Document Type : Original Article
1 Fellowship in Minimally Invasive Gynecologic Surgery (FMIG), Endometriosis Research Center, Iran University of Medical Sciences (IUMS), Tehran, Iran
2 Fellowship in Infertility, Endometriosis Research Center, Iran University of Medical Sciences (IUMS), Tehran, Iran
3 Endometriosis Research Center, Iran University of Medical Sciences (IUMS), Tehran, Iran
4 4Rasoul Akram Hospital, Iran University of Medical Sciences (IUMS), Tehran, Iran
Endometriosis, characterized by abnormal presence of endometrial tissue outside the uterus, is a major cause of discomfort in women (1, 2). This disease which occurs primarily in women of reproductive ages, seems to be an estrogen-dependent phenomenon (1-3). Although clinical symptoms are not seen in all women, the impact of endometriosis on physical, psychological and social performance is obvious in many other women (4). Endometriosis-associated pain includes dysmenorrhea, dyspareunia, dyschezia and dysuria, as well as chronic pelvic pain. Endometriosis patients at some time points endure debilitating pain which is worse than the pain experienced by women suffering from cancer (2). Moreover, ovarian endometriosis may have clinical and paraclinical manifestations of ovarian carcinoma (5). The mainstay of treatment of endometriosis consists of surgery accompanied by ovarian suppressive therapy (6, 7). Full consultation with patients and use of various types of analgesics, oral contraceptive pills, progestins or gonadotropin-releasing hormone agonists (GnRHa) are often required (8-12).
There is sufficient evidence showing the efficacy of progestins and GnRHa against endometriosis- associated pain (13, 14), however, their side effects and patient tolerance, particularly in the long term, should not be overlooked (10, 13, 14). Based on molecular studies, changes in the function of immunologic cells like monocytes, macrophages, natural killer cells (NK), cytotoxic T cells and B cells have been detected in the peritoneal fluid of women with endometriosis. This alteration of immunologic defense which is not capable of removing the ectopic endometrial cells, leads to implantation of endometriosis lesions. Furthermore, the paramount role of NK cells was highlighted in many studies (15-20). According to Oosterlynck et al. (17), decreased activity of NK cells is remarkably associated with the severity of endometriosis. Previous studies led to the hypothesis that lack of ectopic endometrial clearance by NK cells in the peritoneal fluid contributes to the development of the disease. Therefore, any agent that stimulates the immune cells or increases the cytotoxicity of NK cells could be beneficial in treatment of endometriosis (21-24).
Sashihara et al. (21-23) showed that a kind of lactobacillus
called Lactobacillus gasseri (OLL2809),
which is of probiotic type, stimulates the production
of interleukin 12 (IL-12) from murine spleen cells.
IL-12, a cytokine secreted by antigen presenting
cells, triggers the production of cytotoxic lymphocytes
by activating NK cells and T cells (25, 26).
Lactobacillus species including
This was a pilot randomized triple-blind placebo-controlled trial carried out in a referral center for endometriosis in a university-based hospital in Tehran, Iran from October 2016 to October 2017. Enrolled participants were women with endometriosis (diagnosed based on pathologic report) who had undergone laparoscopic surgery due to pain and were randomly allocated into one of the two groups at a 1:1 ratio. The study was approved by the Institutional Review Board of Iran University of Medical Sciences (IUMS) by the Ethical Committee number IR.IUMS.REC1395.9311290013. All participants were patients with stage 3 and 4 of endometriosis (according to the revised American fertility society (AFS) classification of endometriosis (33). Patients were between 18 to 45 years old with menstrual cycle ranging from 21 to 35 days, with initial overall pain score higher than 4 [based on the visual analogue scale (VAS) scoring system]. The overall pain score was defined as the sum of dysmenorrhea, dyspareunia, chronic pelvic VAS pain scores. A scale of 0 (without any pain) to 10 (most severe pain), by the use of a 10-cm ruler in the questionnaire filled by the physician at the initial visit and each follow up visit at 8 and 12 weeks post-treatment, was used in the VAS scoring. Patients had at least 3 months interval from surgery and in this period, they were not supposed to use hormonal treatment; also, the participants were asked not to take any pain-killer medications other than NSAIDs which have short-term effects and do not have interference with lactobacillus effects. Those with history of hormonal replacement after surgery, hepatic or renal disturbances, cancer, diarrhea after taking dairy products, or consuming any type of probiotic products were excluded. Written informed consent was obtained from all patients eligible for the trial. Data including demographic findings, medical history and medication use were recorded in questionnaires by a physician in the first visit and completed in the follow-up visits at 8 and 12 weeks post-treatment visits. The participants were asked to mention any kind of excessive GI upset, nausea, vomiting, or any other non-specific side effects.
