The relationship between alterations to the lactobacilli-dominated vaginal flora and reproductive disorders is well established. Overgrowth of anaerobic and facultative bacteria in the vagina, or bacterial vaginosis, and the increase in proinflammatory cytokines, is implicated as a risk factor for the development of infectious and obstetric complications.1–6
Evidence for protective microbial communities, specifically those of the gastrointestinal tract, has led to investigations of the role played by the human microbiome in health and well-being.7,8 Such research includes the identification of a biodiverse urinary microbiome that is not isolated by conventional culture.9 Similar to the influence of microbial communities in the vagina and gastrointestinal tract, alterations to the urinary microbiome may have an effect on the development of lower urinary tract dysfunction.
Characterized by suprapubic pain and urinary frequency, interstitial cystitis is a debilitating condition with an estimated prevalence of 3–8 million U.S. women.10 The etiology of interstitial cystitis remains elusive, treatment is variable and often unsatisfactory,11 and affected individuals exhibit higher rates of psychiatric diagnoses and other chronic pain conditions.12,13 Although an inflammatory component of interstitial cystitis has been suggested, little is known regarding the possible role played by the urinary microbiome and its effect on the immune response.14,15
Our primary outcome was to investigate measurable differences in the urinary microbiota and cytokine levels that exist among women with interstitial cystitis compared with healthy women. Furthermore, we sought to determine whether the presence of particular bacteria or cytokines was associated with worsening symptom severity or screening positive for psychiatric comorbidities.
MATERIALS AND METHODS
The institutional review board of Northwestern University approved this cross-sectional study. Female patients, 21 years and older, presenting to the Women's Integrated Pelvic Health Program and diagnosed with interstitial cystitis were approached for participation. The diagnosis of interstitial cystitis was based on symptoms of urinary frequency and suprapubic or bladder pain for at least 6 months. Exclusion criteria included recurrent or current urinary tract infection, urethral strictures, iatrogenic cystitis, bladder augmentation, autoimmune disorder, neuromuscular disease, nonskin cancer, high-grade cervical dysplasia, current pregnancy, or current antibiotic use. Women in a control group were recruited from the same institution among women without genitourinary or pelvic floor complaints, age-matched to within 3 years, and met all exclusion criteria.
After obtaining consent, demographic information was collected and participants were administered validated questionnaires. The Pain Disability Index and Catastrophizing Scale describe the degree to which pain disrupts activities and affects catastrophic thinking, respectively. The Urinary Distress Inventory addresses bother associated with urinary symptoms. Effect on sexual function was obtained with the Female Sexual Function Index. Specific to interstitial cystitis, the Female Genitourinary Pain Index, Interstitial Cystitis Symptom Index, and Interstitial Cystitis Problem Index outlined the degree of experienced symptoms as well as the extent to which they were felt to be problematic. Finally, the Beck Depression and Anxiety Inventories screened for severity of associated symptoms.
A transurethral catheterized urine sample was obtained, transferred to the laboratory, and centrifuged. Supernatant was isolated for cytokine analysis leaving an intact pellet. Both samples were then stored at −80°C.
Bacterial DNA was isolated from the pellet and ribosomal RNA libraries were amplified with polymerase chain reaction. Sequences were generated using Illumina MiSeq technology and filtered for contaminants per standard protocols.16,17 Using the bioinformatics program QIIME, sequences were aligned against an established database to identify the bacterial families and genera and clustered into bins, or operational taxonomic units, based on similarity.18 Sequences were also assigned a taxonomic lineage using proprietary Resphera Insight technology.19 A commercially available immunoassay using antibody detection was used to identify the presence of an established panel of cytokines.20
Statistical analysis was completed using SPSS 19. The Wilcoxon rank-sum and Fisher exact tests were used to compare continuous and categorical variables, respectively. Multiple regression analysis was used as appropriate to determine relative associations. Spearman rank correlation was used to describe the relationship between continuous variables. Results were considered significant with a P value of ≤.05.
Between November 2013 and April 2014, 40 participants (20 patients, 20 women in the control group) were enrolled. Median age was 34 years (range 21–65 years) and the majority of women were premenopausal (75%) and nulliparous (68%). There were no differences in demographic data between the two groups (Table 1).
