity is
not sterile in most women undergoing hysterectomy and that the
presence of low levels of bacteria in the uterus is not associated
with significant inflammation.
Key words: endometritis, endometrium, intrauterine bacteria, repro-
ductive tract microbiota, sterile, upper genital tract infection, uterine
cavity

Cite this article as: Mitchell CM, Haick A, Nkwopara E, et al. Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women. Am J Obstet
Gynecol 2015;212:611.e1-9.

B acterial colonization of the uterus is

associated with adverse reproduc-
tive health outcomes, including preterm
delivery and chorioamnionitis,1 pelvic
inflammatory disease and endome-
tritis,2,3 and miscarriage.4 Upper genital
tract infection has been presumed to be
due to the pathological ascent of vaginal
bacteria in to the upper genital tract. The
physical barrier of cervical mucous,
its high concentrations of antimicrobial
peptides and inflammatory cytokines,5-9
and possibly immunoglobulins10 or
matrix-degrading enzymes11 in the mu-
cous plug is thought to provide a defense
against bacterial ascent, and the uterine
cavity of healthy women has long been
considered sterile.

From the Departments of Obstetrics and Gynecology (Drs Mitchell and Eschenbach, Ms Haick, Ms Nkwopara, and Ms Agnew) and Pathology (Drs Garcia
and Rendi), University of Washington School of Medicine, and Vaccine and Infectious Diseases Division, Fred Hutchinson Cancer Research Center
(Dr Fredricks), Seattle, WA.
Received Oct. 1, 2014; revised Nov. 1, 2014; accepted Nov. 24, 2014.
Dr Mitchell is now with the Vincent Center for Reproductive Biology, Massachusetts General Hospital, Boston, MA.
This work was supported by grants from the National Institute of Allergy and Infectious Diseases (1K08AI087969-01) and the University of Washington
Royalty Research Fund (both to C.M.).
The views expressed herein are those of the authors, and neither funding source had any role in collection, analysis, presentation, or decision to publish
the data.
The authors report no conflict of interest.
Presented in part at the 39th annual meeting of the Infectious Diseases Society for Obstetrics and Gynecology, Whistler, BC, Canada, Aug. 9-11, 2012.
Corresponding author: Caroline M. Mitchell, MD, MPH. caroline.mitchell@mgh.harvard.edu
0002-9378/$36.00  ª 2015 Elsevier Inc. All rights reserved.  http://dx.doi.org/10.1016/j.ajog.2014.11.043