The present study was a pilot placebo-controlled
randomized clinical trial which recruited 20 patients
for each arm (Fig.1). After exclusion of 3 patients,
thirty-seven patients with endometriosis were randomly
assigned (by simple randomization method using
table of random numbers) to one of the two groups
receiving either LactoFem®, Zist Takhmir Co. Tehran,
Iran (one capsule per day) or placebo (as the control
group). Each LactoFem® capsule contains 109 colony
of four different lactobacillus strains (
Flow diagram of the trial
All women had undergone complete laparoscopic removal of endometriosis lesions including deep infiltrating endometriosis (DIE). The procedures had been performed with similar extent of resection including ovarian cystectomy (endometrioma), salpingectomy, ureteral dissection, uterosacral ligament ablation or DIE removal. The interval between surgery and commencement of intervention was at least 3 months. At the beginning of the study, patients were evaluated for the intensity of pelvic pain, dysmenorrhea, and dyspareunia based on the VAS score rated from zero (no pain) to 10 (the most severe pain). Patients in the two groups continued taking medication for 8 weeks and then, the pain intensity was evaluated again 8 and 12 weeks following intervention by a follow-up visit or a phone call. During the time of follow-up, patients were allowed to use NSAIDs only as the rescue therapy. Patients who were not willing to continue the trial due to personal reasons were excluded from the study. This study was conducted as a triple-blind trial in which the researcher, the subjects, and the statistician were all unaware of the allocation of the two groups.
Results are presented as mean ± SD for quantitative variables and as absolute frequencies and percentages for categorical variables. Normal distribution of data was assessed using the Kolmogorov-Smirnoff test. Categorical variables were compared using chi-square test or Fisher’s exact test. Quantitative variables were also compared using t test or Mann U test. ANOVA test was also used to analyze more than two means. For statistical analysis, the statistical software SPSS version 20 for windows (SPSS Inc., Chicago, IL) was used. P.0.05 were considered statistically significant.
The main outcome of the study was the mean pain score (for dysmenorrhea, dyspareunia and pelvic pain) after 8 and 12 weeks of intervention as assessed by VAS scoring system. The secondary outcome was the change in VAS scores during the first 8 weeks of intervention and from 8 to 12 weeks post medication.
The two groups were comparable regarding mean age (P=0.955), body mass index (BMI) (P=0.14), history of infertility (P=0.669), irregular menstrual cycle (P=0.264), underlying disorders (P=0.307), and history of medications (P=0.600). Demographic characteristics of the subjects are demonstrated in Table 1. All patients had undergone laparoscopy beforehand and endometriosis was pathologically diagnosed in all participants. According to revised American fertility society (AFS) classification of endometriosis (33), stage III was found in 25 and 0% and stage IV was observed in 75 and 100% of intervention and control groups, respectively (P=0.101).
Baseline characteristics of the participants
|Parameter||Lactobacillus group||Control group||P value|
|Age (Y)||33.81 ± 6.85||33.69 ± 5.63||0.955|
|BMI (Kg/m2)||26.16 ± 5.46||23.64 ± 4.03||0.14|
|History of infertility||3 (18.8)||4 (25.0)||0.669|
|Irregular menses||7 (43.8)||4 (25.0)||0.264|
|Family history of endometriosis||3 (18.8)||1 (6.2)||0.600|
|Stage III||4 (25.0)||0 (0.0)|
|Stage IV||12 (75.0)||16 (100)|
Data are presented mean ± SD or n (%). BMI; Body mass index and *; Based on revised AFS classification.
As shown in Table 2, the mean pain scores at baseline as well as 8 and 12 weeks after intervention were not different between the groups. Using ANOVA analysis, the trend of the changes in pain intensity for dysmenorrhea, dyspareunia, and chronic pelvic pain during 12 weeks were evaluated. Concerning dysmenorrhea, the mean pain score decrease observed in the LactoFem® group was significantly larger than that of the control group during 8 weeks of treatment (3.46 ± 2.97 vs. 2.18 ± 1.06, P=0.018). The decreases in mean pain scores from week 0 to 12 and from week 8 to 12 were not however statistically significant (P=0.051 and 0.191 respectively). Concerning chronic pelvic pain, the mean pain score decrease from week 0 to 8 was 3.35 ± 2.18 for the LactoFem® group and 3.03 ± 0.37 for the placebo group (P=0.119). The decrease in chronic pelvic pain score from week 0 to 12 was not significant (P=0.458). The change in pain scores from week 8 to 12, however, was significantly larger in the control group (1.09 ± 1.00 vs. 1.34 ± 0.06, P=0.02). Concerning the overall pain scores, the mean pain score decreased significantly in the LactoFem® group during 8 weeks of intervention in comparison to the placebo group (7.33 ± 7.00 vs. 4.11 ± 1.68, P=0.017). Moreover, the change in pain scores between week 8 and 12 was statistically different between the groups (P=0.015). No serious side effects following ingestion of these capsules were reported.