Participants diagnosed with interstitial cystitis scored significantly higher on all symptom severity indices (Table 2). Not only did they report higher levels of genitourinary and interstitial cystitis-specific pain, but also reported a greater degree of disability and catastrophizing related to pain symptoms. They were more likely than healthy women to meet the score criteria for mild anxiety (P=.019) and mild depression (P=.008). Additionally, participants with interstitial cystitis trended toward lower scores on the Female Sexual Function Index, indicating more sexual dysfunction compared with women in the control group (P=.05), and were more likely to have dysfunction related to pain (P=.032). These associations remained significant after controlling for age, menopausal status, and use of hormone therapy or oral contraceptives.
Initial analysis of the sequenced ribosomal RNA amplicons resulted in a normalized data set with 13 patients and 18 women in the control group and identified 54 different operational taxonomic units and 37 different bacterial genera. QIIME output is included in Appendix 1 (available online at http://links.lww.com/AOG/A922), which illustrates the relative abundance of identified genera and operational taxonomic units. Cases were not dominated by a common genus. Sequences specific to Lactobacillus species were observed in 5 of 13 (38%) patients and 14 of 18 (78%) women in the control group (P=.06). Participants from whom lactobacilli were isolated in their urine scored lower on the Interstitial Cystitis Symptoms Index (P=.005), Interstitial Cystitis Problem Index (P=.007), and the Female Genitourinary Pain Index (P=.03) (Table 3).
The median number of distinct operational taxonomic units found among the urine samples was three (range 20, interquartile range 4). Fewer distinct operational taxonomic units were found among patients compared with women in the control group (2, interquartile range 1 compared with 3.5 interquartile range 5.25, P=.015).
Species-level data were available for 14 patients and 18 women in the control group. Again, no specific taxa with a higher prevalence among patients were identified. However, the lactobacillus genus enrichment in women in the control group was supported with certain species of lactobacillus identified. Specifically, Lactobacillus acidophilus was observed in 1 of 14 (7%) patients compared with 7 of 18 (39%) control samples (P=.05, odds ratio [OR] 0.12). An additional lactobacillus assignment (operational taxonomic unit) not distinguished to the species level was found in 0 of 14 patients and 6 of 18 (33%) of women in the control group (P=.023, OR 0.07). Participants with evidence of L acidophilus in their urine scored lower on the Interstitial Cystitis Symptoms Index (1.3, interquartile range 1.25) compared with those without (7, interquartile range 12.25, P=.01). Similarly, Female Genitourinary Pain Index scores were lower among patients with L acidophilus (0, interquartile range 1.25) compared with those without (17, interquartile range 36, P=.04).
Cytokine analysis identified 25 of the 41 tested cytokines. The panel of included cytokines is presented in Supplemental Digital Content 1. On cytokine analysis, patients with interstitial cystitis demonstrated higher levels of macrophage-derived chemokine (P=.037) and interleukin-4 (IL-4) (P=.029) (Table 4). On bivariate analysis, higher levels of IL-4 correlated with higher scores on the Interstitial Cystitis Symptoms Index (r=0.406, P=.008) and the Pain Disability Index (r=0.302, P=.05). After controlling for the diagnosis of interstitial cystitis, the only significant association that remained was between IL-4 and the Interstitial Cystitis Symptoms Index (P=.013). There was no association between the presence of lactobacillus genera and cytokine levels (Table 5).
This investigation of the urinary microbiome demonstrated differences in the urinary microbiome of women with and without a clinical diagnosis of interstitial cystitis. Our findings suggest the possibility of a protective role played by a more diverse, lactobacillus-dominated urinary microbiome because the urine of women with interstitial cystitis was found to have fewer distinct operational taxonomic units and was less likely to contain Lactobacillus species, specifically L acidophilus, than that of healthy women. Furthermore, the presence of Lactobacillus species was associated with improved scores on two interstitial cystitis-specific symptom severity indices, suggesting that the urinary microbiome may influence lower urinary tract symptoms. It is unclear whether these associations could be mediated by an inflammatory component because no differences were found between the presence of Lactobacillus species and cytokine levels in the urine.
