International Urology and Nephrology (2019) 51:1677–1697 https://doi.org/10.1007/s11255-019-02225-y

UROLOGY - REVIEW

The impact of microbiome in urological diseases: a systematic review

Joseph K. M. Li1 · Peter K. F. Chiu1 · Chi‑Fai Ng1

Received: 1 June 2019 / Accepted: 4 July 2019 / Published online: 12 July 2019 © Springer Nature B.V. 2019

Abstract Objective The term microbiome is used to signify the ecological community of commensal, symbiotic, and pathogenic microorganisms that share our body space, in which there were increasing evidences to suggest that they might have poten- tial roles in various medical conditions. While the study of microbiome in the urinary system is not as robust as the systems included in the Project, there are still evidences in the literature showing that microbiome may have a role in urological diseases. Therefore, we would like to perform a systematic review on the topic and summarize the avail- able evidence on the impact of microbiome on urological diseases. Methodology This review was performed according to the Preferred Reporting Items for Systematic Review and Meta- analysis (PRISMA) statement. After screening 589 abstracts and including additional studies (such as references from review papers), 76 studies were included for review and discussion. Results Studies had suggested that there were correlations of microbiome of diferent body cavities (e.g., fecal, urinary and seminal fuid) with urological diseases. Also, diferent diseases would have diferent microbiome profle in diferent body cavities. Unfortunately, the studies on the association of microbiome and urological diseases were still either weak or inconsistent. Conclusion Studies suggested that there might be some relationship between microbiome and various urological diseases. However, further large-scale studies with control of confounding factors should be performed under a standardized method- ology in order to have better understanding of the relationship. Also, more standardized reporting protocol for microbiome studies should be considered for better communications in future studies.

Keywords Microbiota · Urology · Prostate cancer · Urolithiasis · Lower urinary tract symptom

Introduction The Human Microbiome Project (HMP) was started since 2007 and one of the aims of HMP was to characterize the Our human body is inhabited by at least ten times more human microbiome and its infuence on disease [6]. Five than the number of human cells in the body [1], body sites (the gastrointestinal tract, the mouth, the vagina, and the term “microbiome” was initially used to signify the the skin and the nasal cavity) were chosen for kickof of the ecological community of commensal, symbiotic, and patho- HMP, yet the urinary tract was not chosen because urine genic microorganisms that literally share our body space [2]. was considered sterile. The study of microbiome in the uri- Currently, the term microbiome has various defnitions, with nary tract started in male urethral microbiome in patients some groups limiting its use to the genes of bacteria, and with and without sexually transmitted infection [7]. Later, other groups refer this to the metagenome, i.e., all genetic the study of microbiota in urine by culture-independent [8, materials of a population including plasmids [3]. In turn, 9] and culture-dependent [10, 11] methods was started and microbiota was defned as the microorganisms in a particular disproved the dogma of urine being sterile. environment [4, 5]. It was noted that the microbiota plays an important role in disease development and progression in diferent parts of the body. For example, infammatory bowel disease, * Chi‑Fai Ng Clostridium difcile-associated colitis and colorectal cancer [email protected] are associated with gut dysbiosis. However, the association 1 SH Ho Urology Centre, Department of Surgery, The Chinese of dysbiosis and urological diseases is not well studied in the University of Hong Kong, Shatin, Hong Kong

Vol.:(0123456789)1 3 1678 International Urology and Nephrology (2019) 51:1677–1697 literature, with mainly observational studies or cohort stud- overactive bladder syndrome, urinary incontinence, infer- ies with small number of subjects involved. Therefore, the tility”. The initial search identifed 589 studies. Additional aim of this review is to give urologists an overview of how manuscripts, such as those referenced by reviews, were also microbiome is associated with urologic diseases by sum- reviewed (Fig. 1). marizing the available evidence of the role of microbiota in urologic disease and outlining the future prospects for microbiome studies for urologic disease. Sample collection, microorganism detection and data analysis Methodology It is well known that diferent collection methods will reveal diferent microbiota even within the same environment. Rec- A systematic literature search was performed using Pub- tal swab will be diferent from the stool sampling, which will Med and Medline databases from inception until April 2019. also be diferent from the mucosal biopsy. Therefore, it is Papers written in English were selected following the Pre- very important to identify the methods for sample collection ferred Reporting Items for Systematic Review and Meta- before we use the results from the studies. analysis (PRISMA) statement. A fowchart of the Systematic search process is shown in Fig. 1. The following keywords Urine samples were included in this systematic review: “microbiome” or “microbiota” in combination with “prostate cancer, blad- der cancer, kidney cancer, urolithiasis, urinary tract stone, For urine samples, the gold standard is suprapubic aspira- Oxalobacter formigenes, urinary tract infection, bladder pain tion (SPA) of urine because it will avoid the contamination syndrome, painful bladder syndrome, interstitial cystitis, from the urethra and external genitalia. There are studies chronic pelvic pain syndrome, lower urinary tract syndrome, showing that catheterized urine has similar microbiota with urine sampling from SPA, and mid-stream clean catch urine (MSU) has microbiota similar to that of the external geni- talia [8]. Although there is a higher chance of contamina- n Records identified tion by external genitalia, MSU has the advantage of being through Pubmed and Medline searching convenient and less invasive, when compared to SPA and (n=589) catheterized urine. Identificatio Microorganism detection

g Records screened by Records excluded title and abstract (n=447) (n=589) Microorganisms can be detected by culture-dependent meth-

Screenin ods and culture-independent methods. Culture-dependent methods include those of standard culture, which are used to identify common uropathogens, and expanded quantitative Full-text articles Full-text articles urine culture (EQUC), which aims to detect organisms that accessed excluded (n=142) (n=99) are difcult to be cultured in standard culture using a greater Eligibility volume of urine, incubation in varied conditions, and the use of extended incubation times [11]. Culture-independent methods include the sequencing of the 16S ribosomal RNA Studies included by Additional studies (rRNA), which has highly conserved regions with 9 difer- database search included (n=42) (n=35) ent variable regions (V1–V9). By detecting the diference in

d the variable regions, diferent genus of the bacteria can be identifed. The disadvantage of culture-independent methods

Include is that they can only tell the bacteria has been present, but Studies included in cannot tell whether the bacteria are viable. Also, the use systematic review of diferent variable regions cannot be compared directly; (n=77) therefore, if two similar studies are using diferent variable regions in identifying bacteria, the results can be diferent. Results from 16S rRNA sequencing and EQUC can be Fig. 1 Flow diagram of evidence acquisition in a systematic review complementary for the identifcation of bacteria in urine, on role of microbiome in urological diseases instead of being mutually exclusive [12].

