Effects of supplementation with two probiotic strains (Lactobacillus helveticus and Bifidobacterium longum) on hormonal status, oxidative stress, and clinical symptoms in women with polycystic ovary syndrome: a randomized clinical trial.

A total of 86 out of the 90 participants completed the 8-week clinical trial (Fig.  1 ). One participant in the intervention group was excluded due to pregnancy, and another person was lost to follow-up due to unwillingness to continue the study. Additionally, two people in the placebo group were excluded from the study because they did not take the supplements. Nonetheless, an intention-to-treat (ITT) approach was used to include all participants in the final analysis. Fig. 1 Flowchart of the study Flowchart of the study Table 1 provides a summary of the baseline characteristics of the study participants. Statistical analysis showed no significant differences between the two groups in variables such as age, body weight, BMI, marital status, education level, physical activity, disease duration, and use of medications or supplements ( P  > 0.05). However, there were significant differences between the groups regarding infertility status and the use of laser therapy for hair removal ( P  < 0.05). Additionally, as detailed in Table  2 , dietary profiles did not differ significantly between the groups in terms of energy intake, macronutrients (carbohydrates, proteins, and fats), fiber, and minerals ( P  > 0.05). The consumption of antioxidants and anti-inflammatory nutrients, including polyunsaturated fatty acids (PUFAs), EPA, DHA, vitamins C, E, A, and D, as well as zinc, magnesium, and selenium, was also comparable between the groups ( P  > 0.05). Table 1 General characteristics of study patients Variables Probiotic group (N:45) Placebo group (N:45) P -value* Age, years 30.16 ± 6.63 31.80 ± 7.10 0.26 Weight (kg) 69.17 ± 14.95 67.65 ± 11.49 0.59 Height (cm) 163.37 ± 6.30 162.82 ± 5.82 0.67 BMI (kg/m 2 ) 25.90 ± 5.52 25.55 ± 4.35 0.74 Disease duration (Year) 6.09 ± 5.56 5.88 ± 5.73 0.86 Physical activity (Met/h/day) 27.67 ± 0.39 27.61 ± 0.40 0.52 Job  Employee 14 (31.11%) 10 (22.22%) 0.14  Housewife 14 (31.11%) 23 (51.1%)  Freelance 17 (37.77%) 12 (26.66%) Marital status  Married 28 (63.6%) 32 (72.7%) 0.36 Education  Diploma 12 (26.66%) 10 (22.22%) 0.66  Bachelor's degree 24 (53.33%) 27 (60%)  Master's or Ph.D 9 (20%) 8 (17.77%) Infertility history  Yes 8 (17.8%) 20 (44.4%) 0.006  No 37 (82.2%) 25 (55.6%) Smoker  Yes 4 (8.88%) 1 (2.22%) 0.058  No 41 (91.11%) 44 (97.77%) Alcohol abuse  Yes 3 (6.66%) 1 (2.3%) 0.30  No 42 (93.33%) 44 (97.7%) Medication History  Metformin Yes 17 (37.77%) 15 (33.33%) 0.65 No 28 (62.22%) 30 (66.66%) Spironolactone  Yes 6 (13.33%) 6 (13.33%) 0.99  No 39 (86.66%) 39 (86.66%) Clomiphene citrate  Yes 0 (0.0%) 2 (4.44%) 0.31  No 45 (100%) 43 (95.55%) Letrozole  Yes 2 (4.44%) 5 (11.11%) 0.16  No 43 (95.55%) 40 (88.88%) Contraceptives  Yes 4 (8.88%) 9 (20%) 0.21  No 41 (91.11%) 36 (80%) Supplementation (Antioxidants, Anti-inflammatory compounds)  Yes 15 (33.33%) 18 (40%) 0.50  No 30 (66.66%) 27 (60%) Laser therapy (Hair removal)  Yes 26 (57.8%) 12 (26.66%) 0.003  No 19 (42.2%) 33 (73.33%) Data are shown as means ± standard deviation (SD) or frequencies (percentage) P -values were obtained from the Chi-Square test or independent sample t-test* Table 2 Dietary intakes of participants, obtained from four dietary recalls Variables Probiotic groupP (N:45) Placebo group (N:45) P -value* Energy (Kcal/day) 1974.69 ± 255.50 1962.07 ± 241.73 0.81 Carbohydrate (g/day) 244.47 ± 40.43 248.90 ± 32.25 0.57 