This study was a prospective, randomized, unblinded, controlled phase III clinical trial aimed at evaluating the efficacy of pollen extract and vitamins in patients affected by CP/CPPS and their levels of pro-inflammatory mediators. All consecutive patients with a clinical and instrumental diagnosis of inflammatory CP/CPPS (NIH class IIIa-inflammatory CP/CPPS) attending the same urologic centre between January and December 2015 were screened for enrolment in the study. The treatment group received pollen extract with vitamins (Deprox 500®), while the control group received bromelain. The choice of bromelain as control group treatment was due to the lack of a goldstandard treatment for CP/CPPS [57]. Bromelain was also chosen primarily for the fact that it is a phytotherapeutic compound with an anti-inflammatory effect, considered safe as a long-term therapy, with high patient compliance [14].

The main outcome measures of our study were as follows: an improvement in quality of life, defined as an improvement in the NIH-CPSI score (i.e., a reduction of the NIH-CPSI total score by ≥25%) and a decrease in pro-inflammatory mediators (i.e., a reduction of at least 65% from the baseline) by the end of the complete study period.

The inclusion criteria were the presence of pelvic pain symptoms for at least 3 months during the 6 months before study enrolment in accordance with the European Association of Urology guidelines, a score in the pain domain of the NIH-CPSI of >4 and a negative result for the Meares-Stamey 4-glass test [111516]. We excluded all patients with the following characteristics: subjects <18 and >65 years of age affected by major concomitant diseases with known anatomical abnormalities of the urinary tract or with evidence of other urological diseases with residual urine volume >50 mL resulting from bladder outlet obstruction, subjects with a reported allergy to pollen extract who had recently (within <4 weeks) undergone oral or parental treatment or who were currently using prophylactic antibiotic drugs, and all patients who tested positive for Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, herpesviruses (HSV 1/2), and human papillomavirus (HPV).

Upon arrival at our centre, all eligible individuals provided their written informed consent, completed baseline questionnaires, underwent a urological examination with the Meares-Stamey test, and provided a seminal sample on site [1516]. All seminal plasma samples were used in the pro-inflammatory cytokine evaluation. All patients who met the inclusion criteria were randomized to either the treatment or control group by using a computer-generated allocation sequence. All patients were assigned to 1 of the 2 groups (group A: pollen extract in association with vitamins; and group B: bromelain) according to a 1:1 randomization. Those patients assigned to group A ingested a daily oral administration of 2 tablets of pollen extract and vitamins in a single dose in the evening. All patients assigned to group B underwent daily oral administration of 2 tablets of 40 mg of bromelain in a single daily dose in the evening. Neither the physicians nor the patients were blinded to the treatment type. All patients were contacted by telephone on day 30 of the therapy to ensure that they were correctly administering the drug, in order to ensure uniform treatment interval and dosage. A follow-up visit was scheduled at 3 months from the start of therapy, with urological and microbiological examinations, questionnaire collection, and pro-inflammatory cytokines evaluation. Fig. 1 shows the study schedule.

The validated Italian versions of the NIH-CPSI [18] and Short Form-36 (SF-36) questionnaires were self-administered to all patients [19].

All samples from the Meares-Stamey test and seminal plasma were collected at the time of the urological visit and immediately taken to the laboratory under refrigerated conditions, analysed for cultures, and aliquoted for DNA extraction and polymerase chain reaction for C. trachomatis, U. urealyticum, N. gonorrhoeae, HSV 1/2 and HPV detection [20]. All microbiological evaluations and DNA extraction and purification were carried out in accordance with the methods described by Mazzoli et al [20]. In addition, natural human-produced IL-6, IL-8, and IL-10 concentrations were identified in the seminal samples of all patients and controls with the solid-phase enzyme-linked immunosorbent assay Quantikine IL-6, IL-8, and IL-10 Immunoassay (R&D Systems, Minneapolis, MN, USA) [20]. All samples were tested in duplicate by using independent analysis in accordance with the manufacturer's recommendations and in order to avoid errors. The medium minimal detectable doses of the IL-6, IL-8, and IL-10 assays were 0.70 pg/mL, 3.5 pg/mL, and 3.9 pg/mL, respectively [20].