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Analysis of microbiome data pathways of natural B-vitamins may provide a reasonable preventive measure for prostate cancer, echoing the study Diversity of the microbiome is used to standardize the result from Golombos et al. that micronutrient may have a role in and to measure the diferences between two groups. Alpha- the prevention of prostate cancer. and beta-diversities are two commonly used parameters. Apart from the looking into the pathogenesis of pros- Alpha-diversity measures the species richness within a fne tate cancer, the efect of treatment on gut microbiome was and homogeneous extent, whereas beta-diversity measures evaluated by Sfanos et al. [19], in which they found that men the extent of species composition among diferent commu- without prostate cancer had a more diverse . nities [13]. The gut microbiota of patients taking oral androgen recep- tor axis-targeted therapies had enriched steroid/hormone biosynthesis pathway and more abundant Akkermansia Malignant urological conditions muciniphila, which is involved in the treatment response to anti-PD-1 in patients with epithelial tumors. The rising interests in relating microbiome with diferent In addition to the gut microbiome, prostate tissue micro- aspects of cancer management started since the populari- biome is also a focus for the study of pathogenesis. Studies zation of the next generation sequencing (NGS). Focuses have shown that there is also a community of bacteria in the of the feld are how the microbiota contribute to the can- tissue of prostate cancer using NGS [20–22]. They found no cer development and progression, and also how it afects signifcant diference between the composition of microbi- the treatment of cancer [14]. In this section, we will also ome between cancer and benign tissue [21, 22]. However, summarize the evidence available in the literature on how these studies identifed possible associations of specifc microbiota afects the pathogenesis and treatment of com- bacteria with prostate cancer. For example, there is gradual mon urological cancer. increase in the richness of Streptococcus and decrease in the richness of Staphylococcus from tumor, peri-tumour to non- Prostate cancer tumor tissue [21], and Pseudomonas infection is negatively associated with the risk of metastasis [22]. The risk of prostate cancer is shown to be associated with Urinary microbiome was also studied using urine sam- obesity and infammation [15]. As well, certain foods and ple after prostatic massage. The two studies showed no sig- nutrients may be associated with increased risk of prostate nifcant diferences in microbiota composition. However, cancer [16]. The above risk factors are also associated with many species were signifcantly and diferentially abundant the gut microbiome [17] and, therefore, a hypothesis of the among cancer and non-cancer samples. For example, Veil- diference of gastrointestinal microbiome between healthy lonella, Streptococcus, and Bacteroides were more abundant men, those with latent prostate cancer, and those with inva- in patients with prostate cancer, whereas Faecalibacterium, sive prostate cancer was made [16]. Recently, there is evi- Lactobaccili, and Actinectobacter were less abundant [23]. dence to support the above hypothesis. Golombos et al. [18] Conversely, a cluster of pro-infammatory microbials (e.g., have shown that there are more Bacteroides massiliensis, Streptococcus anginosus, Actinobaculum schaalii and Pro- and less Faecalibacterium prausnitzii and Eubacterium rec- pionimicrobium lymphophilum) contained more cancer sam- talie in the gut microbiota of patients with prostate cancer ples than samples outside this cluster [24]. when compared with that of patients with benign prostate. They also suggested that butyrate, an anti-infammatory Bladder cancer micronutrient produced by a range of bacteria including F. prausnitzii and E. rectalie, can be one of the pathways for The use of Bacillus Calmette–Guérin (BCG, live-attenuated the prevention of prostate cancer (Table 1). Mycobacterium bovis) in the treatment of non-muscle inva- In another study, Liss et al. [15] have shown that despite sive bladder cancer was long before the study of urinary there is overlap of gut bacterial community between patients microbiome. Until recently, a preliminary report of the uri- with and without prostate cancer, there were altered meta- nary microbiome in eight patients with bladder cancer by bolic pathways. The authors have derived a microbiome Xu et al. from their mini-review revealed that there were score from 10 diferent metabolic pathways and showed diferences of urinary microbiome between normal subjects statistical signifcant diferences between patients with and and patients with bladder cancer [25], although they did not without prostate cancer. Although statistically signifcant, include their methodology. They found that Streptococcus the microbiome score has an area under curve (AUC) of was signifcantly elevated in bladder cancer. Two other stud- only 0.64, which is suboptimal for a biomarker to look ies were found in the search of the association of bladder for patients at high risk for prostate cancer. On the other cancer with urinary microbiome. Bučević Popović et al. hand, authors suggested that further studies into metabolic presented a negative result which showed that the diversity

1 3 1680 International Urology and Nephrology (2019) 51:1677–1697 between malignant between and benign tissue PCR of Akkerman - sia muciniphila was derived was No comparisonNo made Included quantitative Included quantitative Microbiome score score Microbiome Remarks - - - - ADT): mone biosynthesis, cafeine metabolism and glycosaminogly can degradatoin hydrate metabolism hydrate pathways min production biosynthesis super class in carbohy involved acid and fatty drate, lipid, and aminoacid biosynthesis and metabolism metabolisms and biosynthesis glycan N/A Oral ATT group (vs no group (vs ATT Oral enriched steroid/hor Prostate cancer group: Prostate abundance in carbo - B-vita natural - lacking Prostate cancer group: Prostate enriched carbohydrate Benign group: pathways enriched for enriched in energy Functional profling Functional tissue by V4: tissue by Pseudomonadaceae, Coma - Pseu - monadaceae, Ralstonia, domonas, Acinetobacter V2-3: tissue by coccaceae, Staphylococcus, - Propi Moraxella, Escherichia, - acnes and Strep onibacterium pseudopneumoniae tococcus nospiraceae iphila ADT)—less: - , Strepto Erysipelotrichaceae coccaceae cus iensis , Eubacterium prausnitzii rectale ‘Core’ community of prostate community of prostate ‘Core’ Esherichia, Enterobacteriaceae, community of prostate ‘Core’ - Strepto Enterobacteriaceae, Oral ATT group—more: ATT Oral - , Lach Ruminococcaceae genus— species- Akkermansia mucin - no (vs ADT Men receiving , Brevibacteriaceae family— Prostate cancer group—more: Prostate - Streptococ Bacteroides, genus— Prostate cancer group—more: Prostate massil - species— Bacteroides Benign group—more: species— Faecalibacterium Relevant microbials Relevant diference diference N/A Signifcant No signifcant No N/A Beta-diversity - - diference cant difer ence N/A Signifcant No signif - No N/A Alpha-diver sity sequencing sequencing (V2–V3 and V4 regoin) sequencing (V6 sequencing region) sequencing (V1, sequencing V2 regions) - sequenc Shotgun ing 16S rDNA 16S rDNA 16S rDNA 16S rDNA Microbial identif - Microbial cation 16S rDNA 16S rDNA Whole-genome Whole-genome tissue Prostate Prostate Rectal swab Rectal Sample Rectal swab Rectal Fecal swab Fecal - prostatec radical tomy the same speci - men prostate or prostate cancers, prostate no who have medicaations or or ADT having ​ ATT oral ing transrectal biopsy prostate patients undergone patients undergone Bengin tissue from Patients Prostate cancer Prostate Benign Men with benign - undergo Patients Summary of original articles cancer on prostate Agent Cancer, Cancer, Agent 2017 [ 20 ] Urology, Urology, 2018 [ 18 ] Prostate Cancer Prostate Dis, Prostatic 2018 [ 19 ] 2018 [ 15 ] Yow et al. Infect Infect et al. Yow 1 Table Study Golombos et al. Golombos et al. Sfanos et al. et al. Sfanos Liss et al. Eur Urol, Eur Urol, Liss et al.