Protein (g/day) 65.94 ± 11.33 66.00 ± 10.23 0.98 Fat (g/day) 75.86 ± 15.23 72.27 ± 17.38 0.30 Cholesterol (mg/day) 310.23 ± 154.50 333.97 ± 120.09 0.42 SFA (g/day) 17.13 ± 4.48 17.32 ± 4.73 0.85 PUFA (g/day) 21.60 ± 5.99 19.37 ± 7.08 0.11 MUFA (g/day) 26.62 ± 6.32 25.59 ± 6.24 0.44 DHA (g/day) 0.039 ± 0.08 0.028 ± 0.06 0.49 EPA (g/day) 0.016 ± 0.03 0.012 ± 0.02 0.49 Fiber (g/day) 14.10 ± 3.87 13.85 ± 2.58 0.71 Folate (mcg/day) 184.17 ± 47.29 180.48 ± 42.70 0.70 Vitamin C (mg/day) 59.14 ± 20.75 61.64 ± 18.91 0.55 Vitamin A(RE/day) 4.96 ± 1.93 4.41 ± 1.48 0.13 Vitamin E (mg/day) 3.23 ± 2.31 2.89 ± 2.26 0.49 Vitamin D (mcg/day) 20 ± 18.8 23.6 ± 18.8 0.36 Zinc (mg/day) 7.15 ± 1.34 7.10 ± 1.43 0.87 Calcium (mg/day) 608.43 ± 110.70 618.64 ± 141.63 0.70 Magnesium (mg/day) 217.69 ± 55.40 213.62 ± 62.83 0.74 Selenium (mcg/day) 0.013 ± 0.01 0.016 ± 0.02 0.55 Data are shown as means ± standard deviation (SD) SFA Short-chain fatty acid, PUFA Poly-unsaturated fatty acid, MUFA Mono-unsaturated fatty acid, DHA Docosahexaenoic acid, EPA Eicosapentaenoic acid, RE Retinol Equivalents, mcg Microgram, mg Milligram P -values were obtained from the independent sample t-test* General characteristics of study patients Data are shown as means ± standard deviation (SD) or frequencies (percentage) P -values were obtained from the Chi-Square test or independent sample t-test* Dietary intakes of participants, obtained from four dietary recalls Data are shown as means ± standard deviation (SD) SFA Short-chain fatty acid, PUFA Poly-unsaturated fatty acid, MUFA Mono-unsaturated fatty acid, DHA Docosahexaenoic acid, EPA Eicosapentaenoic acid, RE Retinol Equivalents, mcg Microgram, mg Milligram P -values were obtained from the independent sample t-test* The effects of supplementation with two probiotic strains on hormonal status, oxidative stress, and clinical symptoms are presented in Table  3 . Compared to the placebo, the intervention group revealed a reduction in testosterone total levels following probiotic supplementation ( P  = 0.03), however the between-group difference did not reach statistical significance after adjustment for baseline values and other potential confounders (−3.37; 95% CI (−11.85, 5.11) vs. 6.48; 95% CI (3.43, 9.53) ng/dL, P  = 0.08). Additionally, compared to the placebo, SHBG levels showed a significant increase in the intervention group (24.39; 95% CI (15.23,33.55) vs. −11.99; 95% CI (−20.12, −3.86) nmol/L, P  < 0.001), while FAI levels significantly decreased after adjusting for baseline values and other confounding variables (−57.05; 95% CI (−80.33, −33.76) vs. 49.86; 95% CI (28.81,70.92), P  < 0.001). Table 3 The effects of 8 weeks’ probiotic supplementation on oxidative stress, selected inflammatory markers, and clinical symptoms in women with polycystic ovary syndrome Outcome variable Probiotic Group ( N =45) Placebo Group ( N =45) Pb Pc Baseline 8thweek Mean difference (95% CI) Pa Baseline 8thweek Mean difference (95% CI) Pa Testosterone total (ng/dl) 36.37±16.76 33.36±28.0.9 −3.37 (−11.85,5.11) 0.42 31.33±13.65 37.81±15.26 6.48 (3.43, 9.53) <0.001 0.03 0.08 SHBG (nmol/l) 37.71±24.85 62.11±38.89 24.39 (15.23,33.55) <0.001 52.35±33.58 40.35±21.72 −11.99 (−20.12, −3.86) 0.005 <0.001 <0.001 FAI 131.40±95.07 74.34±59.67 −57.05 (−80.33, −33.76) <0.001 78.87±51.67 128.74±108.40 49.86 (28.81,70.92) <0.001 <0.001 <0.001 CRP (mg/l) 3.96± 2.75 3.45±1.55 −0.50 (−0.95, −0.04) 0.03 4.01±2.25 4.06±2.09 0.05 (−0.39,0.50) 0.80 0.08 0.046 TAC (nmol/ml) 2562.05.09±300.43 2687.59±297.31 125.53 (53.78,197.28) 0.001 2645.93±467.47 2603.02±441.49 −42.90 (−78.32, −7.48) 0.019 <0.001 0.002 SOD (U/ml) 1.17±0.31 1.37±0.24 0.19 (0.09,0.30) <0.001 1.26±0.21 1.15±0.21 −0.11(−0.18, −0.04) 0.002 <0.001 <0.001 MDA (nmol/ml) 81.98±25.23 58.28±14.84 −23.69 (−30.02, −17.35) <0.001 86.29±26.60 83.58±25.31 −2.70 (−9.19,3.77) 0.40 <0.001 <0.001 Acne Score 13.07±9.25 11.95±9.69 −1.11 (−2.21, −0.02) 0.04 12.31±10.72 10.83±9.87 −1.48 (−3.32, 0.35) 0.11 0.73 0.62 Alopecia 1.96±0.76 1.82±0.82 −0.13 (−0.24, −0.03) 0.009 1.76± 0.80 1.61±0.83 −0.14 (−0.31, 0.03) 0.11 0.98 0.92 Hirsutism 5.49±2.32 5.20±2.13 −0.28 (−0.76,0.18) 0.22 5.55±2.34 5.39±2.19 −0.18 (−0.49,0.13) 0.25 0.70 0.70 Variables are expressed as mean ± SD, and mean difference (95% CI) for changes from baseline to post-intervention Abbreviations SHBG Sex hormone binding globulin, FAI Free androgen index, TAC Total antioxidant capacity, MDA Malondialdehyde, SOD Superoxide dismutase, CRP C-reactive protein a Paired t-test was used to compare outcomes in pre- and post-test interventions b Independent samples t-test was used to compare the mean change between the two groups c Obtained from ANCOVA in the adjusted models (adjusted for baseline value, Infertility status, Laser therapy) The effects of 8 weeks’ probiotic supplementation on oxidative stress, selected inflammatory markers, and clinical symptoms in women with polycystic ovary syndrome Variables are expressed as mean ± SD, and mean difference (95% CI) for changes from baseline to post-intervention Abbreviations SHBG Sex hormone binding globulin, FAI Free androgen index, TAC Total antioxidant capacity, MDA Malondialdehyde, SOD Superoxide dismutase, CRP C-reactive protein a Paired t-test was used to compare outcomes in pre- and post-test interventions b Independent samples t-test was used to compare the mean change between the two groups c Obtained from ANCOVA in the adjusted models (adjusted for baseline value, Infertility status, Laser therapy) Regarding Inflammatory markers, after adjusting for baseline values and other confounding variables, CRP levels showed a significant reduction in the intervention group, compared to the placebo group (−0.50; 95% CI (−0.95, −0.04) vs. 0.05; 95% CI (−0.39,0.50), P  = 0.046). Considering oxidative stress, after adjusting for baseline values and other confounding variables, the intervention group compared to the placebo demonstrated a significant increase in TAC levels (125.53; 95% CI (53.78,197.28) vs. −42.90; 95% CI (−78.32, −7.48) nmol/mL, P  = 0.002), and SOD activity (0.19; 95% CI (0.09,0.30) vs. −0.11; 95% CI (−0.18, −0.04) U/mL, P  < 0.001), while for MDA levels, a significant higher reduction was observed in the intervention group compared to the placebo after adjusting for baseline values and other cofounding variables, (- 23.69; 95% CI (−30.02, −17.35) vs. −2.70; 95% CI (−9.19,3.77), P  < 0.001). Furthermore, regarding clinical symptoms including acne score and alopecia, a significant decrease was only observed in the intervention group, (acne, P  = 0.04) and (alopecia, P  = 0.009), although, after adjusting for baseline values and other cofounding variables, the between-group changes were not statistically significant (acne score: −1.11; 95% CI (−2.21, −0.02) vs. −1.48; 95% CI (−3.32,0.35), P  = 0.62), (alopecia: −0.13, 95% CI (−0.24, −0.03) vs. −0.14; 95% CI (−0.31,0.03), P  = 0.92). Additionally, no statistically significant change was observed for hirsutism in none of the groups (−0.28; 95% CI (−0.76,0.18) vs. −0.18; 95% CI (−0.49,0.13), P  = 0.70) (Table  3 ). None of the participants reported any significant side effects during the intervention period.