The appreciation of varied urinary microbiomes among healthy and symptomatic women has been implicated in other analyses.21,22 Pearce et al21 evaluated the microbiota of catheterized urine samples from 60 women with urgency urinary incontinence compared with healthy women and found that the urgency incontinence group had fewer lactobacillus sequences compared with women in a control group. Specific to bladder pain syndromes, a Norwegian study compared sequences of clean catch urine samples from eight women with interstitial cystitis to healthy women.23 Although their analysis noted a relative increase in lactobacilli in the urine of women with interstitial cystitis, our study used straight catheterized specimens, which better reflect the urinary microbiome than voided specimens.9 It is plausible that their collection technique affected the analyzed microbial composition. Furthermore, knowledge regarding the protective benefits of lactobacillus species, specifically L acidophilus, in the vaginal microenvironment supports a similar relationship in the lower urinary tract.24
In our study, urine samples from participants with interstitial cystitis had less microbial diversity, implicating that a “healthy microbiome” may be a more diverse microbiome. This is substantiated by a recent analysis among women with urgency urinary incontinence in which increased symptom severity was associated with less microbial diversity.22
Our finding of relative increases in two proinflammatory cytokines among symptomatic women correlates with other studies supporting the role of urinary cytokines in syndrome development.14,15 Although these studies did not find an association with macrophage-derived chemokine or IL-4, the number of specific cytokines evaluated in each study was small (seven and three, respectively) and cytokine levels were not correlated with symptom severity. Biologically, macrophage-derived chemokine and IL-4 are active in the inflammatory response. We know that the human microbiota influences varied conditions, including obesity, asthma, irritable bowel syndrome, hypertension, and multiple sclerosis, by regulating the immune inflammatory response, because many of these diseases are linked to a reduction in bacteria that produce chemicals to suppress inflammation.8 Thus, anti-inflammatory bacteria such as lactobacillus species may be important for sustaining well-being and could play a role in the development and severity of interstitial cystitis.
This study has several limitations. The cross-sectional design prevents distinction of differences as causal or resulting from epiphenomena. Given the use of a convenience sample, we did not establish an a priori sample size and thus our study may have been underpowered to detect differences among some bacterial communities, cytokine levels, and associations with questionnaire data. Furthermore, several of the sequencing reads were not interpretable as a result of incomplete bacterial lysis or primer bias. Such loss of data is inherent in sequencing techniques, and our rate of 80% is in line with, if not improved on, other recent studies.9,21 Finally, we know that the vaginal microbiome temporarily varies depending on hormonal fluctuations.25,26 Our single-sample acquisition does not account for changes related to these fluctuations, the effect of transient systemic bacteremia, or prior antibiotic use.
Despite these limitations, we were able to detect a difference in several important and biologically plausible variables. Similar to the effect of vaginal dysbiosis (an imbalance of the microbiota) on pathologic states,27 it is plausible that a dysbiotic urinary microbiome, mediated through a specific immune response, may be associated with certain urinary symptoms and pelvic floor disorders. Our study highlights the need to fully characterize a healthy urinary microbiome and represents a step toward better understanding of lower urinary tract symptoms and the potential development of more targeted therapies.
1. Li J, McCormick J, Bocking A, Reid G. Importance of vaginal microbes in reproductive health. Reprod Sci 2012;19:235–42.
2. Taylor BD, Darville T, Haggerty CL. Does bacterial vaginosis cause pelvic inflammatory disease? Sex Transm Dis 2013;40:117–22.
3. Gallo MF, Macaluso M, Warner L, Fleenor ME, Hook EW, Brill I, et al. Bacterial vaginosis, gonorrhea, and chlamydial infection among women attending a sexually transmitted disease clinic: a longitudinal analysis of possible causal links. Ann Epidemiol 2012;22:213–20.
4. Gillet E, Meys JF, Verstraelen H, Bosire C, De Sutter P, Temmerman M, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis. BMC Infect Dis 2011;11:10.
5. Giakoumelou S, Wheelhouse N, Cuschieri K, Entrican G, Howie SE, Horne AW. The role of infection in miscarriage. Hum Reprod Update 2016;22:116–33.
6. Laxmi U, Agrawal S, Raghunandan C, Randhawa VS, Saili A. Association of bacterial vaginosis with adverse fetomaternal outcome in women with spontaneous preterm labor: a prospective cohort study. J Matern Fetal Neonatal Med 2012;25:64–7.
7. Silva MJ, Carneriro MB, dos Anjos Pultz B, Pereira Silva D, Lopes ME, dos Santos LM. The multifaceted role of commensal microbiota in homeostasis and gastrointestinal disease. J Immunol Res 2015;2015:321241.