1 3 International Urology and Nephrology (2019) 51:1677–1697 1681 analysis suggested suggested analysis associa - negative Pseu - tion between domonas infection and metastasis Transcriptome Transcriptome Remarks N/A N/A N/A N/A Functional profling Functional plasma urealyticum species contained cancer more thansamples those out of cluster: Anae - lactolyticus, rococcus - obesiensis, Actinob rococcus Varibaculum aculum schaalii, - cambriense, Propionimicro bium lymphophilum cus, Bacteroides baccili, Actinectobacter , Pseudomonas Acinetobacter Prostate cancer—more: Prostate - parvum, Urea Ureaplasma group of bacterialThis cluster anginosus, Anae - Streptococcus Prostate cancer—more: Prostate Bacteroides genus— Prostate cancer—more: Prostate - Streptococ genus—Veillonella, cancer—less Prostate - Lacto genus—Faecalibacterium, , , Propionibacterium Escherichia Tumor tissue—more: Tumor Staphylococcaceae family— Staphylococcus genus— tissue—less: Tumor order—Lactobacillales Streptococcaceae family— Streptococcus genus— Relevant microbials Relevant diference diference diference, diference, but trended cluster to separately diference diference No signifcant No No signifcant No No signifcant No No signifcant No No signifcant No Beta-diversity - - - - - cant difer ence cant difer ence cant difer ence cant difer ence No signif - No No signif - No No signif - No No signif - No N/A Alpha-diver sity sequencing (V6) sequencing sequencing (V3– sequencing V5) sequencing sequencing (V3– sequencing V5) 16S rDNA 16S rDNA 16S rDNA 16S rDNA Whole-genome Whole-genome 16S rDNA 16S rDNA Microbial identif - Microbial cation ples after DRE urine after prostate massage tissue tissue urine sam - rectal swab frst void void frst Prostate Prostate Sample Prostate Prostate prostate biopsy prostate transrectal MRI prostate targeted biopsy - prostatec radical tomy - prostatec radical tomy Patients undergoing undergoing Patients patients undergoing patients undergoing Patients undergoing undergoing Patients Patients Patients undergone undergone Patients (continued) Urol, 2018 [ 24 ] Urol, tate, 2018 [ 23 ] Genomics, 2019 [ 22 ] Urol, 2017 [ 21 ] Urol, Shrestha et al. J et al. Shrestha - Pros Alanee et al. Feng et al. BMC et al. Feng Study 1 Table Cavarretta et al. Eur et al. Cavarretta

1 3 1682 International Urology and Nephrology (2019) 51:1677–1697 and overall urinary microbiome composition were not sig- Limitations nifcantly diferent between patients with or without bladder cancer [26], whereas Wu and colleagues showed that there is There are common limitations to the studies of micro- increased richness, but not species diversity, in the bladder biome in malignant urological conditions, the studies of cancer group and in those with high risk of disease recur- which are usually having relatively small sample size, rence and progression according to the EORTC risk strati- unavailability of a standardized sample collection and fcation [27]. Also, there are signifcant diferences in the lack of longitudinal samples for the confirmation for the beta-diversity between the cancer and non-cancer subjects, cause–effect relationship. Further studies should include a as well as those with high risk and low risk of recurrence larger sample size with a standardized, longitudinal sam- and progression [27]. A point to note is that Bučević Popović pling of specimen from the gut, tissue and urine from et al. have found that Streptococcus was more abundant in human subjects. normal subjects [26], on the contrary with the preliminary results of Xu et al. Therefore, this signifes the importance Conclusion of mentioning the method of urine sampling and bacterial identifcation (Table 2). There is evidence that the microbiome may have a role in the pathogenesis of malignant urologic conditions. Further large-scale, longitudinal studies are required to identify a Treatment of malignant urological conditions causal relationship between cancer and the changes in micro- biome. After understanding the pathogenesis, it may provide In recent years, immunotherapy with immune check point a foundation to develop biomarkers for diagnosis and risk inhibitors like the PD-1/PD-L1 inhibitors became a hot topic stratifcation, novel treatments for cancer and regimes for in the treatment of malignant urological conditions, as it pro- modulating microbiota to help with treatment responses. vides a new treatment options with encouraging results with overall survival. However, the response rate of this thera- peutic option is suboptimal [28]. Biomarkers like PD-L1 expression and mutational load were suggested to predict Benign urological conditions the response of immunotherapy [29]. Routy et al. [30] have found that antibiotics given around Another large group of urological conditions are the the initiation of the immunotherapy reduced the efective- benign conditions, which afect the quality of life (QOL) ness of the treatment signifcantly, in which antibiotics is a of the patients. Better understanding of the pathophysiol- predictor of resistance to PD-1 blockade in renal cell carci- ogy of these diseases is useful for the treatment, which in noma. On the other hand, the response of PD-1 blockade was turn improves the QOL of the patient. restored after fecal microbiota transplantation of responder cancer patients into germ-free or antibiotic-treated mice. The authors also noted that Akkermansia muciniphila was Urolithiasis signifcantly associated with favorable clinical outcome, and the colonization of A. muciniphila in antibiotic-treated The role of microorganism in the formation of urinary stone mice reversed the non-responsiveness to PD-1 blockade. is well documented. It is well known that urea-splitting However, the presence of A. muciniphila does not impli- organisms, like Proteus species, can increase the pH of urine cate a good response, and a hypothesis suggesting that A. and, therefore, favor the formation of struvite stones, also muciniphila involves in only one of the multiple steps in the known as “infection stones”. Apart from being responsible cancer-immunity cycle [31]. This also suggested that gut creating an environment for stone formation, microbials microbiota is not a perfect biomarker for predicting response can also create an environment to treat stone disease [33] to immunotherapy, as variations of the gut microbiota could (Table 3). be associated with other factors, like the histological type of Not only the local microbiome afects the formation of tumor, environmental factors, study populations, or analyti- urinary stones, but also microbiome from the gut. Recent cal methods [32]. studies have shown that the imbalance of the gut microbiome Future studies on the involvement of gut microbiome in (gut dysbiosis) is associated with metabolic diseases such immunotherapy and the study of their functional pathway as obesity, hypertension, diabetes mellitus and metabolic could provide us with more insights into integrating gut syndrome. Coincidentally, these metabolic diseases are also microbiome into a composite of biomarker as well as devel- associated with the increased incidence of urinary stones. oping an adjuvant therapeutic option for modulating gut There were a few attempts in the literature trying to correlate microbiome to improve the response rate of immunotherapy. gut dysbiosis and urinary stone formation.

