A Urine-Based Biomarker for Chronic Prostatitis/Chronic Pelvic Pain Syndrome: A Retrospective Multi-Center Study

Background There are currently no objective diagnostic criteria for chronic prostatitis or chronic pelvic pain syndrome and no accepted therapies that cure the disease. Methods We identied 372 patients with chronic prostatitis diagnosed from 2015 to 2018 and collect the information of age, routine urinary test, EPS and NIH-CPSI. Results Our study demonstrated a correlation between the increase of PSEP level and NIH-CPSI scores. At meantime, the correlation was found between the PSEP level and EPS-white blood cells. Conclusions These ndings highlight the potential of PSEP as a viable indicator of symptomatic progression of CP/CPPS. Applications of PSEP assay may guide drug discovery and lead to the better treatment to improve patient’s quality of life. PSEP positive/negative status and concentration with Chi-square test statistics by SAS9.4 (The SAS software was developed by The State University of North Carolina, U.S.A in 1966) for each individual factor including WBC and lecithin corpuscle and NIHCPSI. Data were stratied by counting method to minimize potential confounding factors when testing for association between PSEP and CP/CPPS status. The differences were considered signicant when p < 0.05. We conduct power analyses for the assessment of our sample size by G power software.

of in ammatory atrophy. In ammatory atrophy can provide a favorable breeding ground for PCa development. 7 Exosomes are small, membrane-bound storage vesicles that mediate transport of a cytosolic cargo between the cells and to the extracellular space. 8 Exosomes are produced in many cell types including the prostate epithelial cells where they are termed prostasomes. 9 They can also be excreted to the interstitial tissue compartments when in ltrating leucocytes accumulate in response to in ammation. Thus, prostasomes can be found in seminal plasma and urine. 10 Prostasomes have been reported to elicit antioxidant effects, antibacterial activity, and immunomodulation. 11,12 It has been proposed that prostasomes may have the ability to reduce the production of reactive oxygen species (ROS). 13 Studies also suggested that prostasomes inhibit the NADPH oxidase activity of polymorph nuclear neutrophils by lipid transfer from prostasomes to the plasma membrane of this cells. 14 The molecular composition of human prostasomes is varied and consists of hundreds of known and unknown proteins. Prostate diseases such as prostate cancer, benign prostatic hyperplasia (BPH), and prostatitis present unique phenotypes at the level of their respective prostasomal proteomes. 15 Recently, antibodies against human prostasomes were generated and found to be reactive to urine samples of CP/CPPS patients. The proteins that are immune reactive to the antibodies were designated as prostatic exosomal proteins (PSEPs). 16,17 A multi-center clinical trial performed in China indicated that CP/CPPS patients present elevated PSEP in the void urine when compared to the healthy men. 18 Subsequent applications of PSEP test con rmed the utility in many clinics across China; however, these applications have not addressed the relationship between PSEP test and current methods of diagnosing CP/CPPS. In this study, we intended to be the rst to elucidate the potential relationship between PSEP in urine samples and EPS indexes such as white blood cells (WBC) and lecithin corpuscles as well as NIH-CPSI. Our studies highlight the potential value of PSEP as an indicator for CP/CPPS symptoms and disease progression in clinical practice.  19 For inclusion into this study, CP/CPPS patients must meet the following criteria: male with CP/CPPS history and clinical symptoms, such as urinary frequency, urgency, and retention, the in ammatory reaction or re ective (perineal pain, abdominal bulge, and discomfort). Some patients presented with premature ejaculation or other symptoms such as infertility. Upon rectal exam, CP/CPPS patients con rmed changes of EPS nding over the normal person, such as WBC and lecithin corpuscle (phosphatidylcholine) in secretion. In addition, routine urinary test or culture showed no signi cant anomaly of acute in ammatory cell types or other urinary tract infection. NIH-CPSI questionnaire survey was used to report pain, symptoms of abdominal discomfort, nding of urination symptom and quality of life to give rise to a total score.

Subjects
All research analyses were approved by the Institutional Research Ethics Committee. The written informed consent was obtained from all individuals and case-report-forms (CRF) were administered during outpatient visits to collect the information of age, routine urinary test, EPS and NIH-CPSI etc.

