{"paper_id":"6975e367-e573-4316-a686-c4f64b9e24f4","body_text":"Endometriosis, characterized by abnormal presence \nof endometrial tissue outside the uterus, is a major \ncause of discomfort in women (1, 2). This disease \nwhich occurs primarily in women of reproductive ages, \nseems to be an estrogen-dependent phenomenon (1-3). \nAlthough clinical symptoms are not seen in all women, \nthe impact of endometriosis on physical, psychological \nand social performance is obvious in many other \nwomen (4). Endometriosis-associated pain includes \ndysmenorrhea, dyspareunia, dyschezia and dysuria, \nas well as chronic pelvic pain. Endometriosis patients \nat some time points endure debilitating pain which is \nworse than the pain experienced by women suffering \nfrom cancer (2). Moreover, ovarian endometriosis may \nhave clinical and paraclinical manifestations of ovarian \ncarcinoma (5). The mainstay of treatment of endometriosis \nconsists of surgery accompanied by ovarian \nsuppressive therapy (6, 7). Full consultation with patients \nand use of various types of analgesics, oral contraceptive \npills, progestins or gonadotropin-releasing \nhormone agonists (GnRHa) are often required (8-12).\nThere is sufficient evidence showing the efficacy \nof progestins and GnRHa against endometriosis-\nassociated pain (13, 14), however, their side effects \nand patient tolerance, particularly in the long term, \nshould not be overlooked (10, 13, 14). Based on \nmolecular studies, changes in the function of immunologic \ncells like monocytes, macrophages, natural \nkiller cells (NK), cytotoxic T cells and B cells have \nbeen detected in the peritoneal fluid of women with \nendometriosis. This alteration of immunologic defense \nwhich is not capable of removing the ectopic \nendometrial cells, leads to implantation of endometriosis \nlesions. Furthermore, the paramount role of \nNK cells was highlighted in many studies (15-20). \nAccording to Oosterlynck et al. (17), decreased activity \nof NK cells is remarkably associated with the \nseverity of endometriosis. Previous studies led to the \nhypothesis that lack of ectopic endometrial clearance \nby NK cells in the peritoneal fluid contributes \nto the development of the disease. Therefore, any \nagent that stimulates the immune cells or increases \nthe cytotoxicity of NK cells could be beneficial in \ntreatment of endometriosis (21-24).\nSashihara et al. (21-23) showed that a kind of lactobacillus \ncalled Lactobacillus gasseri (OLL2809), \nwhich is of probiotic type, stimulates the production \nof interleukin 12 (IL-12) from murine spleen cells. \nIL-12, a cytokine secreted by antigen presenting \ncells, triggers the production of cytotoxic lymphocytes \nby activating NK cells and T cells (25, 26). \nLactobacillus species including  Lactobacillus acidophilus, \nLactobacillus plantarum, Lactobacillus \nfermentum  and  Lactobacillus  gasseri, constitute the \npredominant normal microbial flora of genitourinary \nand gastrointestinal (GI) tract of healthy individuals. \nThe effectiveness of these probiotics in maintenance \nof the normal pH of vagina and prevention of genital \ninfections has been well-studied (27). Host immunity \nmodification and interference with colonization of \nexternal pathogens are considered their main mechanisms \nof action (28, 29). There is also evidence that \nalterations in the normal flora within the gastrointestinal \n(GI) tract caused by administration of probiotics, \nantibiotics or even transplantation of feces into \nthe GI tract, could result in pain relief by affecting \nneurologic pathways (30). In this regard, some recent \nstudies indicated the use of lactobacillus-mediated \nmedications in the treatment of endometriosis-\nrelated lesions (31, 32). Considering the hypothesis \nthat lactobacillus may have immunogenic properties, \nthe present study was conducted to assess the efficacy \nof oral lactobacillus-based pills on pain relief \nin patients diagnosed with endometriosis.