1 3 International Urology and Nephrology (2019) 51:1677–1697 1683 on high risk of recurrence and high risk- of pro gression Remarks Included analysis Included analysis domonas , Anaerococcus , , Facklamia Actinobaculum Campylobacter massil - nis , Actinobaculum anthropi iense , Jonquetella coccus , Corynebacterium , cristatus Streptococcus Corynebacterium appendicis rococcus, Rubrobacter, Sphin - Rubrobacter, rococcus, Atopostipes, gobacterium, Geobacillus - Ruminiclostrid Roseomonas, - - xylano ium - 6, Eubacterium philum Relavant microbials Relavant Bladder cancer—more: , Pseu - Streptococcus genus— Bladder cancer—more: , Fusobacterium Genera— homi - Species— Campylobacter Control—more: - , Strepto Veillonella Genera— dispar , Species— Veillonella Bladder cancer—more: Anae - Acinetobacter, genus— Control—more: , , Proteus Serratia genus— - ence bacterial profles Beta-diversity N/A No signifcant difer No Signifcantly diferent diferent Signifcantly bacterial richness in cancer patients Alpha-diversity N/A No signifcant diference No Signifcantly increased increased Signifcantly sequencing sequencing (V4) sequencing sequencing (V4) Microbial identi - Microbial fcation N/A 16S rDNA 16S rDNA 16S rDNA 16S rDNA Sample N/A MSU MSU patients tumor der cancer Patients Urothelial carcinoma carcinoma Urothelial individuals Healthy Non-muscle-invasive Non-muscle-invasive Control Male patients with blad - Male controls Summary of original articles on bladder cancer Urol, 2014 [ 25 ] Urol, Sci Rep, 2018 [ 26 ] Sci Rep, Infect Microbiol, Microbiol, Infect 2018 [ 27 ] 2 Table Study Xu et al. Am J Clin Exp et al. Xu Bučević Popović et al. et al. Popović Bučević Wu et al. Front Cell Front et al. Wu

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Stern et al. [34] were one of the frst groups in trying more protection to the prevention of stone disease [39]. With to characterise the diferences between gut microbiome of this in mind, it is not surprising that with prepara- kidney stone patients with that of non-stone formers. They tions including Oxalobacter, Lactobacillus and/or Bifdo- noted that kidney stone patients had more Bacteroides and bacterium species have been disappointing [40]. less Prevotella as compared with the control. The use of probiotics for the prevention of oxalate stone From an Indian human study, there were more Firmicutes formation can be further investigated, and the use of OMBS and Lachnospiraceae, and less Bacteroidetes, Prevotella as a dissolution treatment of formed stone should also be and Eubacterium in kidney stone patients [35]. The authors considered. explained that the diference in the abundance of Bacteroi- detes compared with Stern et al. was because of the recruit- Urinary tract infection ment of female, inclusion of mixed types of stones and stone formers with other disease-associated complications. The As urinary tract infection (UTI) involves bacteria, the micro- other reason for the diference may lie in sequencing the biome would certainly have a role in UTI. Therefore, we diferent variable region of the 16S rRNA. would just highlight some of the application of microbiome A more recent study recruited patients from Southern in UTI. Firstly, not using antibiotic in patients with asymp- China [36]. There were signifcant diferences in the com- tomatic bacteriuria is justifed by the fact that commensals position of gut microbiome between kidney stone patients have a protective efect on active UTI [41]. Secondly, NGS and those healthy controls. The study also showed that there can be used to identify the causative pathogen, as well as were signifcantly higher levels of pro-infammatory bacte- identify the resistant pattern to antibiotics [42, 43]. Lastly, ria, and a trend toward reduced diversity within samples in the use of probiotics is a prevention or treatment of recur- those with kidney stones. rent UTI, either by instilling a single strain into the bladder All the above studies were great pilot studies in the [44–46] or the whole community by fecal microbiota trans- understanding of the gut microbiome in kidney stone dis- plantation [47–49]. ease. However, there were common limitations, including small sample size and the lack of diet history. Further larger- Bladder pain syndrome scale studies with better control of confounding factors are required to characterize whether gut dysbiosis is associated The etiology of bladder pain syndrome (BPS; or interstitial with kidney stone disease. cystitis or painful bladder syndrome) remains unknown, but by defnition, it excludes the presence of infection, which it was traditionally described as chronic sterile bladder infam- Oxalate metabolizing bacterial species mation [50, 51] (Table 4). With the help of the 16S rRNA gene sequencing, it was As more than 70% of the calcium-containing renal stone is found that Lactobacillus was represented in a higher abun- made up with oxalate, the oxalate metabolism is explored dance in the MSU samples in patients with BPS, with a in hope to reduce the recurrence rate. Oxalate metabolizing decrease of microbial diversity when compared with healthy bacterial species (OMBS) in the gut microbiome is one of women [51]. Another recent study using the National Insti- the focuses in the oxalate metabolism, and they are divided tute of Health (NIH) Multidisciplinary Approach to the into specialist and generalist OMBS. Generalist OMBS, Study of Chronic Pelvic Pain (MAPP) Research Network like Lactobacillus, Bifidobacterium, Enterococcus and showed that the urine samples had less Corynebacterium Clostridium species, can utilize other compounds as carbon and more Lactobacillus gasseri in BPS patients [52]. One and energy source, whereas specialist OMBS, like Oxalo- of the limitations for the above studies was the use of MSU bacter formigenes, utilizes oxalate as the sole carbon and samples, which may have contamination from the vaginal energy source. microbiome. In contrast to the above studies, another study Oxalobacter formigenes is a Gram-negative, obligate using catheterized urine samples showed lower abundance of anaerobic bacterium frst discovered in 1985 [37]. It was Lactobacillus acidophilus, but similarly decreased microbial found that O. formigenes metabolises ingested oxalate and diversity [53]. Furthermore, they have investigated the pro- stimulate oxalate secretion from the colon. Healthy sub- infammatory cytokine levels in the urine and showed that jects have higher colonization rate than subjects with kid- it was higher in patients with BPS [53]. ney stones [35]. In subjects with kidney stones, those who Not only bacteria are studied in this disease, but also are colonized with O. formigenes have 70% reduced risk fungi and viruses. Nickel and colleagues have shown that of stone recurrence [38]. Having said that, a recent meta- there was no diference in the species composition, but a analysis showed that the role of a specifc OMBS may not be signifcantly greater prevalence of fungi in those with a fare that important and having a diverse gut microbiota provides in symptoms [54]. On the other hand, polyomaviruses and