MAY 2015 American Journal of Obstetrics & Gynecology 611.e1

Research Gynecology
However, radioactively labeled albu-
min spheres placed in the vagina ascend
into the uterus as early as 2 minutes after
instillation,12 suggesting that fluid and
particles move between the vagina and
uterus relatively freely. Studies of osten-
sibly healthy women report a variable
rate of uterine bacterial colonization by
culture, ranging from 0% to 82%.13-22
This wide range is due in part to the
differences in sample collection: studies
using hysterectomy or transfundal sam-
pling had lower rates (0-24%)13-16,22
compared with those using trans-
cervical sampling (33-82%).17,18,21
Many studies using molecular char-
acterization of the microbiota have
demonstrated the ubiquitous presence of
bacteria throughout the body and their
influence on health.23,24 We hypothe-
sized that bacterial colonization of the
upper genital tract may be quite com-
mon and not pathological in many cases.
We undertook this study to assess the
prevalence and concentrations of bacte-
ria in the upper genital tract (UGT) us-
ing sensitive molecular methods in
sterilely sampled hysterectomy speci-
mens. Additionally, we measured the
endometrial immune response to de-
termine whether intrauterine bacterial
colonization was associated with epi-
thelial inflammation, which could sug-
gest an adverse effect of the bacteria.
M ATERIALS AND M ETHODS
Study cohort and sample collection
Women undergoing hysterectomy for
noncancer indications were eligible.
Exclusion criteria included presence of
an intrauterine device, use of antibiotics,
endometrial biopsy, intrauterine device
removal or hysteroscopy in the past 30
days, or concern for cervical or endo-
metrial neoplasia. Total laparoscopic or
laparoscopically assisted vaginal hyster-
ectomy specimens were collected only if
the surgeon was able to complete the
procedure using a noninvasive vaginal
fornix delineator (Colpo-Probe; Cooper
Surgical, Trumbull, CT) or a vaginal
sponge stick rather than an intracervical
manipulator.
The University of Washington Human
Subjects Division approved the study.
All subjects signed informed consent.
611.e2 American Journal of Obstetrics & Gynecology MAY 2015
All patients received standard preopera-
tive antibiotic prophylaxis at least 30
minutes prior to surgery.
Prior to vaginal examinations or
preparation, flocked swabs (Copan Di-
agnostics Inc, Murrieta, CA) were
inserted 3-4 cm into the vagina for 5
seconds. One was smeared on a glass
slide for Gram stain and Nugent
scoring.25 The uterus was removed,
wrapped in a sterile towel, taken to pa-
thology without fixation, and incised
sagitally under sterile conditions,
beginning at the fundus. Swabs were
collected first from the endometrium
and then from the upper endocervix by
rolling the swab 2-3 times across the
epithelium and frozen at e80 C.
In a subset of participants (n ¼ 30,
52%), swabs were collected in the Port-
A-Cul anaerobic system (Beckton,
Dickinson and Co; Franklin Lakes, NJ)
and cultured in standard fashion, in-
cluding selective broth to allow growth
of mycoplasma species and isolates
identified by routine biochemical
methods. Tissue sections were collected
from the endometrium contralateral to
the swab collection, cut into 1  1 cm
blocks, placed in RNALater (Life Tech-
nologies, Grand Island, NY) at 4 C for
24 hours, and then placed at e80 C.
Bacterial polymerase chain reaction
assays
Frozen swabs were thawed and 400 mL
of phosphate-buffered saline added,
mixed by a vortex shaker for 1 minute,
then the swab removed, and the sample
spun at 17,000  g for 10 minutes (all at
4 degrees). The pellet underwent DNA
extraction with the MoBio Bacteremia
DNA isolation kit (MoBio, Carlsbad,
CA), whereas the supernatant was ali-
quoted and frozen for Luminex analysis.
DNA underwent taxon-directed 16S ri-
bosomal ribonucleic acid (rRNA)
gene TaqMan format quantitative poly-
merase chain reaction (qPCR) assays for
the following bacterial species: Lactoba-
cillus crispatus, L jensenii, L iners, Gard-
nerella vaginalis, Atopobium vaginae,
Megasphaera genus, Prevotella genus,
bacterial vaginosiseassociated bacte-
rium (BVAB)-1, BVAB2, BVAB3, and
ajog.org
an assay detecting two closely related
bacteria (Leptotrichia and Sneathia).26,27
For the Prevotella genus assay, the
forward primer 384F (50 - GC CTG AAC
CAG CCA AGT A-30 ), reverse primer
513R (50 - GGA ATT AGC CGG TCC
TTA TT-30 ), and a taxon-specific probe
(6FAM-GTG CAG GAI GAC GGC
C-MGBNFQ) were used. The thermo-
cycler program (ABI 7500 Thermocycler;
Applied Biosystems, Foster City, CA) was
2 minutes at 50 C, 10 minutes at 95 C,
and then 45 cycles of 15 seconds at 95 C,
39 seconds at 59 C, and 30 seconds at
72 C. UGTswabs were also tested using a
broad-range 16S rRNA gene assay to
assess for the presence of any bacteria.
Limits of detection for the assays were as
follows: L crispatus 75 gene copies/swab,
L jensenii 125 gene copies/swab, all other
species-specific assays 150 gene copies/
swab, and broad-range 16S 6400 gene
copies/swab.28 Negative assays were
assigned a value of half the lower limit of
detection for that assay.
Measurement of cytokines,
chemokines, and antimicrobial
peptides
Supernatant from endometrial swabs
was submitted for Luminex analysis
(Luminex Corp, Austin, TX). Seven of
the 14 analytes (interleukin [IL]-4, IL-
10, IL-17, interferon-g, interferon-a,
tumor necrosis factor-a, macrophage
inflammatory protein-1a) were unde-
tectable in more than 95% of the
samples and were not included in
the final analysis. An enzyme-linked
immunosorbent assay for human beta
defensin (HBD)-2, HBD3 (Alpha Di-
agnostics International, San Antonio,
TX) and human alpha defensins 1-3
(Hycult Biotech, Plymouth Meeting,
PA) was performed. Homogenized
endometrial tissue sections underwent
ribonucleic acid (RNA) extraction using
the RNEasy fibrous tissue kit (QIAGEN
Inc, Valencia, CA). RNA was reverse
transcribed using the iScript cDNA
synthesis kit (Bio-Rad Laboratories,
Waltham, MA) and amplified using
primers and probes from Applied Bio-
systems for HBD2, HBD3, cathelicidin,
and IL-1b as well as the housekeeping
gene b-actin.
ajog.org
Statistical analysis
All analyses were performed using Stata
(version 10; StataCorp, College Station,
TX). Prevalences were compared be-
tween groups using the c2 test. Quanti-
ties of bacteria and concentrations of
cytokines were not normally distributed
so were compared across groups using
Wilcoxon rank-sum or Kruskall Wallis
tests.
R ESULTS
Cohort
We enrolled 58 women with mean age
of 43
7 years. Participants were pri-
marily white (n ¼ 46; 79%), with a small
proportion of African American (n ¼ 6;
10%) and Hispanic (n ¼ 4; 7%) women
(2 declined to answer the question). All
underwent hysterectomy for benign
disease: primarily bleeding (n ¼ 20;
34%), fibroids (n ¼ 15; 26%), and pain
(n ¼ 17; 29%). Most had a normal
Nugent score (n ¼ 43; 74%), whereas
6 (10%) had bacterial vaginosis, 7 (12%)
had an intermediate score, and 2 (3%)
could not be scored. Most (37; 64%)
were on no hormonal medications, 5
(9%) were taking oral contraceptives,
13 (22%) were using Lupron, and 2
(3%) were using a different hormonal
medication (testosterone or hormone
replacement therapy).
Eight women (14%) reported being
menopausal. Only 37 of 50 (74%) pre-
menopausal women provided informa-
tion about their last menstrual period
(LMP). The median number of days
since LMP was 28 (interquartile range
[IQR], 12e64), and of the 24 women
reporting less than 40 days since their
LMP, only 8 (33%) were in the first
14 days of their cycle. Most women had
never douched (n ¼ 32; 55%) or
douched more than 1 week prior to
surgery (n ¼ 10; 17%), with a minority
who had douched within the past week
(n ¼ 2; 3%) and 14 (24%) who did not
answer the question. Nineteen women
(33%) reported sexual intercourse in the
week prior to surgery.
UGT colonization
By species-specific qPCR, 55 women
(95%) had UGT colonization (ie, in the
Gynecology Research
endometrium or upper endocervix)
with at least 1 of the assayed species (n ¼ FIGURE 1
52) or were positive by broad-range 16S Distribution of upper genital tract
PCR (n ¼ 3). The most commonly bacterial colonization
detected species in the vagina were Pre-
votella spp (76%) L iners (61%), and L
crispatus (56%). These were also the
most commonly detected species in the
UGT: L iners (45%), Prevotella spp
(33%), and L crispatus (33%) (Figure 1,
A). G vaginalis, A vaginae, and L jensenii
were detected in the vagina in more than
40% of the women but were detected less
frequently in the UGT (in 19%, 10%,
and 20%, of women). Mean quantities
of bacteria detected in the UGT were
lower than levels in the vagina by 2e4
log10 rRNA gene copies (Figure 1, B).
When detected in the vagina, A
vaginae was the least likely species to
also be detected in the UGT, whereas
BVAB1 and L iners were the most likely
(Figure 1, C). The mean vaginal quantity
of L crispatus and G vaginalis was
significantly higher in women who had
UGT colonization with those species: 7.7