\n\nThis was a pilot randomized triple-blind placebo-controlled \ntrial carried out in a referral center for endometriosis \nin a university-based hospital in Tehran, Iran from \nOctober 2016 to October 2017. Enrolled participants \nwere women with endometriosis (diagnosed based on \npathologic report) who had undergone laparoscopic surgery \ndue to pain and were randomly allocated into one \nof the two groups at a 1:1 ratio. The study was approved \nby the Institutional Review Board of Iran University of \nMedical Sciences (IUMS) by the Ethical Committee \nnumber IR.IUMS.REC1395.9311290013. All participants \nwere patients with stage 3 and 4 of endometriosis \n(according to the revised American fertility society \n(AFS) classification of endometriosis (33). Patients were \nbetween 18 to 45 years old with menstrual cycle ranging \nfrom 21 to 35 days, with initial overall pain score higher \nthan 4 [based on the visual analogue scale (VAS) scoring \nsystem]. The overall pain score was defined as the sum \nof dysmenorrhea, dyspareunia, chronic pelvic VAS pain \nscores. A scale of 0 (without any pain) to 10 (most severe \npain), by the use of a 10-cm ruler in the questionnaire \nfilled by the physician at the initial visit and each follow \nup visit at 8 and 12 weeks post-treatment, was used in \nthe VAS scoring. Patients had at least 3 months interval \nfrom surgery and in this period, they were not supposed \nto use hormonal treatment; also, the participants were \nasked not to take any pain-killer medications other than \nNSAIDs which have short-term effects and do not have \ninterference with lactobacillus effects. Those with history \nof hormonal replacement after surgery, hepatic or \nrenal disturbances, cancer, diarrhea after taking dairy \nproducts, or consuming any type of probiotic products \nwere excluded. Written informed consent was obtained \nfrom all patients eligible for the trial. Data including \ndemographic findings, medical history and medication \nuse were recorded in questionnaires by a physician in \nthe first visit and completed in the follow-up visits at \n8 and 12 weeks post-treatment visits. The participants \nwere asked to mention any kind of excessive GI upset, \nnausea, vomiting, or any other non-specific side effects.\nThe present study was a pilot placebo-controlled \nrandomized clinical trial which recruited 20 patients \nfor each arm (Fig.1). After exclusion of 3 patients, \nthirty-seven patients with endometriosis were randomly \nassigned (by simple randomization method using \ntable of random numbers) to one of the two groups \nreceiving either LactoFem ® , Zist Takhmir Co. Tehran, \nIran (one capsule per day) or placebo (as the control \ngroup). Each LactoFem ®  capsule contains 10 9  colony \nof four different lactobacillus strains ( Lactobacillus \nacidophilus, Lactobacillus plantarum, Lactobacillus \nfermentum  and  Lactobacillus gasseri ). The lactobacilli \ncontents and placebo contents were packed in the \nsimilar packing with 30 capsules in each pack by the \nmanufacturer; two packs were given to each patient \nin the first visit, to be used during the 8 weeks. The \nlactobacilli packs and placebo packs were named A \nor B by the manufacturer. After completion of the \nanalysis, the manufacturer revealed which one was \nlactobacilli or placebo.\nFlow diagram of the trial\nAll women had undergone complete laparoscopic removal \nof endometriosis lesions including deep infiltrating \nendometriosis (DIE). The procedures had been performed \nwith similar extent of resection including ovarian cystectomy \n(endometrioma), salpingectomy, ureteral dissection, \nuterosacral ligament ablation or DIE removal. The \ninterval between surgery and commencement of intervention \nwas at least 3 months. At the beginning of the study, \npatients were evaluated for the intensity of pelvic pain, \ndysmenorrhea, and dyspareunia based on the VAS score \nrated from zero (no pain) to 10 (the most severe pain). Patients \nin the two groups continued taking medication for 8 \nweeks and then, the pain intensity was evaluated again 8 \nand 12 weeks following intervention by a follow-up visit \nor a phone call. During the time of follow-up, patients \nwere allowed to use NSAIDs only as the rescue therapy. \nPatients who were not willing to continue the trial due \nto personal reasons were excluded from the study. This \nstudy was conducted as a triple-blind trial in which the \nresearcher, the subjects, and the statistician were all unaware \nof the allocation of the two groups.\nResults are presented as mean ± SD for quantitative \nvariables and as absolute frequencies and percentages for \ncategorical variables. Normal distribution of data was assessed \nusing the Kolmogorov-Smirnoff test. Categorical \nvariables were compared using chi-square test or Fisher’s \nexact test. Quantitative variables were also compared using \nt test or Mann U test. ANOVA test was also used to \nanalyze more than two means. For statistical analysis, the \nstatistical software SPSS version 20 for windows (SPSS \nInc., Chicago, IL) was used. P.0.05 were considered statistically \nsignificant.