1 3 International Urology and Nephrology (2019) 51:1677–1697 1685 - O formi for region be low; may genes - the use of Oxalo bacter specifc 16S primars gene rRNA 100% demonstrated 17% coloniza - vs tion of Oxalobacter formigenes Sensitivity of V1-3 Remarks diferent in kidney stone stone in kidney disease synthesis, glycan metabolism of and cofactors vitamins disease stone ney carbohydrate - metabolism, xeno degradation biotic and metabolism Not statistically statistically Not N/A Down regulated regulated Down metabolism, energy in kid - Up-regulated lipid metabolism, Functional profling Functional Erysipelotrichales Erysipelotrichaceae Erysipelatoclostridium, - unidentifed_Lachno - Phascolarcto spiraceae, Megamonas, bacterium, - Escheri Acinetobacter, Sutterella - Shigella, chia , lii _group, Dorea _5, Ruminiclostridium - , Fusicateni Anaerostipes , , Subdoligranulum bacter – ruminanEubacterium - tium _group, Holdemania , - , Ruminococ Dialister , cus _1, Parasutterella Biliphila more: TM7 teria, Faecalibacterium prausnitzii Nephrolithiasis—more: Pseudomonadales , Order— , Moraxellaceae Family— Alloprevotella, genus— nephrolithiasis—less: - Eubacterium_hal genus— Kidney stone patients— stone Kidney Bacteroides Control—more: Prevotella Kidney stone—more: Kidney Firmicutes, Proteobacteria, stone—less: Kidney - Cyanobac Bacteroidetes, only: Control , Dialister Prevotella Relavant microbials Relavant - - ences noted ences noted Signifcant difer Signifcant difer N/A Beta-diversity ference ference No signifcant dif - No N/A No signifcant dif - No Alpha-diversity sequencing sequencing (V4 region) sequencing sequencing (V4 region) sequencing sequencing (V3 region) 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA Microbial Microbial identifcation samples sample samples Fecal Fecal Fecal Fecal Fecal Fecal Sample patients patients ney stone diseased stone ney Nephrolithiasis Nephrolithiasis control Healthy Kidney stone stone Kidney Control Symtpomatic kid - control Healthy Patients Summary of original articles on urolithiasis asis, 2018 [ 36 ] asis, 2016 [ 34 ] Sci Rep, 2016 [ 35 ] Sci Rep, 3 Table - Urolithi et al. Tang - Stern Urolithi et al. Suryavanshi et al. et al. Suryavanshi Study

1 3 1686 International Urology and Nephrology (2019) 51:1677–1697 associated with less on scores severe questionnaires cystitis interstitial demonstrated of higher levels pro-infammatory cytokines was 1. L acidophilus was with 2. Patients Remarks N/A N/A N/A Functional profling Functional acidophilus (not statistically statistically (not gasseri signifcant) Pseudomonas mirabilis, Francisella Aeruginosa, Mycoplasma tularensis, helicpbacter hyorhinis, hepaticus, Clostridium Candida perfringens, dubliniensis reduced: - , Fusobacte Bacteroidetes ria , Actinobacteria Proteus, Atopbium, Cronobacter Interstitial cystitis—less: Interstitial species— Lactobacillus IC/BPS—less: Corynebacteriumgenera— IC/BPS—more: species— Lactobacillus IC/BPS only: coli, Proteus - Escherichia Interstitial cystitis—more: Interstitial Firmicutes phylum— Lactobacillales order— Lactobacillus genera— cystitis— Interstitial , Proteobacteria phylum— only: cystitis Interstitial Nitrospirae phylum— Sapropsiraceae order— Enterococcus, genera— Relavant microbials Relavant diference diference diference noted N/A No signifcant No Signifcant Beta-diversity ference signifcantly signifcantly diversity lower than healthy females N/A No signifcant dif - No Interstitial cystitis cystitis Interstitial Alpha-diversity ionization—time- of-fight—mass spectrometry sequencing sequencing (V1V2 and V6 regions) rRNA sequencing rRNA Electrospray Electrospray 16S rDNA 16S rDNA Microbial identif - Microbial cation - ized urine Catheter MSU MSU Sample col - Sample lection - - - stitial cystitis stitial stitial cystitis/ stitial bladder pain (IC/ syndrome BPS) stitial cystitis stitial Controls Female withFemale inter Controls Female withFemale inter Healthy female Healthy Female withFemale inter Patients Summary of original articles on bladder pain syndrome Obstet Gynecol, Obstet 2017 [ 53 ] Med, 2019 [ 52 ] BMC Microbiol, BMC Microbiol, 2012 [ 51 ] 4 Table Abernethy et al. et al. Abernethy Nickel et al. J Clin et al. Nickel Siddiqui et al. et al. Siddiqui Study

1 3 International Urology and Nephrology (2019) 51:1677–1697 1687

Epstein–Barr virus were noted in the bladder samples of patients with BPS, and both were thought to be linked with the pathogenesis of BPS [50]. It was noted that patients with BPS often have other con- ditions like irritable bowel syndrome, Sjogren’s syndrome

used to identify theused to DIPP species cystitis interstitial by biomarkers DIPP species and metabolites stool and mental disorders like depression. Yet, these conditions 1. Quantitative PCR 1. Quantitative 2. Stool-based Remarks were shown to have gut dysbiosis. On the other hand, in a study for biomarkers of BPS, authors have found reduced levels of some bacterial species in stool samples from patients with BPS [55]. Whether gut dysbiosis is associ- ated with BPS should be investigated, and results from trans-MAPP II study of Urologic Chronic Pelvic Pain functional pathways: thesis bination mide metabolism Signifcantly diferent diferent Signifcantly acid biosyn - - Fatty - - Homologous recom - - Nicotinate/nicotina Functional profling Functional (NCT02898220) are awaited.

Chronic pelvic pain syndrome in men

The etiology of chronic pelvic pain syndrome (CPPS) in men is unknown, and it is a syndrome that is diagnosed by exclusion of infection, anatomic abnormalities, malignant neoplasm, or neurological disorders [50]. Other than being cystitis Pelvic Pain (DIPP) Pain Pelvic cystitis species E. nitzii, C. aerofaciens, sinensis, L. longoviformis Defcient in Interstitial Defcient in Interstitial - praus F. - O. splanchnicus, Relavant microbials Relavant difcult to diagnose, it is also difcult to treat (Table 5). Although it was shown long ago that microorganisms was isolated from seminal fuid, there is no single causative microbial species that has been identifed as the main cause

diference of CPPS [56]. One of the frst studies of the microbiota of No signifcant No Beta-diversity seminal fuid in patients with CPPS defned the microbial communities in the seminal fuid of patients with CPPS and healthy men by decomplementary activity, and showed that the culprit could be normal commensals that were regarded as contaminants [56]. A more recent study compared the seminal microbiome in men with and without CPPS using N/A Alpha-diversity 16S rRNA gene sequencing, and they confrmed that seminal fuids were having polymicrobial communities. Furthermore, they demonstrated higher species diversity, as well as less Lactobacilli, in particular Lactobacillus iners, in patients with CPPS [57].