1 vs 5.5
2.5 gene copies/swab for
L crispatus (P ¼ .006) and 7.8
1.2 vs
4.9
1.5 gene copies/swab for G vagi-
nalis (P < .001).
There were no significant differences
in vaginal quantity between women with
and without UGT colonization with
other bacteria (data not shown). The
median number of species detected in
the UGT was 2 (IQR, 1e3; range, 0e8),
whereas the median number of species
detected in the vagina was 3 (IQR, 2e5;
range, 0e9). There was no correlation
between number of species detected in
the vagina and the UGT (correlation
coefficient, 0.21, P ¼ .12). Of note, in
several cases an organism present in A, Proportion of participants with detection of
the UGT was not present in the vagina bacteria by species-specific qPCR in the vagina
(Figure 2). alone, both vagina and UGT, and UGT alone.
Because almost all women had at least B, Comparison of the mean quantity of bacteria in
1 species detected in the UGT, we were the vagina and UGT of women with either vaginal or
unable to evaluate the risk factors for upper genital tract detection of that species.
colonization in general. We divided Asterisk indicates comparisons that are significantly
women into those with no bacteria different (P < .05) by Student t test. C, Proportion of
detected in the UGT (n ¼ 3), Lactoba- women with vaginal detection of a given species by
cillus species only (n ¼ 18; 31%), any qPCR who also had UGT detection of that species.
non-Lactobacillus species (n ¼ 34; 59%), qPCR, quantitative polymerase chain reaction; UGT, upper genital
tract.
and 16S positive only (n ¼ 3). The only Mitchell. Intrauterine bacterial colonization. Am J Obstet
demographic difference between these Gynecol 2015.
groups was race: UGT colonization with
MAY 2015 American Journal of Obstetrics & Gynecology 611.e3
Research Gynecology ajog.org