\nThe main outcome of the study was the mean pain score \n(for dysmenorrhea, dyspareunia and pelvic pain) after 8 \nand 12 weeks of intervention as assessed by VAS scoring \nsystem. The secondary outcome was the change in VAS \nscores during the first 8 weeks of intervention and from 8 \nto 12 weeks post medication.\n\nThe two groups were comparable regarding mean age \n(P=0.955), body mass index (BMI) (P=0.14), history of \ninfertility (P=0.669), irregular menstrual cycle (P=0.264), \nunderlying disorders (P=0.307), and history of medications \n(P=0.600). Demographic characteristics of the subjects \nare demonstrated in Table 1. All patients had undergone \nlaparoscopy beforehand and endometriosis was \npathologically diagnosed in all participants. According to \nrevised American fertility society (AFS) classification of \nendometriosis (33), stage III was found in 25 and 0% and \nstage IV was observed in 75 and 100% of intervention and \ncontrol groups, respectively (P=0.101).\nBaseline characteristics of the participants\nData are presented mean ± SD or n (%). BMI; Body mass index and *; Based on revised \nAFS classification.\nAs shown in Table 2, the mean pain scores at baseline \nas well as 8 and 12 weeks after intervention were not different \nbetween the groups. Using ANOVA analysis, the \ntrend of the changes in pain intensity for dysmenorrhea, \ndyspareunia, and chronic pelvic pain during 12 weeks \nwere evaluated. Concerning dysmenorrhea, the mean pain \nscore decrease observed in the LactoFem ®  group was significantly \nlarger than that of the control group during 8 \nweeks of treatment (3.46 ± 2.97 vs. 2.18 ± 1.06, P=0.018). \nThe decreases in mean pain scores from week 0 to 12 and \nfrom week 8 to 12 were not however statistically significant \n(P=0.051 and 0.191 respectively). Concerning \nchronic pelvic pain, the mean pain score decrease from \nweek 0 to 8 was 3.35 ± 2.18 for the LactoFem ®  group \nand 3.03 ± 0.37 for the placebo group (P=0.119). The decrease \nin chronic pelvic pain score from week 0 to 12 was \nnot significant (P=0.458). The change in pain scores from \nweek 8 to 12, however, was significantly larger in the control \ngroup (1.09 ± 1.00 vs. 1.34 ± 0.06, P=0.02). Concerning \nthe overall pain scores, the mean pain score decreased \nsignificantly in the LactoFem ®  group during 8 weeks of \nintervention in comparison to the placebo group (7.33 ± \n7.00 vs. 4.11 ± 1.68, P=0.017). Moreover, the change in \npain scores between week 8 and 12 was statistically different between the groups (P=0.015). No serious side effects \nfollowing ingestion of these capsules were reported.\nPain scores (VAS) at 3 different time points\nData are presented mean ± SD.\n\nThe aim of this study was to assess the therapeutic effects \nof oral lactobacillus on endometriosis-associated \npain (including pain caused by dysmenorrhea, dyspareunia, \nand chronic pelvic pain). Few studies were conducted \nuntil now on the effects of lactobacilli on pain complaints \nrelated to endometriosis. A review of these few studies \nindicated the beneficial impact of lactobacilli on endometriosis \n(24, 31, 32). This possible effectiveness could result \nfrom increases in interleukin-12 levels and NK cells \nactivity (15-18). Also, decrement of the activity of natural \nlethal cells seems to be related to the severity of endometriosis, \nand the inability to clear the ectopic endometrial \nlesions by the NK cells in the peritoneal space, contributes \nto development of disease (16-19, 22-24) which could be \nprevented by the use of probiotics. In a study done by \nUchida and Kobayashi (32), lactobacillus therapeutic effect \nwas evaluated in animal models following four weeks \nof treatment. It was finally observed that administration of \nlactobacillus was associated with a significant reduction \nin the volume of induced endometriosis in rats.