This study is a parallel, double-blind, randomized, placebo-controlled trial examining the effects of two probiotic strains on clinical symptoms and hormonal status in PCOS patients. This paper constitutes a part of a comprehensive research project. The study protocol received approval from the Research Ethics Committee of the School of Pharmacy and Nutrition, Isfahan University of Medical Sciences, on March 19, 2024 (Approval No: IR.MUI.PHANUT.REC.1402.098). The trial protocol was registered in the Iranian Registry of Clinical Trials on 30 March 2024 (ID: IRCT20121216011763N62) ( https://irct.behdasht.gov.ir/trial/76067 ). The trial was conducted in accordance with the principles of the Declaration of Helsinki. The study population was selected from Shahid Beheshti Hospital, Isfahan, Iran. An experienced gynecologist (H.G.T) evaluated the symptoms of PCOS and distinguished them from other endocrine disorders, such as endometriosis, fibroids, uterine polyps, and various menstrual cycle irregularities. The eligibility was determined based on the Rotterdam criteria, designed for PCOS diagnosis [ 52 ]. Initially, the principal researcher (M. Shi.) explained the objectives of the study along with its pros and cons to the participants. Participants were asked to provide written informed consent prior to the commencement of the study. Participants had the right to withdraw from the experiment at any time without limiting their access to other services. Participants with a documented history of chronic conditions, such as cardiovascular disease, hepatic or renal disorders, and poorly managed diabetes, were not included in the study. Pregnant or breastfeeding women were also excluded. Furthermore, the study excluded menopausal women, those with a history of hyperprolactinemia, and individuals who had taken probiotic supplements within the previous four weeks. Other disqualifying factors included following a specific diet for at least three months before the study, having a known sensitivity to the supplements used, a history of laparoscopic ovarian surgery, or undergoing in vitro fertilization (IVF). Participants were also excluded if they altered their treatment regimen during the study or chose to withdraw from the intervention. The sample size for the current study was determined using an appropriate formula based on SHBG as the key variable. The mean and standard deviation of SHBG were extracted from Ji X et al.’s study [ 28 ]. In this calculation, we considered the type 1 error of 5%, the type 2 error of 20%, and the study power of 80%. Based on these considerations, the required sample size was 37 participants for each group (total: 74). Accounting for a 20% dropout rate, we included 45 participants in each group. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathrm N=\left[\left(\mathrm Z1-\mathrm\alpha/2+\mathrm Z1-\mathrm\beta\right)^2\times\left(\mathrm\delta1^2+\mathrm\delta2^2\right)\right]/\left(\mathrm\mu1-\mathrm\mu2\right)^2$$\end{document} Participants were allocated into two groups using a stratified block randomization technique based on their BMI status conditions (under 25 kg/m 2 and 25 kg/m 2 or above). Using six blocks with a size of four, they were randomly assigned in a 1:1 ratio to receive the intervention or placebo. A single individual who was not participating in the study performed random allocation. In this approach, one block was randomly selected (using dice), and then participants with the same BMI status condition were allocated to each group based on the selected block. Due to the double-blind nature of the study, patients, laboratory personnel, and researchers were unaware of group assignments until the completion of the data analysis. Additionally, probiotic capsules were packed in the same color and shape as the placebo to maintain blinding. Alongside standard care, participants in the treatment group took a capsule daily before lunch, containing 3 × 10 9 colony-forming units (CFU) of two specific strains, Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 (Psychobiotic, PersiLife company, Iran), for eight weeks. The control group was instructed to consume a matching placebo capsule containing 300 mg of maltodextrin for the same duration. To ensure blinding, both the probiotic and placebo packages were identical in color and shape. An independent individual labeled the packages as A or B, without knowledge of the study details. Participants were instructed to take one capsule before their main meal. New packages were distributed every four weeks, and follow-up calls and text messages, along with clinic visits, were conducted to monitor and cover side effects and treatment adherence. During the intervention period, participants were not prevented from receiving their usual medical treatments, and probiotics or a placebo were given as a supplement alongside other medications prescribed by the physician. Laboratory