FIGURE 2
Comparison of vaginal and upper genital tract detection of bacteria

10,000-1,000,000
> 1,000,000
100-10,000
0-100
Nugent score - - 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 2 0 0 2 0 0 0 0 1 0 0 1 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 5 4 6 4 4 4 6 8 8 7 8 8 8 L. crispatus
L. jensenii
L. crispatus ## ## # # # # # # # # # # # # # # # # 0 0 0 0 0 # # # # # # # 0 0 0 0 0 0 0 0 0 0 0 0 0 # # # 0 0 0 0 # # # 0 0 0 L. iners
L. jensenii ## ## # # # # # # # # # # 0 # 0 # 0 # # # # # # 0 0 0 0 0 0 # # # # 0 0 0 0 0 0 0 0 0 0 0 # 0 # 0 0 0 0 0 0 0 0 0 G. vaginalis
L. iners ## ## # # # # # # # # # # # 0 # 0 # 0 # # # # # 0 0 0 0 0 0 0 0 0 0 # # # # # # # # # 0 0 # 0 0 0 # 0 0 # 0 # 0 0 A. vaginae
G. vaginalis 0 ## # 0 # 0 # # # 0 0 0 0 # 0 # 0 0 # 0 # 0 0 # 0 0 0 0 0 # 0 # 0 # # # # # 0 # 0 0 0 0 0 # # 0 0 0 0 # # # # # # # Megasphaera
Vagina

A. vaginae 0 0 0 # 0 # # # # 0 0 0 # 0 0 0 0 # 0 0 0 0 # 0 0 0 0 0 0 # 0 0 0 # # # 0 # 0 0 # 0 0 # # 0 0 0 0 # # # 0 # # # Leptotrichia/Sneathia
Megasphaera 0 0 # 0 0 0 0 # # 0 0 0 0 # 0 0 0 0 # 0 0 0 0 0 0 0 # 0 0 0 0 # 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # # 0 # 0 0 Prevotella 92 ## ## ##
Leptotrichia/Sneathia 0 0 # 0 0 0 0 0 # # 0 0 0 0 # # 0 0 # 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 # # 0 0 0 # # # 0 0 0 0 BVAB1 3 ## ## ##
Prevotella ## 0 # # # # # # # # 0 # # 0 0 # # # # # # # # # # 0 # 0 # 0 # # # # 0 # # # # # # 0 # # 0 # # # 0 # 0 # # # # 0 0 # BVAB2 10 ## ##
BVAB1 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 BVAB3 1 ## ## ##
BVAB2 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 # 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 # # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0
BVAB3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

L. crispatus ## 0 # # # 0 # # 0 0 0 0 # 0 0 0 # 0 0 0 0 0 0 # # # 0 0 # 0 0 # 0 0 0 # 0 0 0 0 # 0 0 0 0 # 0 0 # # # 0 0 0 0 0 0 0
L. jensenii ## 0 # 0 0 0 0 # # 0 0 0 0 # 0 0 0 # # 0 0 0 0 0 0 0 0 # 0 0 # # 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0
L. iners 0 0 # 0 0 # # 0 # 0 0 0 0 0 # 0 0 0 # 0 # # # # 0 # # # # 0 # 0 # # # # # # 0 # 0 # 0 0 0 # 0 0 0 # 0 0 0 0 0 0
G. vaginalis ## 0 # 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 # # 0 0 0 0 0 0 # 0 # # #
A. vaginae 0 0 # 0 0 0 0 0 0 # # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0
UGT