sequencing (V3– sequencing V5 regions) Nickel et al. compared the urinary microbiota of healthy 16S rRNA 16S rRNA Microbial identif - Microbial cation controls and patients with CPPS, and they found that the bacteria Burkholderia cenocepacia, Propionibacterium acnes and Staphylococcus capitis/caprae were more preva- inal swabs - ± vag Stool Sample col - Sample lection lent in the initial stream urine specimen in patients with CPPS [58]. In the era of NGS, 16S rRNA gene sequencing - showed that the microbiota of mid-stream urine in patients with CPPS has a higher bacterial diversity than in healthy controls, as well as having higher prevalence of anaerobic bacteria [59]. There are also signifcant diferences in the stitial cystitis stitial Healthy controls Healthy Female withFemale inter Patients microbiome in diferent symptom severity and clinical phe- notypes, and the authors expected an action of functional pathways by the diference in microbiome [59]. It is known that CPPS is associated with intestinal symp- (continued) toms [60], and the intestinal symptoms are also associated with gut dysbiosis. Shoskes et al. have shown that the gut ing et al. Sci Rep, Sci Rep, ing et al. 2016 [ 55 ] - Braundmeier-Flem 4 Table Study microbiome was less diverse and having less Prevotella

1 3 1688 International Urology and Nephrology (2019) 51:1677–1697 - subjects appeared subjects appeared (no be lower to tests statistical done) were signifcant difer the ences between groups two Diversity of healthy of healthy Diversity VB2/VB3 showed no VB2/VB3 showed Remarks CP/CPPS: controls: system lism N/A N/A Overexpressed in Overexpressed sporulation chemotaxis in overexpressed glycolysis phosphotransferase metabo - pyruvate N/A - profl Functional ing - - more: cilli holderia cenoce - - pacia, Propioni acnes, bacterium Staphylococcus capitis/caprae (in VB1): - ria, Propionibac - Staphylo terium, coccus detes - iales, Bacteroi dales Blautia, roides, - Faecalibacte rium,- Ruminococ cus, Coprococcus coccus lales, Bacillales terium comicrobia Porphyromonas healthy men— healthy - Lactoba genus— species— L. iners CP/CPPS—more CP/CPPS—more - Burkholde genus— species— Burk CP/CPPS—more: - Bacteroi phylum— Clostrid order— - - Bacte genus— Control—more: - Staphylo genus— - Lactobacil order— CP/CPPS—more: - Phyllobac genus— Control—more: Verruphylum— Prevotella, genus— - microbi Relavant als - - ence ence N/A N/A Signifcant difer Signifcant difer Beta-diversity in CP/CPPS group in CP/CPPS N/A N/A Statistically higher Statistically Statistically lower lower Statistically Alpha-diversity sequencing (V6 sequencing region) ionization—time- of-fight—mass spectrometry sequencing (V3 sequencing and V4 regions) sequencing (V3 sequencing and V4 regions) Microbial identif - Microbial cation 16S rDNA 16S rDNA Electrospray Electrospray 16S rDNA 16S rDNA 16S rDNA 16S rDNA - bation urine) urine)massage glove tip after glove - rectal examina tion in sterile saline Sample collection Sample Semen by mastur Semen by VB1 (initial stream VB1 (initial stream VB2 (MSU) VB3 (post-prostate MSU Immersing soiled CP/CPPS CPPS CPPS consulted for consulted for coml - prostatitis paints, fertility or status check prophylactic purposes Patients Male patients with controls Healthy Patients with CP/ Patients Controls Patients with CP/ Patients Controls Male patients Summary pain syndrome of original articles pelvic on chronic 2015 [ 58 ] ogy, 2016 [ 59 ] ogy, Urol, 2016 [ 61 ] Urol, Urol, 2017 [ 57 ] Urol, 5 Table Study Nickel et al. J Urol, J Urol, et al. Nickel - Urol et al. Shoskes Shoskes et al. J et al. Shoskes Mändar et al. Int J Mändar et al.

1 3 International Urology and Nephrology (2019) 51:1677–1697 1689

(which has an anti-infammatory role) in patients with CPPS symptoms, and increased diversity was associated with a [61]. To fght against gut dysbiosis, several strategy could lower frequency of Lactobacillus in hormone-negative been considered, like the use of bacterial therapy (live probi- women [67]. otic bacteria) and hydrocolon therapy, which aims to restore There are encouraging results from the above studies, a healthy gut microbiota by a gentle and targeted fow of but they all contained sequence-negative urine samples. water entering the intestine during the treatment sessions Karstens and colleagues have shown contradicting result and, therefore, reducing infammation [60]. with the previous studies, in which lower diversity of urinary The role of the microbiota of partners of patients of microbiome is associated with increased symptom sever- CPPS should also be taken into account. A study has shown ity in women with UUI [68]. The authors explained the that the presence of Gardnerella vaginalis as a predomi- contradicting result by the diference in small sample size, nant organism in the vaginal microbiota is associated with diferences in participant populations, diferences in urine the presence of leukocytospermia in the male partner [62]. sample volumes, and preprocessing/fltering techniques used Moreover, another study has revealed that the vaginal, cer- on the data. The diference in result could as well be due vical or urethral swab from the partners of patients sufer- to the older subjects. Although having only 19 subjects in ing from CPPS shows the same pathogen isolated in male this study, it has the advantage of using catheterized urine patients [60]. Further studies in associating the relationship for urine sampling and a higher urine volume for sequenc- between the genital microbiota of patients with CPPS and ing, which may be reasons for having all samples sequence their partners are warranted. positive. This study also refects that a standard protocol for the collection and extraction of bacterial DNA from urine is Lower urinary tract dysfunction important for future studies. Urgency urinary incontinence is not only a physical dis- Lower urinary tract symptom (LUTS) is a board term and ease; it also correlates with the psychological wellbeing describes the diferent symptoms associated with the dys- of the patients. Wu and colleagues found that the urinary function of the lower urinary tract. LUTS is one of the most microbiome of OAB patients was having a lower diversity commonly encountered complaints in the urology clinic and than normal controls, and OAB patients with depression it afects both male and female patients. Due to the difer- have further reductions in bacterial diversity and richness ence in anatomy, male patients usually complain of voiding [69]. Furthermore, they showed that some genera were dif- symptoms, whereas female patients more usually complain ferent between OAB patients with and without anxiety and of storage symptoms. Other than the diference in anatomy, depression. The authors suggested the existence of a brain- the male and female urinary microbiota are also noticed to bladder-microbiome axis and suggested further studies on be diferent, in which females are predominantly by Lacto- the axis. bacillus and males predominantly by Corynebacterium [9, The treatment response of oral medications for UUI can 63] (Table 6). be predicted from the pre-treatment urinary microbiome. Thomas-White et al. (from Brubaker’s group) showed that Female urinary microbiome and LUTS apart from the association with the UUI symptoms; urinary microbiome diversity is noted to be higher in patients UUI Overactive bladder syndrome (OAB) and urinary inconti- who do not respond to solifenacin [70]. nence (UI) are the most commonly seen lower urinary tract Despite encouraging results in associating urinary micro- symptoms in female patients. Urinary incontinence can be biome and OAB and UUI, it seems that there is no associa- urgency urinary incontinence (UUI), stress urinary inconti- tion between urinary microbiome and SUI symptoms [67], nence (SUI) or mixed. Overactive bladder syndrome is also presumably due to the diferent pathophysiology. associated with UUI, and studies have been performed to To sum up, whether the OAB symptoms is associated correlate urinary microbiome and OAB or UUI. with higher or lower diversity of urinary microbiome still Sequence-positive urine samples are shown to be associ- require further analysis as contradicting results were pre- ated with more severe symptoms [64–66], as well as younger sent. However, through these studies, we can conclude that age, higher body mass index (BMI), more UUI episodes at urinary microbiome is associated with OAB and UUI, and baseline, better treatment response and fewer post-treatment further study is required to determine the association and the UTI risk [65]. Moreover, two specifc bacterial species, clinical implications. Atopobium vaginae and Finegoldia magna, were associated with urinary symptom severity by the overactive bladder Male urinary microbiome and LUTS questionnaire, suggesting etiology implications of OAB symptoms [66]. The diversity of the urinary microbiome Although the urinary microbiota within the female urinary is greater in patients with higher BMI and increased UUI tract is better studied, the study in male patients is picking