Sample Collection
Mid segment urine samples were obtained in the morning and were immediately frozen. They were stored at -20℃ until ready for use. Subjects were excluded when there were incomplete clinical data, inadequate quantity of urine samples, or grossly bloody and thick urine or with alcohol consumption.

Psep Assay
The double-blinded PSEP-ELISA assay was performed essentially as described according to the manufacturer's instruction (Onco Biomedical Technology [Suzhou] CO., Ltd). 18 Void human urine samples were added to the 96-well micro plate trays and incubated at 37℃ for one hour. After antibody incubation, the reaction was visualized by the addition of chromogenic tetramethylbenzidine (TMB).The resulting color development indicates the amount of PSEP in urine samples. The absorbance of the samples was read at 450 nm/630 nm.

Statistical Analysis
The statistical analysis was performed in a blinded manner. For the cross-sectional study analysis, a database was established to collect all information from each patient including age, routine urinary test, EPS such as WBC and lecithin corpuscle in secretion as well as NIH-CPSI. Data were strati ed by WBC and lecithin corpuscle in secretion and NIH-CPSI respectively according to different classi cation methods. The mean of PSEP concentration and detection rate of PSEP were calculated respectively. Contingency tables and Spearman's correlation coe cient were used to test for independence between PSEP positive/negative status and concentration with Chi-square test statistics by SAS9.4 (The SAS software was developed by The State University of North Carolina, U.S.A in 1966) for each individual factor including WBC and lecithin corpuscle and NIHCPSI. Data were strati ed by counting method to minimize potential confounding factors when testing for association between PSEP and CP/CPPS status. The differences were considered signi cant when p < 0.05. We conduct power analyses for the assessment of our sample size by G power software.

Results
Relationship between urine PSEP level and EPS-WBC number with"+/-" as indicator of disease severity All 372 patients were documented with EPS-WBC number in their case report forms (CRFs). They were strati ed by this method of documenting WBCs as showed in Table 1.

Relationship Between Urine Psep Level And Eps-lecithin Corpuscles
Although the vitality EPS examination has been questioned in clinical practice, EPS is still widely used clinically because there is no ideal speci c diagnostic marker. We therefore examined EPS-lecithin corpuscles for all patients. All 372 patients had records of EPS-lecithin corpuscle in their CRFs. They were strati ed by the grade of EPS-lecithin corpuscle density as show in Table 2. In the normal EPS, a full eld of lecithin corpuscles was being seen under the high power microscope, which was designated as ++++.
The density of EPS-lecithin corpuscles lower than 50% (++) per vision eld under the high power microscope is viewed as a sign of CP/CPPS in urological clinics. Form Table 2, the data showed that there was no statistical signi cance (χ2 = 0.003, p = 0.999) between the density of lecithin corpuscles and PSEP concentration in the urine of CP/CPPS patients when we analyzed them with contingency tables chi-square test. Also the Spearman's correlation coe cient showed no signi cant rank correlation between the two either (rs = 0.001, p = 0.994).