Megasphaera 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 # 0 # 0 0
Leptotrichia/Sneathia 0 0 # 0 0 0 0 0 0 # 0 0 0 0 # 0 # 0 # 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 # 0 0 # # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # # 0 0 0
Prevotella 0 92 # 0 # 0 0 0 0 # 0 0 0 0 0 0 # 0 0 # 0 0 # 0 0 0 0 0 0 0 0 # 0 # 0 0 0 # # 0 0 # # 0 # 0 0 # 0 # # 0 0 # 0 0 0 #
BVAB1 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
BVAB2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
BVAB3 0 0 0 0 0 0 0 0 0 0 0 0 0 # 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Detection and quantity of each species in the vagina and UGT for each participant, organized by the Nugent score. Each bacterium is represented by
a row, and the quantity is represented by a gradient of color, with darker colors representing higher quantities. The color gradients represent the
grouping of 1e100, 101e10,000, 10,000 to 1 million, and greater than 1 million 16S rRNA gene copies/swab. A white space means that the bacterium
was not detected in that sample.
rRNA, ribosomal ribonucleic acid; UGT, upper genital tract.
Mitchell. Intrauterine bacterial colonization. Am J Obstet Gynecol 2015.

a non-Lactobacillus species was more had bacteria detected by culture. Both in the UGT. The 1 woman with high
common in African American women women with negative qPCR results were (>100,000 gene copies/swab) of L iners
(5 of 6; 83%) and Hispanic women (3 of also negative by culture. The most in the UGT had relatively high levels
4; 75%) than white women (25 of 46; commonly cultured organisms were of several inflammatory markers but
54%) (P ¼ 0.01). Rates of bacterial Diphtheroids (n ¼ 15; 50%), followed by the lowest levels of gene expression
vaginosis were slightly different between anaerobic Gram-positive cocci (12; for the beta defensins, cathelicidin, and
these groups: 17% for African American, 40%), Proprionibacterium spp (n ¼ 9; IL-1b.
0% for Hispanic, and 11% for white 30%), and Lactobacillus species (n ¼ 8 When compared between women
women. species from 5 women; 17%) (Appendix; who had surgery for fibroids, bleeding,
There was a trend to increasing UGT Supplemental Table). pain, or other reasons, the only analyte
colonization by non-Lactobacillus spe- that was significantly different between
cies with increasing Nugent score: with Immune response the groups was IL-6: median, 6 pg/mL
Nugent score 0-3, the rate was 51% (22 Soluble markers of inflammation were (IQR, 1e28) in women having surgery
of 43), score of 4-6, 71% (5 of 7) and measured from endometrial swabs by for fibroids, 21.9 pg/mL (IQR, 9e154) in
score 7-10, 83% (5 of 6) (P ¼ 0.24). Luminex, antimicrobial peptides by an women having surgery for bleeding, 32
However, the 6 women with the highest enzyme-linked immunosorbent assay, pg/mL (IQR, 14e323) in women having
levels of non-Lactobacillus species and gene expression for defensins, surgery for pain, and 1 (IQR, 1e7.8).
detected in the UGT all had a Nugent cathelicidin, and IL-1b from tissue RNA, There was no difference in the distribu-
score less than 7. Age, menopausal sta- and results were compared between tion of women with only Lactobacillus
tus, treatment with a gonadotropin- women with no bacteria, only Lactoba- spp in the UGT vs non-Lactobacillus
releasing hormone agonist, gravidity, cillus species, or any non-Lactobacillus species between the surgical indications
parity, and douching or sex in the past species detected in the upper genital tract (data not shown).
week were not significantly different (Figure 3). There were no significant
between the groups (data not shown). differences in the median values of these C OMMENT
Of the subset of 30 women who also markers between groups. However, We detected UGT bacteria by PCR in
had cultures performed of upper genital the lowest quantities of beta-defensin 95% of women undergoing hysterec-
tract swabs, 28 (93%) had bacteria proteins seemed to be samples from tomy for benign gynecological condi-
detected by qPCR, and 26 of 30 (87%) women with non-Lactobacillus species tions. These results confirm the growing