evaluations were conducted at the beginning and end of the study. After a 12-h fast, a 10-ml blood sample was collected from each participant and centrifuged at 2500 rpm for 10 min at room temperature. The serum was aliquoted into microtubes and immediately frozen at −80°C. Serum levels of total testosterone and SHBG were assessed using the immunoassay (ELISA) method, and the Free androgen index (FAI) was calculated using the following formula: FAI = 100 × (Total Testosterone)/SHBG. Serum levels of quantitative C-reactive protein (CRP) were measured using the turbidometry method. Total Antioxidant Capacity (TAC) levels were assessed with the CUPric Reducing Antioxidant Capacity method. Malondialdehyde (MDA) levels were quantified using the Thiobarbituric Acid Reactive Substances assay (TBARS), while superoxide dismutase (SOD) activity was determined by evaluating Mn-SOD's ability to prevent the conversion of resazurin to resorufin. All oxidative stress markers were measured following the instructions provided by the Kiazist company (Iran). Data on demographic variables, including age, education, marital status, medical history, medication and supplement use, PCOS onset, and history of infertility, were collected from each participant at baseline using validated tools. Body weight was recorded using a Seca scale, precise to 0.1 kg, while participants wore minimal clothing. Height was measured with a portable stadiometer accurate to 0.5 cm, with participants standing barefoot. To assess individuals' dietary intake, four one-day food recalls were completed at baseline and at weeks 3, 5, and 8 of intervention. Dietary food recalls were completed by a dietitian who was blinded to the study protocol. The quantity of each food was converted into grams per day using Iranian Household Measures [ 18 ] and subsequently converted into nutrient and calorie values using Nutritionist 4 software. The physical activity level among study participants was evaluated by the International Physical Activity Questionnaire (IPAQ) (This assessment included the hours spent sitting, sleeping, and engaging in physical activities of varying intensities—mild, moderate, and vigorous—throughout the day. Physical activity was measured over a 24-h period, with results reported as metabolic equivalent hours per day (MET/h/day). Previous studies have confirmed the validity and reliability of this 24-h physical activity assessment method [ 53 ]. To measure the severity of hirsutism, the Simplified Ferriman-Gallwey Score questionnaire was used in this study. This questionnaire includes three visual questions evaluating hirsutism. Participants scored hair growth intensity from 1 to 4 in three areas: upper lip, lower abdomen, and groin (1 representing the least intensity and 4 representing the most intensity of hair growth in the mentioned areas). A total score equal to or more than 4 was considered a predictor of hirsutism [ 51 ]. The reliability and validity of this questionnaire have already been confirmed in Iran [ 39 ]. The severity of acne was assessed using the global acne grading system [ 40 ]. The reliability and validity of this method have been previously confirmed in Iran [ 13 , 24 ]. In this scoring system, six areas—forehead, nose, right cheek, left cheek, chin, chest, and upper back—are evaluated for acne [ 40 ]. Additionally, to evaluate the clinical signs of alopecia, the Sinclair scale was used at the beginning and end of the study [ 14 ]. This method has been validated by previous studies in Iran [ 30 ]. This visual questionnaire consists of five stages that indicate the severity of alopecia [ 48 ]. SPSS version 21.0 (IBM Corp., Released 2012. IBM SPSS Statistics for Windows, Version. 26.0. Armonk, NY: IBM Corp) was used for all statistical analyses, and a P -value < 0.05 was considered statistically significant. Data normality was assessed using a one-sample Kolmogorov–Smirnov test along with an evaluation of skewness coefficients. Categorical variables were presented as counts and percentages, whereas continuous variables were reported as means accompanied by standard deviations. Baseline comparisons of qualitative and quantitative variables between the two intervention groups were conducted using the chi-square test and independent samples t-tests, respectively. Changes from baseline to the end of the intervention were presented as mean differences with 95% confidence intervals (CI). Independent samples t-tests were used for between-group comparisons, and paired t-tests assessed within-group changes over the two-month intervention period. To control for potential confounding factors, analysis of covariance (ANCOVA) was applied when comparing primary outcomes between groups. The research utilized an intention-to-treat analysis (ITT) using the expectation–maximization (EM) algorithm, encompassing all randomized participants regardless of protocol adherence [ 44 ].