8. Althani AA, Marei HE, Hamdi WS, Nasrallah GK, El Zowalaty ME, Al Khodor S, et al. Human microbiome and its association with health and diseases. J Cell Physiol 2016;231:1688–94.
9. Wolfe AJ, Toh E, Shibata N, Rong R, Kenton K, Fitzgerald M, et al. Evidence of uncultivated bacteria in the adult female bladder. J Clin Microbiol 2012;50:1376–83.
10. Berry SH, Elliott MN, Suttorp M, Bogart LM, Stoto MA, Eggers P, et al. Prevalence of symptoms of bladder pain syndrome/interstitial cystitis among adult females in the United States. J Urol 2011;186:540–4.
11. Hanno PM, Burks DA, Clemens JQ, Dmochowski RR, Erickson D, Fitzgerald MP, et al. Diagnosis and treatment of interstitial cystitis/bladder pain syndrome. J Urol 2011;185:2162–70.
12. Suskind AM, Berry SH, Suttorp MJ, Elliott MN, Hays RD, Ewing BA, et al. Health-related quality of life in patients with interstitial cystitis/bladder pain syndrome and frequently associated comorbidities. Qual Life Res 2013;22:1537–41.
13. Nickel JC, Tripp DA, Pontari M, Moldwin R, Mayer R, Carr LK, et al. Interstitial cystitis/painful bladder syndrome and associated medical conditions with an emphasis on irritable bowel syndrome, fibromyalgia and chronic fatigue syndrome. J Urol 2010;184:1358–63.
14. Tyagi P, Killinger K, Tyagi V, Nirmal J, Chancellor M, Peters KM. Urinary chemokines as noninvasive predictors of ulcerative interstitial cystitis. J Urol 2012;187:2243–8.
15. Lamale LM, Lutgendorf SK, Zimmerman MB, Kreder KJ. Interleukin-6, histamine and methylhistamine as diagnostic markers for interstitial cystitis. Urology 2006;68:702–6.
16. Jumpstart Consortium Human Microbiome Project Data Generation Working Group. Evaluation of 16S rDNA-based community profiling for human microbiome research. PLoS One 2012;7:e39315.
17. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 2012;6:1621–4.
18. Kuczynski J, Stombaugh J, Walters WA, Gonzalez A, Caporaso JG, Knight R. Using QIIME to analyze 16S rRNA gene sequences from microbial communities. Curr Protoc Bioinformatics 2011;Chapter 10:Unit 10.7.
19. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 2007;73:5261–7.
20. Multiplex map human cytokine/chemokine magnetic bead panel—immunology multiplex assay. Available at: https://www.emdmillipore.com
. Retrieved November 18, 2016.
21. Pearce MM, Hilt EE, Rosenfeld AB, Zilliox MJ, Thomas-White K, Fok C, et al. The female urinary microbiome: a comparison of women with and without urgency urinary incontinence. MBio 2014;5:e01283–14.
22. Karstens L, Asquith M, Davin S, Stauffer P, Fair D, Gregory WT, et al. Does the urinary microbiome play a role in urgency urinary incontinence and its severity? Front Cell Infect Microbiol 2016;6:78.
23. Siddiqui H, Lagesen K, Nederbragt AJ, Jeansson SL, Jakobsen KS. Alterations of microbiota in urine from women with interstitial cystitis. BMC Microbiol 2012;12:205.
24. Martin R, Soberón N, Vaneechoutte M, Flórez AB, Vázquez F, Suárez JE. Characterization of indigenous vaginal lactobacilli from healthy women as probiotic candidates. Int Microbiol 2008;11:261–6.
25. Chaban B, Links MG, Jayaprakash TP, Wagner EC, Bourque DK, Lohn Z, et al. Characterization of the vaginal micriobiota of healthy Canadian women through the menstrual cycle. Microbiome 2014;2:23.
26. Gajer P, Brotman RM, Bai G, Sakamoto J, Schütte UM, Zhong X, et al. Temporal dynamics of the human vaginal microbiota. Sci Transl Med 2012;4:132ra52.
27. Kovachev SM. Obstetric and gynecological diseases and complications resulting from vaginal dysbacteriosis. Microb Ecol 2014;68:173–84.
Supplemental Digital Content
© 2017 by The American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.