611.e4 American Journal of Obstetrics & Gynecology MAY 2015

ajog.org Gynecology Research
consensus that the endometrial cavity
is not sterile. However, the quantity of FIGURE 3
bacteria present in the uterus and high Endometrial immune markers
endocervix was significantly lower than Lactobacillus Non-Lactobacillus
that in the vagina, suggesting that either None Low High Low High 16S only
the cervix serves as a partial filter to N 3 17 1 28 6 3
Elafin 1.29 1.03 2.06 0.74 0.98 2.45
ascent or that the endometrial immune
IL-1ra 0.29 1.53 0.29 1.14 2.09 0.76
response clears bacteria that do ascend IL-1a 1.17 2.67 3.33 0.67 2.33 0.67

Luminex
or a combination of both. IL-1b 1.14 1.43 33.00 0.57 1.29 0.43
We found a much higher prevalence IL6 0.33 0.44 0.83 1.22 1.00 0.06
of UGT colonization but less correlation IL-22 0.94 1.06 0.24 0.76 0.95 1.07
between vaginal and UGT samples than IL8 3.43 1.00 10.47 0.98 0.57 0.12
MCP-1 7.47 1.13 3.80 0.94 0.82 1.51
we anticipated. In women with vaginal
colonization by a given species, rates of HBD2 1.92 0.33 34.92 1.00 0.67 34.92

ELISA
UGT colonization varied widely, sug- HBD3 5.20 3.20 16.90 1.00 1.00 12.60
gesting differences in microbial ability HNP1-3 0.74 1.41 0.46 1.00 0.94 2.46
to evade cervical immunity or greater
permissiveness to some species. Many HBD2 0.82 0.91 0.82 1.00 1.27 1.27

groups have shown that the vaginal mi-
crobial community is dynamic.27,29 Our
results suggest that microbes may
remain in the UGT after they disappear
from the vagina and/or have better
growth in the UGT than the vagina.
Studies using a similar strategy of
incising a hysterectomy specimen to
collect samples but using culture to
identify bacterial colonization report
rates of intrauterine bacterial coloniza-
tion ranging from 0% among 10 women
from Finland22 to 31% in a cohort of
100 women from England.16 Studies
using transcervical sampling report
higher rates of intrauterine bacterial
colonization, ranging from 33%18 to
60%,17 but the degree of cervical or
vaginal contamination of the endome-
trial specimen is unknown.30
Our qPCR results from surgically
obtained samples suggest an even higher
rate of low-level bacterial presence in the
upper genital tract than culture-based
studies using transcervical sampling.
Many of the bacteria identified by qPCR
in this study, such as BVAB1-3 and
Leptotrichia/Sneathia, are fastidious and
difficult to culture, which may account
for the differences between our data
and previous reports. The bacteria we
identified by culture from a subset of
women include several taxa that have
been identified in vaginal communities
but were not targeted by our PCR
assays: Corynebacteria (Diptheroids),
Proprionibacteria, Ureaplasma, and
coagulase-negative Staphylococcus as well
qRT-PCR
HBD3 0.80 1.10 0.40 1.10 1.10 0.80
Cathelicidin 1.15 1.00 0.54 1.00 0.92 0.85
IL1b 1.38 1.13 0.50 1.00 1.38 1.00
Comparison of markers of the immune response in the upper genital tract between women with
no bacteria detected by PCR in the upper genital tract, only Lactobacillus species detected, or any
non-Lactobacillus species detected. Numbers in boxes are multiples of the median, calculated by
taking the median value for the whole cohort and dividing the individual group value by that number.
There were no significant differences between these 3 groups. Values highlighted in red are higher
than the group median, and those in blue are lower than the group median.
PCR, polymerase chain reaction.
Mitchell. Intrauterine bacterial colonization. Am J Obstet Gynecol 2015.
as several anaerobic colonies that could common vaginal species does not
represent any number of other common induce a strong inflammatory stimulus
vaginal species. All women in this study in most cases.
received preoperative antibiotics intra- This is an exploratory analysis with a
venously, which likely affected our cul- small sample size, which limits our
ture results. ability to detect small associations or to
Surprisingly, we saw few differences in perform well-powered subgroup ana-
endometrial immune markers between lyses to look at factors associated
women with and without upper genital with UGT colonization for different
tract colonization bacterial vaginosis- species. However, it is the first study
associated microbes. This could be using molecular methods to assess upper
due to trauma-induced cytokine release genital tract colonization in nonpreg-
at the time of surgical removal of the nant women.
tissue, but our median values are similar Our analysis is cross-sectional, which
to reported median values from endo- limits our ability to make conclusions
metrial aspirates in women with intact about causation or direction of associa-
uteri undergoing in vitro fertilization tions. However, opportunities to sterilely
procedures,31 suggesting this is not the collect endometrial samples with mini-
case. Alternatively, cytokines may have mal risk of contamination from the
an impact by hormonal status, the un- lower genital tract are becoming scarce
derlying pathology leading to hysterec- and preclude longitudinal sample
tomy, or by viral or fungal pathogens collection from the UGT. Changing
not measured in our study. Our data patterns of surgery mean that fewer
suggest that a low quantity of upper hysterectomies are being performed,
genital tract bacterial colonization by and many are now performed using