The study was conducted in line with Good Clinical Practice guidelines, in compliance with the ethical principles published in the latest version of the Declaration of Helsinki. Written informed consent was obtained from all patients prior to treatment. Furthermore, this study was conducted in line with the Consolidated Standards of Reporting Trials statement (URO-TN-2015). The homogeneity of the 2 groups at the baseline evaluation was carried out by using the Student t-test and Mann-Whitney U-test for continuous variables and by the chi-square test for categorical variables. General characteristics of the study participants were expressed using descriptive statistics (means, standard deviations, or ranges). Randomization based on a single sequence of random assignments (simple randomization) was performed using a pseudo-random number generator program (Research Randomizer ver. 4.0; Social Psychology Network, Wesleyan University, Middletown, CT, USA). ANOVA was used for comparing the means. The Bonferroni adjustment test was also used at the second stage of the ANOVA. The differences between the groups regarding the NIH-CPSI results and mean concentration of pro-inflammatory cytokines were obtained using an ANOVA test. The calculation of the sample size needed for enrolment was based on the first outcome measure (improvement of quality of life), due to the fact that we had no data on the effects of pollen extract in association with vitamins on pro-inflammatory cytokines. In this sense and in line with the literature results, the required sample size was calculated under the following conditions: difference between the groups, 35% of patients who reach a reduction in 25% of the NIH-CPSI total score; α error level=0.05, 2-sided; statistical power, 80%; and anticipated effect size, Cohen d=0.5. Our calculation indicated that 32 individuals would be needed in each of 2 groups. Statistical significance was achieved when p<0.05. All reported p-values were 2-sided. Statistical analyses were performed using SPSS software ver. 11.0 (SPSS Inc., Chicago, IL, USA) for Macintosh (Apple, Cupertino, CA, USA).

At the end of the treatment period, 24 of 32 patients (75.0%) in the pollen extract and vitamins group reported an improvement in quality of life, as did 8 of 33 patients (24.2%) in the bromelain group (p<0.001). The SF-36 questionnaires confirmed these results (mean SF-36 value in the treatment group: 98.6±2.1; mean SF-36 value in the control group: 94.9±2.9; p<0.001). All questionnaire results at 3 months after treatment are listed in Table 2. The greater improvement in the treatment group compared with the control group was statistically significant (treatment difference in the NIH-CPSI pain domain: −4.8±0.3 vs. −2.1±0.7; p<0.001).

The mean levels of pro-inflammatory cytokines at the follow-up evaluation are detailed in Table 3. No statistically significant differences were identified between the 2 groups in terms of IL-6 and IL-10 levels (p=0.81 and p=0.41, respectively). The mean post-treatment level of IL-8 was significantly lower in group A compared with group B (IL-8, 298 pg/mL vs. IL-8, 736 pg/mL; p<0.001). In group A we found a statistically significant reduction of IL-8 levels between the enrolment period and the follow-up visit (IL-8, 878 pg/mL vs. IL-8, 298 pg/mL; p<0.001) (Table 3). A good correlation between the reduction of IL-8 (at least 65% from the pre-treatment value) and quality of life improvement (receiver operating characteristic, area under curve=0.83; p=0.001) was found. All patients tested negative at the Meares-Stamey evaluation.

One patient out of 32 patients (3.1%) in group A and 2 of 33 patients (6.1%) in group B had mild adverse effects (nausea).

Herein, we have demonstrated the clinical efficacy of pollen extract in association with vitamins for managing patients affected by CP/CPPS, and the results of our study revealed a relationship between the reduction of IL-8 and clinical efficacy. Another important aspect that came to light is that that the reduction of IL-8 (65%) could be considered a good marker for the response to treatment for CP/CPPS and improvement of patient quality of life.