Pain scores (VAS) at 3 different time points
|Parameter||Lactobacillus group||Control group||P value|
|Week 0||4.82 ± 3.76||3.67 ± 2.64||0.402|
|Week 8||2.55 ± 2.77||3.25 ± 2.30||0.513|
|Week 12||3.09 ± 2.59||3.17 ± 2.08||0.939|
|Change between week 0-8||-3.55 ± 2.27||-2.02 ± 0.38||0.117|
|Change between week 0-12||-2.86 ± 1.72||-2.96 ± 0.46||0.301|
|Change between week 8-12||0.93 ± 0.54||-1.97 ± 0.07||0.350|
|Week 0||6.53 ± 2.88||5.60 ± 2.06||0.316|
|Week 8||3.07 ± 2.49||4.47 ± 2.13||0.110|
|Week 12||3.80 ± 2.54||4.60 ± 1.92||0.339|
|Change between week 0-8||-3.46 ± 2.97||-2.18 ± 1.06||0.018|
|Change between week 0-12||-2.73 ± 2.68||-1.66 ± 1.06||0.051|
|Change between week 8-12||1.75 ± 0.73||1.95 ± 0.00||0.339|
|Chronic pelvic pain|
|Week 0||4.19 ± 3.53||2.88 ± 2.80||0.253|
|Week 8||2.00 ± 1.93||2.50 ± 2.34||0.515|
|Week 12||3.00 ± 2.39||2.44 ± 2.13||0.448|
|Change between week 0-8||-3.35 ± 2.18||-3.03 ± 0.37||0.119|
|Change between week 0-12||-3.22 ± 1.18||-2.33 ± 0.43||0.458|
|Change between week 8-12||1.09 ± 1.00||-1.34 ± 0.06||0.02|
|Overall pain score|
|Change between week 0-8||-7.33 ± 7.00||-4.11 ± 1.68||0.017|
|Change between week 0-12||-6.86 ± 4.93||-4.05 ± 1.81||0.127|
|Change between week 8-12||2.47 ± 2.06||2.27 ± 0.12||0.015|
Data are presented mean ± SD.
The aim of this study was to assess the therapeutic effects of oral lactobacillus on endometriosis-associated pain (including pain caused by dysmenorrhea, dyspareunia, and chronic pelvic pain). Few studies were conducted until now on the effects of lactobacilli on pain complaints related to endometriosis. A review of these few studies indicated the beneficial impact of lactobacilli on endometriosis (24, 31, 32). This possible effectiveness could result from increases in interleukin-12 levels and NK cells activity (15-18). Also, decrement of the activity of natural lethal cells seems to be related to the severity of endometriosis, and the inability to clear the ectopic endometrial lesions by the NK cells in the peritoneal space, contributes to development of disease (16-19, 22-24) which could be prevented by the use of probiotics. In a study done by Uchida and Kobayashi (32), lactobacillus therapeutic effect was evaluated in animal models following four weeks of treatment. It was finally observed that administration of lactobacillus was associated with a significant reduction in the volume of induced endometriosis in rats.
In another study (31), 33 patients with clinical diagnosis
of endometriosis were given Lactobacillus gasseri capsules
for 12 weeks. It was shown that 2 and 3 months post-
treatment, use of lactobacillus was associated with significant
improvements in pain intensity during menstruation
in comparison with placebo. This finding was consistent
with ours. The difference in pain scores during the first 8
weeks were apparently more in the mentioned study (31),
and this was due to the lower initial pain scores post-surgical
treatment in the present study. In both studies, no
significant relief in non-menstrual pain was achieved. In
our study, diagnosis of endometriosis was based on pathologic
report and not just based on complaints of dysmenorrhea
or other types of pain, which could be a strength of
the present study. Furthermore, surgical staging was done
based on the revised AFS classification. All the subjects
had gone through laparoscopic surgery because of intolerable
pain. An interval of at least 3 months was given to
each patient before prescribing lactobacillus, to evaluate
the effects of the surgical treatment. Lactobacillus-based
medication used in our study consisted of four different
strains of Lactobacilli including
Furthermore, these capsules modify microbiota of urogenital
and GI tract and prevent from infections by improving
immune system function. LactoFem® capsules
are readily available in our country at a reasonable price.
The finding that the remedial outcome of LactoFem® was
not as significant as expected could be due to the limitations
of our study. The first limitation was the small sample
size which was not large as many patients had received
hormonal therapy during 3 month interval before initiating the study. Also, some patients were not able to refer to
the clinic for participation in the study. Another limitation
that should be mentioned was the lower initial pain scores
of the patients, due to the surgical treatment, which could
affect both the sample size and the results. This trial was
designed as a pilot study and we believe that in a larger
study population, more robust results could be achieved.
The dosage of lactobacillus capsules administered could
be another limitation. Maybe at higher doses, more declines
in pain scores could have been resulted. Moreover,
changes in microbiome caused by lactobacilli were not
evaluated which could be another limitation of this study.
It should also be mentioned that it was not possible to
design a cross-over study because of the limited time that
many of the patients agreed to participate in the study,
since many of them planned for
It 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.