Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women




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)–associated 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 = 46; 79%) and BV negative (n = 43; 74%). By species-specific quantitative PCR, 55 (95%) had UGT colonization with at least 1 species (n = 52) or were positive by 16S PCR (n = 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 log 10 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.


Bacterial colonization of the uterus is associated with adverse reproductive health outcomes, including preterm delivery and chorioamnionitis, pelvic inflammatory disease and endometritis, and miscarriage. 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, and possibly immunoglobulins or matrix-degrading enzymes in the mucous plug is thought to provide a defense against bacterial ascent, and the uterine cavity of healthy women has long been considered sterile.


However, radioactively labeled albumin spheres placed in the vagina ascend into the uterus as early as 2 minutes after instillation, suggesting that fluid and particles move between the vagina and uterus relatively freely. Studies of ostensibly healthy women report a variable rate of uterine bacterial colonization by culture, ranging from 0% to 82%. This wide range is due in part to the differences in sample collection: studies using hysterectomy or transfundal sampling had lower rates (0-24%) compared with those using transcervical sampling (33-82%).


Many studies using molecular characterization of the microbiota have demonstrated the ubiquitous presence of bacteria throughout the body and their influence on health. We hypothesized that bacterial colonization of the upper genital tract may be quite common and not pathological in many cases. We undertook this study to assess the prevalence and concentrations of bacteria in the upper genital tract (UGT) using sensitive molecular methods in sterilely sampled hysterectomy specimens. Additionally, we measured the endometrial immune response to determine whether intrauterine bacterial colonization was associated with epithelial inflammation, which could suggest an adverse effect of the bacteria.


Materials and Methods


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 endometrial neoplasia. Total laparoscopic or laparoscopically assisted vaginal hysterectomy 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. All patients received standard preoperative antibiotic prophylaxis at least 30 minutes prior to surgery.


Prior to vaginal examinations or preparation, flocked swabs (Copan Diagnostics 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. The uterus was removed, wrapped in a sterile towel, taken to pathology 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 –80°C.


In a subset of participants (n = 30, 52%), swabs were collected in the Port-A-Cul anaerobic system (Beckton, Dickinson and Co; Franklin Lakes, NJ) and cultured in standard fashion, including 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 Technologies, Grand Island, NY) at 4°C for 24 hours, and then placed at –80°C.


Bacterial polymerase chain reaction assays


Frozen swabs were thawed and 400 μL 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 aliquoted and frozen for Luminex analysis. DNA underwent taxon-directed 16S ribosomal ribonucleic acid (rRNA) gene TaqMan format quantitative polymerase chain reaction (qPCR) assays for the following bacterial species: Lactobacillus crispatus , L jensenii , L iners , Gardnerella vaginalis , Atopobium vaginae , Megasphaera genus, Prevotella genus, bacterial vaginosis–associated bacterium (BVAB)-1, BVAB2, BVAB3, and an assay detecting two closely related bacteria ( Leptotrichia and Sneathia ).


For the Prevotella genus assay, the forward primer 384F (5′- GC CTG AAC CAG CCA AGT A-3′), reverse primer 513R (5′- GGA ATT AGC CGG TCC TTA TT-3′), and a taxon-specific probe (6FAM-GTG CAG GAI GAC GGC C-MGBNFQ) were used. The thermocycler 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. UGT swabs 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. 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-γ, interferon-α, tumor necrosis factor-α, macrophage inflammatory protein-1α) were undetectable 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 Diagnostics 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 Biosystems for HBD2, HBD3, cathelicidin, and IL-1β as well as the housekeeping gene β-actin.


Statistical analysis


All analyses were performed using Stata (version 10; StataCorp, College Station, TX). Prevalences were compared between groups using the χ 2 test. Quantities of bacteria and concentrations of cytokines were not normally distributed so were compared across groups using Wilcoxon rank-sum or Kruskall Wallis tests.




Results


Cohort


We enrolled 58 women with mean age of 43 ± 7 years. Participants were primarily white (n = 46; 79%), with a small proportion of African American (n = 6; 10%) and Hispanic (n = 4; 7%) women (2 declined to answer the question). All underwent hysterectomy for benign disease: primarily bleeding (n = 20; 34%), fibroids (n = 15; 26%), and pain (n = 17; 29%). Most had a normal Nugent score (n = 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%) premenopausal women provided information about their last menstrual period (LMP). The median number of days since LMP was 28 (interquartile range [IQR], 12–64), 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 = 32; 55%) or douched more than 1 week prior to surgery (n = 10; 17%), with a minority who had douched within the past week (n = 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 endometrium or upper endocervix) with at least 1 of the assayed species (n = 52) or were positive by broad-range 16S PCR (n = 3). The most commonly detected species in the vagina were Prevotella 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 2–4 log 10 rRNA gene copies ( Figure 1 , B).