The efficacy of pollen extract in the treatment of patients affected by CP/CPPS has been demonstrated by several clinical studies [8910111621222324]. All authors agree that pollen extracts significantly improved total symptoms, pain, and quality of life in patients with inflammatory CP/CPPS without severe side effects [8910111621222324]. In particular, Cai et al [9] in a non-randomized clinical study reported a clinical response rate of 90%, demonstrating that pollen extract in association with vitamins significantly improved total symptoms, pain, and quality of life in patients with non-inflammatory CP/CPPS without severe side effects. Moreover, 3 studies by Japanese researchers demonstrated a high clinical response rate to pollen extract treatments in patients with both class IIIa and class IIIb CP/CPPS [242526]. In a randomized control trial involving 139 patients affected by inflammatory CP/CPPS and treated for 12 weeks with flower pollen extract, Wagenlehner et al [11] demonstrated a clinical response rate of 70.6%. In addition, in a cohort of patients randomized to pollen extract or ibuprofen treatment groups, Cai et al [16] reported a response rate of 75.6% in the flower pollen extract group with a low prevalence of adverse effects. All authors hypothesized that the clinical effect of pollen extract was due to an anti-inflammatory anti-proliferative effect on the basis of pre-clinical studies [21]. Up to the present, no clinical study has yet demonstrated the effect of pollen extract on pro-inflammatory cytokines. The only available data come from an animal experiment on a dosedependent anti-inflammatory action of pollen extract in comparison with aspirin in nonbacterial prostatitis in rats. The study findings showed an approximately 10× greater decrease in the levels of IL-1b, IL-6, and tumour necrosis factor in the pollen extract treatment group [21]. Herein, we have demonstrated for the first time that the clinical effect of pollen extract in association with vitamins is associated with a reduction in IL-8 levels. A few years ago, Penna et al [12] demonstrated that, among all the cytokines and chemokines analysed, IL-8 appears to be the most reliable and predictive surrogate marker for diagnosing prostate inflammatory conditions, such as CP/CPPS and benign prostatic hyperplasia. This aspect is very important, because in CP/CPPS patients no biological or molecular markers exist that may be used to evaluate the response to treatment [21]. In fact, Wagenlehner et al [11] found a decrease of leukocytes in post-prostate massage urine samples in both patients and controls; however, they did not find a significant difference between the 2 groups in terms of leukocyte numbers and, for this reason, they concluded that leukocytes cannot be correlated with clinical success [11]. This aspect supports the hypothesis that the presence of inflammatory cells in the post-prostate massage urine sample is not a laboratory characteristic adequately able to predict the response to the treatment. In this sense, our study highlights the feasible role of IL-8 evaluation in predicting the response to treatment in CP/CPPS patients. However, no statistically significant difference in terms of IL-6 or IL-10 levels was observed in our patient population. A few experiments have demonstrated the possible diagnostic role of IL-6 and/or IL-10 evaluation in patients affected by asymptomatic prostatitis (class IV) or benign prostate hyperplasia [2728]. In particular, Miller et al [29] found that IL-10 levels correlated directly to measures of life interference and pain severity, thus highlighting their diagnostic role. However, no studies have ever evaluated changes to levels of IL-6 and/or IL-10 during therapy in CP/CPPS patients. As far as we are aware, ours is the first study to evaluate the changes in levels of IL-6 and IL-10 during treatment. On the basis of these considerations, the efficacy of pollen extract in association with vitamins in CP/CPPS patients is probably due to the anti-inflammatory effects of pollen extract and the neuroprotective role of B vitamins. The superiority of pollen extract in association with vitamins in comparison with bromelain is probably due to the additional role of B vitamins, because bromelain also shows an anti-inflammatory effect [30]. However, the neuroprotective role of B vitamins should be confirmed by future studies.

The present paper reveals important factors that should be taken into account in CP/CPPS treatment: the evaluation of the pro-inflammatory cytokines level and the correlation between these and clinical results. However, some limitations of our study also need to be taken into account, including the small number of enrolled patients, the short follow-up period, the selected patient population, and the non-blinded nature of the study.