MAY 2015 American Journal of Obstetrics & Gynecology 611.e5

Research Gynecology
minimally invasive techniques in
which an intracervical manipulation
device is used. Intracervical instrumen-
tation could introduce an uncontrolled
amount of endometrial contamination,
so these cases were excluded.
Another limitation of our study is
the use of selective qPCRs, which do
not capture the entire microbiota.
However, we did not have sufficient
concentrations of bacterial DNA to
perform broad-range bacterial PCR with
pyrosequencing. We did obtain addi-
tional information using bacterial cul-
ture in some cases, but prophylactic
antibiotics potentially have an impact
on the sensitivity of cultures, and the
culture is not able to identify fastidious
bacterial species. Finally, because these
samples were collected from surgical
specimens, all participants had pathol-
ogy, and thus, the study population
may not reflect the conditions present
in normal, healthy women.
Recent advances in our understanding
of the human microbiome reveal the
important role that microbes play in
many facets of human health.32 The
microbiome plays an important role in
the immunological homeostasis of the
gut, encouraging proper development
of mucosal immunity and preventing
excessive inflammation (reviewed else-
where33). T-regulatory cells in the gut
mucosa maintain a tolerogenic envi-
ronment and appear to be selected by
interactions with commensal gut
microbiota.34 In the uterus, T-regulatory
cells are important for implantation of
the embryo and early placental devel-
opment.35,36 In one study, the presence
of hydrogen peroxideeproducing
Lactobacillus species on the tip of the
embryo transfer catheter for in vitro
fertilization increased the chance of live
birth compared with women who did
not have these bacteria detected by
culture.37
This finding raises the possibility that
the presence of intrauterine commensal
bacteria may have a similar role in the
selection of uterine T-regulatory cells
as commensal gut microbiota do for
colonic T-regulatory cells. Although
many other factors contribute to a suc-
cessful pregnancy, it is worth noting that
611.e6 American Journal of Obstetrics & Gynecology MAY 2015
germ-free mice raised in sterile condi-
tions have lower rates of reproductive
success after embryo transfer than con-
ventional animals,38 suggesting a po-
tential role for intrauterine bacteria in
successful pregnancy. This potentially
critical issue is largely unexplored.
It is clear that intrauterine bacterial
colonization is not always benign, or
positive. In patients undergoing
in vitro fertilization cycles, the presence
of Streptococcus viridans on the embryo
transfer catheter tip was associated
with a decreased chance of live birth
compared with women without S vir-
idans detected.37 Women with preterm
birth are more likely to have intrauterine
placental infection than women who
deliver at term (reviewed elsewhere39).
Women with significant inflammatory
sequelae in the upper genital tract
with pelvic inflammatory disease often
have anaerobic Gram-negative rods and
mixed communities of bacteria in the
uterus and fallopian tubes.3,40 Patho-
logical effects of intrauterine bacteria
may occur only with particularly virulent
strains or species, only with high con-
centrations of bacteria, or only in the
presence of a mixed bacterial commu-
nity at the endometrial surface.
In summary, these data indicate that
the endometrial cavity is not sterile in
most women undergoing hysterectomy
for benign indications. Additionally,
detection of bacteria in the upper
genital tract is not associated with a
significant inflammatory immune
response. Although bacterial concentra-
tions in the endometrium are much
lower than that in the vagina, a low-level
bacterial presence in the uterus appears
common and not pathological. -
ACKNOWLEDGMENTS
We acknowledge Xuezhou Hou, who developed
the Prevotella genus assay in Dr Fredricks’ lab-
oratory at the Fred Hutchinson Cancer Research
Institute as well as the surgeons in the Gyne-
cology Division at the University of Washington,
who facilitated the collection of specimens.
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611.e7
Research Gynecology ajog.org