This study suggests that an 8-week supplementation with Lactobacillus helveticus and Bifidobacterium longum may have beneficial effects on certain hormonal parameters (SHBG and FAI), oxidative stress (TAC, SOD, and MDA), and selected inflammatory markers (CRP) in women with PCOS. However, regarding clinical symptoms, including acne score, alopecia, and hirsutism, the between-group differences were not statistically significant. Overall, these findings imply that 8 weeks of probiotic supplementation may alleviate oxidative stress, modulate certain hormonal factors, and reduce low-grade inflammation in women with PCOS. While these findings are encouraging, confirming them in larger and longer-term studies is essential to more accurately determine the impact of probiotic supplementation in women with PCOS.

This study is a parallel, double-blind, randomized controlled placebo trial examining the effects of two probiotic strains ( Lactobacillus helveticus and Bifidobacterium longum ) on hormonal status, oxidative stress, and clinical symptoms in patients with PCOS. The results of the present study revealed potential beneficial effects of an 8-week intervention on hormonal parameters (SHBG, testosterone total, and FAI), oxidative stress (TAC, SOD, and MDA), and selected inflammatory markers (CRP). However, regarding clinical symptoms, including acne score, alopecia, and hirsutism, the between-group changes were not statistically significant. This study highlights the positive impact of probiotics on inflammation and oxidative stress, crucial factors in the pathophysiology of PCOS, which leads to IR, hyperandrogenism, and menstrual irregularities [ 47 ]. These findings align with previous research showing probiotics help restore hormonal balance and reduce oxidative stress in metabolic disorders [ 6 ]. The two probiotic strains, Lactobacillus helveticus and Bifidobacterium longum, have already shown antioxidant and anti-inflammatory effects, which confirms the findings of the present study [ 43 ]. Probiotics have been suggested to influence the gut microbiota composition, which in turn affects systemic inflammation and metabolic processes [ 3 ]. This is particularly relevant for women with PCOS, where dysbiosis has been implicated in exacerbating metabolic and endocrine disturbances [ 22 ]. In line with our findings, recent studies indicated that supplementation with the same probiotic strains could positively impact levels of CRP and stress-related responses [ 12 , 43 ]. Furthermore, a 12-week course of probiotic supplements in women with PCOS resulted in decreased levels of CRP and MDA [ 29 ]. Additionally, probiotics have been reported to reduce oxidative damage and improve systemic inflammatory profiles in various conditions, such as multiple sclerosis [ 31 ]. According to evidence, elevated oxidative stress and inflammatory cytokines increase the risk of hyperandrogenism, IR, cardiovascular disease, and diabetes in PCOS patients [ 5 , 38 ]. Probiotics may reduce inflammatory cytokines and lipid peroxidation, while modulating oxidative stress markers like MDA, SOD, and TAC via short-chain fatty acid (SCFA) production and decreased hydrogen peroxide radical generation [ 45 ]. Supporting these results, other studies report increased serum TAC levels [ 42 , 55 ], SOD levels [ 19 ], and decreased MDA levels [ 2 , 15 , 55 ] after supplementation with probiotics for at least 6 or 8 weeks. On the other hand, gut dysbiosis has been linked to changes in sex hormone levels and ovarian structure [ 23 ]. Hyperandrogenism, a key feature of PCOS, is linked to various clinical manifestations of the syndrome, including acne, hirsutism, and menstrual irregularities [ 11 ]. Some evidence has shown that lowering androgen levels is associated with enhanced ovulatory function and improved quality of life [ 10 , 36 ]. Although probiotic supplementation improved total testosterone levels and other hormonal markers (SHBG and FAI), regarding clinical symptoms, this trial found no significant between-group changes. One possible explanation is that the probiotic’s impact on visible symptoms—such as acne, alopecia, and hirsutism—may be more subtle and require a longer duration of intervention to become fully apparent. Previous studies on PCOS and probiotic supplementation have yielded mixed results regarding clinical symptoms. For example, some studies have reported improvements in acne and hirsutism with probiotic supplementation [ 21 ], while others have not observed significant clinical changes [ 1 ]. These inconsistent findings could be due to differences in study design, probiotic strains and dose, duration of intervention, or the severity of symptoms in the participants. Furthermore, clinical symptoms like acne and hirsutism