\nIn another study (31), 33 patients with clinical diagnosis \nof endometriosis were given Lactobacillus gasseri capsules \nfor 12 weeks. It was shown that 2 and 3 months post-\ntreatment, use of lactobacillus was associated with significant \nimprovements in pain intensity during menstruation \nin comparison with placebo. This finding was consistent \nwith ours. The difference in pain scores during the first 8 \nweeks were apparently more in the mentioned study (31), \nand this was due to the lower initial pain scores post-surgical \ntreatment in the present study. In both studies, no \nsignificant relief in non-menstrual pain was achieved. In \nour study, diagnosis of endometriosis was based on pathologic \nreport and not just based on complaints of dysmenorrhea \nor other types of pain, which could be a strength of \nthe present study. Furthermore, surgical staging was done \nbased on the revised AFS classification. All the subjects \nhad gone through laparoscopic surgery because of intolerable \npain. An interval of at least 3 months was given to \neach patient before prescribing lactobacillus, to evaluate \nthe effects of the surgical treatment. Lactobacillus-based \nmedication used in our study consisted of four different \nstrains of Lactobacilli including  Lactobacillus gasseri  \nused by Itoh et al. (31). Although the mean pain scores for \ntwo groups (according to VAS) after 8 weeks and 12 weeks \nwere comparable, a larger decrease in dysmenorrhea intensity \nand the overall pain scores in the LactoFem ®  group \nwas seen after 8 weeks of treatment. This improvement in \npain after 8 weeks was not significant for chronic pelvic \npain and dyspareunia comparing with dysmenorrhea and \noverall pain scores. Quite interestingly, during the four \nweeks following cessation of LactoFem ®  (i.e. from week \n8 to 12), the mean pain scores related to chronic pelvic \npain and the overall pain intensity increased significantly \ncompared to the control group. This increase could be due \nto the withdrawal effects of the LactoFem ®  and the fact \nthat the efficacy of the lactobacillus is limited to the treatment \nduration only. Our study was the first randomized \ntrial using lactobacillus-based medication on stage 3 and \n4 of endometriosis regarding three common pain types in \nsuch patients. Given the progressive nature of endometriosis \nand unbearable pain episodes related to this disorder, \nany intervention that could mitigate its symptoms, \nis certainly invaluable. Compared to other conventional \nmedical therapies used for endometriosis-associated pain, \nLactoFem ®  capsules have no remarkable side effects such \nas weight gain, flushing or abnormal uterine bleeding and \nno serious side effects following ingestion of these capsules \nwere reported in our experiment.\nFurthermore, these capsules modify microbiota of urogenital \nand GI tract and prevent from infections by improving \nimmune system function. LactoFem ®  capsules \nare readily available in our country at a reasonable price. \nThe finding that the remedial outcome of LactoFem ®  was \nnot as significant as expected could be due to the limitations \nof our study. The first limitation was the small sample \nsize which was not large as many patients had received \nhormonal therapy during 3 month interval before initiating the study. Also, some patients were not able to refer to \nthe clinic for participation in the study. Another limitation \nthat should be mentioned was the lower initial pain scores \nof the patients, due to the surgical treatment, which could \naffect both the sample size and the results. This trial was \ndesigned as a pilot study and we believe that in a larger \nstudy population, more robust results could be achieved. \nThe dosage of lactobacillus capsules administered could \nbe another limitation. Maybe at higher doses, more declines \nin pain scores could have been resulted. Moreover, \nchanges in microbiome caused by lactobacilli were not \nevaluated which could be another limitation of this study. \nIt should also be mentioned that it was not possible to \ndesign a cross-over study because of the limited time that \nmany of the patients agreed to participate in the study, \nsince many of them planned for  in vitro  fertilization (IVF) \nor pregnancy in the near future. Also, there was no similar \nstudy conducted within a longer time of follow-up to be \nsure how long the effect of lactobacilli could remain on \npain suppression, therefore to avoid a bias in this field, we \npreferred a non-cross over design.\n\nIt seems that lactobacilli have some beneficial effects regarding endometriosis-associated pain including dysmenorrhea and chronic pelvic pain. Regarding the dysmenorrhea, the best results happened after 8 weeks of the lactobacilli consumption, which also caused a significant decrease in the overall pain over the course of lactobacilli use in our study. The findings of our research may be used for sample size estimation for further randomized trials to better evaluate the impact of lactobacilli on endometriosis and its related symptoms.","source_license":"public-domain-us","license_restricted":false}