A PPENDIX

SUPPLEMENTAL TABLE
Comparison of qPCR and culture results of women who had both assays
completed
UGT culture UGT qPCR
1015 Diphtheroids L iners
Prevotella spp
1016 — L crispatus
L iners
16S
1017 Coagulase negative staphylococcus L iners
Anaerobic GPC Prevotella spp
Leptotrichia/Sneathia
1018 Viridans streptococcus (2  102), L iners
Lactobacillus spp 16S
Anaerobic GPC
1026 Coagulase-negative staphylococcus L crispatus
Diphtheroids Prevotella spp
Propionibacterium spp 16S
Anaerobic GNR
Anaerobic GPC
1028 Diphtheroids L iners
Leptotrichia/Sneathia
16S
1032 Propionibacterium spp L iners
Coagulase-negative staphylococcus
1033 Propionibacterium acnes Prevotella spp
16S
1034 — —
1035 Coagulase-negative staphylococcus L iners
1037 — L crispatus
Prevotella spp
16S
1038 G vaginalis (105) G vaginalis
Lactobacillus (2 spp, each 103) Prevotella spp
Anaerobic GPC (102)
1044 Coagulase-negative staphylococcus L crispatus
Leptotrichia/Sneathia
Prevotella spp
16S
1046 Anaerobic GNR L jensenii
16S
1049 Proprionibacterium acnes A vaginae
Leptotrichia/Sneathia
Prevotella spp
BVAB1
16S
1050 Diphtheroids, L iners
Propionibacterium acnes Leptotrichia/Sneathia
Prevotella spp
Mitchell. Intrauterine bacterial colonization. Am J Obstet Gynecol 2015. (continued)

611.e8 American Journal of Obstetrics & Gynecology MAY 2015

ajog.org Gynecology Research

SUPPLEMENTAL TABLE
Comparison of qPCR and culture results of women who had both assays
completed (continued)
UGT culture UGT qPCR
1053 Lactobacillus spp #1 (9  10 ) 2
L jensenii
Lactobacillus spp #2 (103) L iners
Coagulase-negative staphylococcus G vaginalis
Diphtheroids 16S
Lactobacillus spp #3
Anaerobic GPC
1054 — —
1055 Lactobacillus spp (2  10 ) 2
Prevotella
Anaerobic GPR (2spp)
1056 Diphtheroids L crispatus,
Aerobic GPC Leptotrichia/Sneathia
1057 Diphtheroids (2 types) Leptotrichia/Sneathia
1060 Aerobic GPC L iners
Escherichea coli Prevotella spp
Coagulase-negative staphylococcus 16S
Diphtheroids
Anaerobic GPC
Anaerobic GNR
Anaerobic GNC
Anaerobic GPR
1061 Proteus spp (presumptive mirabilis) L jensenii
Diptheroids L iners
1062 Diphtheroids L crispatus
Propionibacterium spp L jensenii
Anaerobic GPR L iners
G vaginalis
BVAB2
1064 Diphtheroids G vaginalis,
Coagulase-negative staphylococcus Megasphaera spp
Anaerobic GPC (3 isolates) 16S
1066 Coagulase-negative staphylococcus (2 isolates) L crispatus
Anaerobic GNR
Anaerobic GPC
1067 Anaerobic GPR L crispatus
Lactobacillus spp
Diphtheroids
1071 Viridans streptococci Prevotella spp
Diphtheroids 16S
Anaerobic GPC
Anaerobic GPR
1073 Diphtheroids G vaginalis,
Propionibacterium spp (2 types) Propionibacterium A vaginae,
acnes Megasphaera spp
Anaerobic GPC Leptotrichia/Sneathia
Prevotella spp
16S
1075 Ureaplasma urealyticum 16S
Unless otherwise noted, all bacteria detected by culture were present at low levels (<102 cfu/swab).
GNC, Gram-negative cocci; GNR, Gram-negative rods; GPC, Gram-positive cocci; GPR, Gram-positive rods; qPCR, quantitative
polymerase chain reaction; UGT, upper genital tract.
Mitchell. Intrauterine bacterial colonization. Am J Obstet Gynecol 2015.

MAY 2015 American Journal of Obstetrics & Gynecology 611.e9