1 3 1690 International Urology and Nephrology (2019) 51:1677–1697 positive and sequence- positive patients negative positive and sequence- positive patients negative positive and sequence- positive patients negative Compared sequence- Compared Compared sequence- Compared Remarks Compared sequence- Compared - adales, Chitinophaga, adales, Chitinophaga, Cadida - Brevundimonas, - Altero tus Planktoluna, monadaceae, Elizabethk ingia, Methylobacterium, Caldicellulosiruptor, Stenotrophomonas - monadaceae, Nocardi oides, Mycobacterium Gardnerella vaginalis, vaginalis, Gardnerella gasseri Lactobacillus score: more: Gardnerella UUI—more: Genus— Sphingomon - UUI—less: Coma - Genus— Prevotella, N/A UUI urinae, Aerococcus without LUTS crispatus Lactobacillus Relavant microbials Relavant higher OABq symptoms symptoms higher OABq vaginae Atopobium magna Finegoldia hormone-positive women— hormone-positive Genus— Lactobacillus, N/A N/A N/A Beta-diversity N/A N/A - ences between groups ences between symptoms 1. No signifcant difer 1. No 2. Reduced in greater 2. Reduced N/A N/A Alpha-diversity N/A Signifcant diference sequencing (V4 sequencing region) sequencing (V4 sequencing region) sequencing (V4 sequencing region) sequencing (V4 sequencing region) 16S rDNA 16S rDNA Quantitative PCR Quantitative Microbial identif - Microbial cation 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA 16S rDNA urine urine urine Catheterized Catheterized Sample collection Sample Catheterized Vaginal swab Vaginal perineal swab catheterized urine Clean catch or Clean catch catheterized urine to severe UUI severe to to severe UUI severe to POP/SUI surgery undergoing surgery undergoing Females with daily UUI withFemales daily Controls Patients Women with moderate Women Women with moderate Women Women undergoing undergoing Women Women with SUI Women Summary of original articles on female lower urinarySummary of original syndrome lower tract articles on female Cell Infect Microbiol, Microbiol, Cell Infect 2016 [ 68 ] gynecol J, 2014 [ 64 ] Obstet Gynecol, Obstet 2015 [ 65 ] cology J, 2018 [ 66 ] Am J Obstet Gynecol, Am J Obstet 2017 [ 67 ] Karstens et al. Front Front et al. Karstens 6 Table Study - Brubaker Int Uro et al. Pearce et al. Am J et al. Pearce - Int Urogyne et al. Fok Thomas-White et al. et al. Thomas-White

1 3 International Urology and Nephrology (2019) 51:1677–1697 1691 between the depression between and both bacterialscore richness and diversity patients in OAB was less in the “5 mg- was responding solifenacin to group” compared “10 mg-solifenacin- group”responding or “non-responding group” Negative correlations Negative Remarks Unique species isolated Unique - - phyromonas, Gallicola, phyromonas, - Alicyclo Leptolyngbya, bacillus, Helcococcus, Actinobactulum - Collinsella, Methanobre Agrobacterium, vibacter, Crycola, Burkholderia Pedobacter, WAL_1855D, Actinobaculum Staphylococcus, Proteus, Proteus, Staphylococcus, Helcococcus, Gemella, Aerococcus Mycoplasma, - Ente Novosphingobium, Ureaplasma, rococcus, - Lacto Pyramidobacter, bacillus, Anaerococcus, Campy Finegoldia, Jonquetella, lobacter, Dialister, Fusobacterium, Prevotella non-anxiety—more Genus— Psychrobacter, Non-depression—more Genus— Clostridium, OAB—more: Actinobacteria Phylum— Bifdobacteriaceae Family— Genus— Sneathia, Control—more: Genus— Lactococcus, Anxiety—more Por Genus— Sneathia, Relavant microbials Relavant diferences diferences present Signifcant Beta-diversity N/A - subjects cases ence 1. Lower in OAB in OAB 1. Lower in depression 2. Lower No signifcant difer No Alpha-diversity sequencing (V4 sequencing region) sequencing (V4 sequencing region) 16S rDNA 16S rDNA 1. 16S rDNA 1. 16S rDNA 2. EQUC Microbial identif - Microbial cation urine urine Sample collection Sample Catheterised Catheterized treatment Patients Patients with OAB Patients Controls Women seeking UUI Women Controls (continued) Infect Microbiol, Microbiol, Infect 2017[ 69 ] Int Urogynecology J, Int Urogynecology 2016 [ 70 ] 6 Table Study Wu et al. Front Cell Front et al. Wu Thomas-White et al. et al. Thomas-White

1 3 1692 International Urology and Nephrology (2019) 51:1677–1697 up. An early attempt in identifying urinary microbiome in male subjects was seen in the study by Nelson et al. [7], by comparing men with sexually transmitted infection (STI) and STI-negative individuals. In fnding the association between urinary microbiota and LUTS, Bajic et al. [71] showed that the severity of lower urinary tract syndrome (measured by

international prostate symptoms score (IPSS)) is associated associated with was higher odds signifcantly of detectable bacteria in catheterized urine Increase in IPSS categoryIncrease with the presence of bacteria in catheterized urine, but not Remarks in MSU. However, there is no single species or genera that is associated with the lower urinary tract symptoms. There are a number of limitations for this study. Firstly, although the number of subjects is reasonable, it still seems too lit- tle; secondly, the diversity parameters were not included in

the analysis; fnally, catheterized urine sampling may not be cant diferences No statistically signif - statistically No accepted by all patients and this afects the generalizability microbials Relavant of the study (Table 7).