Relationship Between Urine Psep Level And Nihcpsi
The chronic prostatitis symptom index developed by the NIH of United States (NIHCPSI) is an established scoring method to record the symptoms of the patients. According to the severity of symptoms, NIH-CPSI is divided into mild (1-14 points), moderate (15-29 points) or severe (30-43 points). 19 In General, increases of NIH-CPSI were used as the indication that CP/CPPS becomes more pronounced with more severe symptoms.
From the 372 CP/CPPS patients, 225 patients had NIH-CPSI records. The correlation between urine PSEP level in urine and NIH-CPSI was examined. As shown in Table 3, the rising NIH-CPSI was correlated with the increase in the number of patient with positive rate of PSEP. We analyzed them with Contingency tables chi-square test (χ2 = 9.149, p = 0.0091). Spearman's correlation coe cient showed a signi cant rank correlation between NIH-CPSI and PSEP concentration (rs = 0.194, P = 0.0035). Although the correlation between NIH-CPSI and PSEP is weak, these data suggest that an increased PESP concentration in urine sample is correlated with the severity of symptoms and the advanced stages of CP/CPPS in clinical practice. Chi-Square test χ2 = 9.149, p = 0.0091 Spearman's correlation coe cient analysis rs = 0.194, P = 0.0035 We have conducted the power analyses by the G power software to assessment the sample size. Power (1-β) = 0.99, which means that the power of the test is very well and the sample size is enough to have the valid results. (Fig. 1) Discussion CP/CPPS is a frustrating clinical condition for both practitioners and patients. In developing countries, the situation is worse because often CP/CPPS is not correct diagnosed using a combination of multiple methodologies such as NIH-CPSI, EPS, 2-and 4 cup tests, and so on. 20 Rather, clinician experience sometimes dictates the treatment of self-described abdominal and urinary discomfort with frequent prescription of antibiotics to observe treatment response to the anticipated CP/CPPS.
The available PSEP -ELISA assay as a simple, objective, and non-invasive urine test provides the clinician a biological surrogate to assist in the diagnosis of CP/CPPS. In the past couple of years, the PSEP test became adopted in hospitals and clinics in China and received some positive responses. However, the correlation of urine PSEP level with EPS and/or NIH-CPSI was only described in meetings and conferences anecdotally. Therefore, the current study is the rst to validate that PSEP level is associated with the increasing WBC counts and NIH-CPSI scores. Thus, an increasing PSEP level in the urine can be an indication of severity of CP/CPPS and may guide the optimal treatment plan. However, it is a pilot study that only elaborated the correlation of urine PSEP level with EPS and/or NIH-CPSI.
Therefore, further study should focus on the cut-off point of PSEP in order to distinguish between IIIa ans IIIb category avoiding prostatic massage.
During the chronic prostatic process, leucocytes exudate and swarm to the in ammation region, leukocytes engulf lecithin, making lecithin bodies decrease.
In our current study, we do not nd a strong correlation between urine PSEP level and density of EPSlecithin corpuscles. We noticed this result and had extensive discussions with other clinicians.
Nevertheless, we believe that the further study may be needed to evaluate this relationship more closely in a study with a larger sample size.
There is intense research ongoing to identify better and more practical biomarkers for CP/CPPS. Studies have shown that in ammatory cytokines in seminal plasma of CP/CPPS patients are increased signi cantly, such as IL-1, IL-6, IL-8, IL-10, and TNF-a. 21 Polymorph nuclear (PMN) elastase in EPS was also shown to be signi cantly higher in IIIa in comparison to that of IIIb. 22,23 The presence of other pathogens than bacteria, such as Chlamydia, is associated with increased WBC counts and pain severity in men with CP/CPPS. Perhaps the most thorough survey of the protein biomarkers came from mass spectrometry of seminal plasma proteome of prostatitis patients. 24 this study identi ed 418 proteins associated with prostatitis versus 280 present in the healthy individuals with 1662 proteins present in both populations. While these are encouraging steps towards the development of vial biomarkers for CP/CPPS, they are either derived from EPS or require sophisticated equipment to perform analysis. Therefore, they are not of practical value for general clinical application at this moment.
While PSEP-ELISA assay is simple to perform on voided urine, much remains to be learned. For example, it would be important to validate our study by more independent hospitals and clinics around the world for different ethnic background. There are reports that in some regions, particular pathogens may be more closely related with CP/CPPS. In addition, the mechanism of PSEP involvement in CP/CPPS is completely unknown. It would be important to understand why PSEP is elevated in CP/CPPS and whether it is causative or it is a mere biomarker surrogate. The understanding of PSEP biology would also be important for drug development as well. For example, there are many animal models that are currently being employed to investigate the etiology and drug response of experimental prostatitis in animal models. 25 PSEP may be used to monitor the disease course and drug treatment outcomes.

Conclusions
Our study demonstrated a correlation between the increase of PSEP level and NIH-CPSI scores. At meantime, the correlation was found between the PSEP level and EPS indexes. These ndings highlight the potential of PSEP as a viable indicator of symptomatic progression of CP/CPPS. Applications of PSEP assay may guide drug discovery and lead to the better treatment to improve patient's quality of life.

Declarations
Ethics approval and consent to participate: Not applicable.
Consent for publication: Not applicable.
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.