Figure 1


Distribution of upper genital tract bacterial colonization

A, Proportion of participants with detection of bacteria by species-specific qPCR in the vagina alone, both vagina and UGT, and UGT alone. B, Comparison of the mean quantity of bacteria in the vagina and UGT of women with either vaginal or upper genital tract detection of that species. Asterisk indicates comparisons that are significantly different ( P < .05) by Student t test. C, Proportion of women with vaginal detection of a given species by qPCR who also had UGT detection of that species.

qPCR , quantitative polymerase chain reaction; UGT , upper genital tract.

Mitchell. Intrauterine bacterial colonization. Am J Obstet Gynecol 2015 .


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 = .006) and 7.8 ± 1.2 vs 4.9 ± 1.5 gene copies/swab for G vaginalis ( 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, 1–3; range, 0–8), whereas the median number of species detected in the vagina was 3 (IQR, 2–5; range, 0–9). There was no correlation between number of species detected in the vagina and the UGT (correlation coefficient, 0.21, P = .12). Of note, in several cases an organism present in the UGT was not present in the vagina ( Figure 2 ).




Figure 2


Comparison of vaginal and upper genital tract detection of bacteria

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 1–100, 101–10,000, 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 .


Because almost all women had at least 1 species detected in the UGT, we were unable to evaluate the risk factors for colonization in general. We divided women into those with no bacteria detected in the UGT (n = 3), Lactobacillus species only (n = 18; 31%), any non- Lactobacillus species (n = 34; 59%), and 16S positive only (n = 3). The only demographic difference between these groups was race: UGT colonization with a non- Lactobacillus species was more common in African American women (5 of 6; 83%) and Hispanic women (3 of 4; 75%) than white women (25 of 46; 54%) ( P = 0.01). Rates of bacterial vaginosis were slightly different between these groups: 17% for African American, 0% for Hispanic, and 11% for white women.


There was a trend to increasing UGT colonization by non- Lactobacillus species with increasing Nugent score: with Nugent score 0-3, the rate was 51% (22 of 43), score of 4-6, 71% (5 of 7) and score 7-10, 83% (5 of 6) ( P = 0.24). However, the 6 women with the highest levels of non- Lactobacillus species detected in the UGT all had a Nugent score less than 7. Age, menopausal status, treatment with a gonadotropin-releasing hormone agonist, gravidity, parity, and douching or sex in the past week were not significantly different between the groups (data not shown).


Of the subset of 30 women who also had cultures performed of upper genital tract swabs, 28 (93%) had bacteria detected by qPCR, and 26 of 30 (87%) had bacteria detected by culture. Both women with negative qPCR results were also negative by culture. The most commonly cultured organisms were Diphtheroids (n = 15; 50%), followed by anaerobic Gram-positive cocci (12; 40%), Proprionibacterium spp (n = 9; 30%), and Lactobacillus species (n = 8 species from 5 women; 17%) ( Appendix ; Supplemental Table ).


Immune response


Soluble markers of inflammation were measured from endometrial swabs by Luminex, antimicrobial peptides by an enzyme-linked immunosorbent assay, and gene expression for defensins, cathelicidin, and IL-1β from tissue RNA, and results were compared between women with no bacteria, only Lactobacillus species, or any non- Lactobacillus species detected in the upper genital tract ( Figure 3 ). There were no significant differences in the median values of these markers between groups. However, the lowest quantities of beta-defensin proteins seemed to be samples from women with non- Lactobacillus species in the UGT. The 1 woman with high (>100,000 gene copies/swab) of L iners in the UGT had relatively high levels of several inflammatory markers but the lowest levels of gene expression for the beta defensins, cathelicidin, and IL-1β.




Figure 3


Endometrial immune markers

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 .


When compared between women who had surgery for fibroids, bleeding, pain, or other reasons, the only analyte that was significantly different between the groups was IL-6: median, 6 pg/mL (IQR, 1–28) in women having surgery for fibroids, 21.9 pg/mL (IQR, 9–154) in women having surgery for bleeding, 32 pg/mL (IQR, 14–323) in women having surgery for pain, and 1 (IQR, 1–7.8). There was no difference in the distribution of women with only Lactobacillus spp in the UGT vs non- Lactobacillus species between the surgical indications (data not shown).




Results


Cohort


We enrolled 58 women with mean age of 43 ± 7 years. Participants were primarily white (n = 46; 79%), with a small proportion of African American (n = 6; 10%) and Hispanic (n = 4; 7%) women (2 declined to answer the question). All underwent hysterectomy for benign disease: primarily bleeding (n = 20; 34%), fibroids (n = 15; 26%), and pain (n = 17; 29%). Most had a normal Nugent score (n = 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%) premenopausal women provided information about their last menstrual period (LMP). The median number of days since LMP was 28 (interquartile range [IQR], 12–64), 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 = 32; 55%) or douched more than 1 week prior to surgery (n = 10; 17%), with a minority who had douched within the past week (n = 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 endometrium or upper endocervix) with at least 1 of the assayed species (n = 52) or were positive by broad-range 16S PCR (n = 3). The most commonly detected species in the vagina were Prevotella 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 2–4 log 10 rRNA gene copies ( Figure 1 , B).


May 6, 2017 | Posted by in GYNECOLOGY | Comments Off on Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women
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