Infertility N/A Beta-diversity Although there are studies that focus on association between male infertility and individual bacteria, there are limited studies on the microbiota in the semen. Polymerase chain reaction (PCR) technique was used to identify bacteria in N/A seminal fuid in early studies [72, 73]. In the era of NGS, Alpha-diversity there are four studies focusing on identifying whether semi- nal microbiome has association with azoospermia using the 16S rRNA sequencing method (Table 8). Some studies that grouped participants by sperm qual- ity showed no signifcant diferences in diversity of seminal microbiome [74, 75]. However, individual bacterial identif- ing (region: N/A) ing (region: 1. EQUC - sequenc 2. 16S rDNA cation noted that the sperm quality was negatively associated identifcation Microbial with Anaerococcus [74], and seminal fuid had more Staphy- lococcus and Lactobacillus, and less Prevotella in partici- pants having normal sperm quality [75]. Chen et al. [76] had semen sample from fertile donors by masturbation (control group) and patients with obstructive or non-obstructive azo- ospermia by percutaneous epididymal sperm aspiration or testicular sperm extraction (study group) showed that there urine MSU and catheterized Sample collection Sample was signifcantly higher number of bacteria in control group. The above studies have specimen collected by masturbation, which is a source of contamination from the distal urethra. To tackle the problem of contamination from distal ure- thra, Alfano et al. [77] had semen sample by microscopic tes- ticular sperm extraction from patients undergoing unilateral

orchidectomy for non-metastatic seminoma (control group) surgery outlet and general urology procedures surgery Men undergoing bladder Men undergoing other Men undergoing and patients with idiopathic non-obstructive azoospermia Patients (study group) and then sequenced the V3-5 regions. There is an increased number of bacteria in the study group, with predominance of Acinobacteria and Firmicutes. They also showed that there is decreased bacterial richness and diver- sity in men with complete germline cell aplasia, which is Summary of original urinary articles syndrome on male lower tract predominated by Acinobacteria, and absence of Clostridia, Focus, 2018 [ 71 ] Focus, Bajic et al. Eur Urol Eur Urol et al. Bajic which may refect an aging process in testes of patients with 7 Table Study

1 3 International Urology and Nephrology (2019) 51:1677–1697 1693 divided into non- divided into and obstructive obstructive Remarks Azoospermia is metabolic, infectious metabolic, infectious and immune disease metabolism, metabo - and lism of cofactors bio - vitamins, glycan synthesis and metabo - lism, enzyme families and metabolism biodegradation biotic and metabolism, metabolism of terpe - noids and polyketides, lipid metabolism and amino acid metabo - lism Functional profling Functional N/A N/A Azoospermia group riskIncreased of having Enhanced nucleotide - xeno Downregulated - - associated with Anaero coccus more: more: (spermLactobacillus morphology) Acidobacteria, Gemma - Acidobacteria, - Plancto timonadetes, Chlorofexi, mycetes, Armati - Crenarchaeota, - Elusimicro monadetes, Euryar bia, Nitrospirae, chaeota, Spirochaetes, Spirochaetes, chaeota, Synergistetes, Chlorobi, - Verrucomi Chlamydiae, crobia Proteus, Prevotella, - Pseudomonas, Veil lonella, Corynebac - Rhodococcus, terium, Bacillus Staphylococcus, more: - Amaricoccus, Anaero Aquicella, myxobacter, Azos - Arsenicicoccus, pirillum, Chitinimonas, - Copro Chlorobaculum, coccus, Desulfovibrio, Dokdonella, Gallionella, Helicobacter, Geobacter, Idiomarina, Kaistia, Kribbella Relavant microbials Relavant Sperm quality negatively Sperm negatively quality Abnormal spermiogram— Prevotella genus— normospermic group— Staphylococcus, genus— Azoospermia group—less: Cyanobacteria, phylum— Lactobacillus, Lactobacillus, genus— Azoospermia group— Bacteroidetes phylum— Alicyclobacillus, genus— - ence have higher degree have of similarity than group control Beta-diversity N/A No signifcant difer No Azoospermia group - cant difer ence increase increase in chao1 in index group with abnormal - total motil ity Alpha- diversity No signif - No Only minor Only N/A (V1–V2 regions) (V1–V2 regions) (V4 region) Microbial identifca - Microbial tion 16S rDNA sequencing sequencing 16S rDNA 16S rDNA sequencing sequencing 16S rDNA 16S rDNA sequencing sequencing 16S rDNA TESE Sample Masturbation Masturbation Azoospermia: PESA/ masturbation Control: spermia nozoospermia and azoospermia spermia Patients Healthy spermHealthy donors asthenozoospermia oligoasthenozoo - oligoasthe - severe Healthy men ( n = 94) Healthy Patients with azoo - Patients Control Summary of original articles on infertility 2013 [ 74 ] Microbiol, 2019 [ 75 ] Microbiol, Med, 2018 [ 76 ] 8 Table Study Hou et al. FertilHou et al. Steril, Baud et al. Front Front Baud et al. Chen et al. Exp Ther Chen et al.

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idiopathic non-obstructive azoospermia. However, as studies in other structures have shown that cancer can change the local microbiota, the control group used in this study may not be reliable. The above studies provide early results on the microbiota Remarks of semen and their potential role in azoospermia, although most have small number of subjects. Further larger-scale studies are required to characterize the normal seminal microbiota, to determine whether seminal dysbiosis is asso- ciated with male infertility and the function of bacteria in the semen. Functional profling Functional N/A

Conclusion

Preliminary evidence showed that microbiome has a role in diferent aspects of urological diseases, including diag- nostic, treatment or prognostic. However, the evidence is Proteobacteria Saprospirae, bacteria, - Bacilli, Alphaproteobac - Betaproteobacte teria, - ria, Gammaproteobac teria Relavant microbials Relavant iNOA group—more: iNOA Actinobacteria phylum— Class—Actinobacteria group—less: iNOA , Bacteroidetes phylum— - Flavo Class— Bacteroidi, either weak or inconsistent, which makes the translation into clinical practice difcult. Standardized methodology, such as sample collection and sequencing the same vari- able region, as well as reporting methods, like including alpha- and beta-diversities, should be developed. After that, further large-scale investigations, like longitudinal analysis presented, presented, analysis but the statistical comparison not presented Beta-diversity Principal component studies or combination of the information to develop risks scores for diagnostic tests, should be carried out to trans- late this knowledge into clinical use, in addition to under- stand how the microbiome is correlated with urological iNOA iNOA group Alpha- diversity Higher in diseases.

Compliance with ethical standards

Conflict of interest All authors declare that they have no confict of

(V3–V5 regions) interest related to this manuscript. Microbial identifca - Microbial tion 16S rDNA sequencing sequencing 16S rDNA

Human and animal rights statement This article does not contain any studies with human participants or animals performed by any of the authors. plastic tissue from tissue from plastic specimen Sample iNOA: microTESE iNOA: non-neo - Control: References

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