are influenced by multiple factors, including genetic predisposition, diet, and hormonal fluctuations, which may limit the effect of probiotics in isolation [ 1 , 34 , 46 ]. Therefore, a more comprehensive approach that includes dietary and lifestyle modifications along with probiotic supplementation may be more effective in managing clinical symptoms. The exact mechanisms by which probiotics improve PCOS outcomes are not fully understood. One key pathway involves the production of SCFAs during intestinal fermentation, which can enhance gut barrier integrity, reduce inflammation, lower microbial endotoxins, and improve IR [ 16 ]. Additionally, probiotic interventions have been demonstrated to reduce PCOS symptoms by modifying gut microbiota, increasing the levels of Bifidobacteria and Lactobacillus , and restoring microbial balance [ 7 ]. Moreover, probiotics may enhance androgenic profiles by promoting insulin sensitivity, supporting nutrient digestion and absorption, modulating the gut microbiota, and exerting effects through the gut-brain axis [ 8 , 37 ]. This study represents the first clinical trial investigating the effectiveness of two specific probiotic strains on PCOS. The use of a double-blind, randomized, placebo-controlled design enhances the validity of the results by minimizing confounding factors through participant randomization. However, several limitations should be noted. Initially, the study did not measure any specific marker to definitively assess the diversity of the gut microbiota population before and after intervention. Additionally, the effects of probiotics as a standalone treatment cannot be assessed for ethical reasons, as it would be unethical to instruct participants to discontinue their standard medications. Lastly, the study is confined to a particular dose and ratio of supplements, which prevents the determination of the optimal dosage. Furthermore, the 8-week intervention duration is relatively brief, which may restrict the observation of long-term effects on PCOS outcomes. For future studies, it is recommended to evaluate the effects of probiotics with a longer intervention period, and if applicable, incorporating a marker to assess gut microbiota composition pre- and post-intervention is advisable.

Polycystic ovary syndrome (PCOS) is the leading cause of infertility in women and ranks among the most common endocrine disorders affecting the female population [ 54 ]. It affects an estimated 5 to 10 percent of women of childbearing age and is often linked to insulin resistance (IR) and obesity [ 49 ]. According to the Rotterdam criteria, the primary diagnostic indicators for PCOS include elevated androgen levels, ovulatory dysfunction, and a polycystic appearance of the ovaries [ 52 ]. Additional characteristics of this syndrome include enhanced secretion of androgen hormones, such as testosterone, coupled with a suppression of the liver's production of sex hormone-binding globulin (SHBG) [ 50 ]. Also, PCOS affects multiple body systems, leading to a range of abnormalities, including menstrual disorders, infertility, alopecia, acne, and hirsutism [ 4 ]. Moreover, chronic inflammation and oxidative stress are two contributing factors in the pathogenesis of PCOS [ 27 , 56 ]. The ovary, as a metabolically active organ, is constantly subjected to various forms of stress. In conditions like PCOS, excessive oxidative stress may lead to ovarian mesenchymal hyperplasia [ 17 ] and induce DNA damage in ovarian epithelial cells, ultimately triggering apoptosis [ 9 ]. Recently, the microbiological hypothesis states that the gut microbiome may play a fundamental role in the pathogenesis of PCOS. The gut microbiota is now viewed as a functional endocrine organ that influences inflammation, oxidative stress, and neural activity [ 33 ]. An imbalance in this microbial community, known as dysbiosis, has been linked to hormonal imbalances, IR, inflammation, and changes in ovarian structure [ 26 ]. Recent studies identified certain Lactobacillus and Bifidobacterium strains as beneficial for regulating sex hormones and metabolism [ 20 , 41 ]. Specifically, Lactobacillus species can metabolize estrogens in the gut through beta-glucuronidase activity [ 32 ]. The choice of Lactobacillus helveticus and Bifidobacterium longum at the selected dose is supported by previous studies showing improvements in mood disorders and anxiety in healthy individuals [ 35 ], as well as reductions in depression, stress, CRP levels, and sleep disturbances in patients with depression following 8-week supplementation [ 43 ]. Furthermore, Carlman et al. demonstrated the potential of these two probiotic strains as a non-pharmacological strategy for alleviating stress-related responses in healthy subjects [ 12 ]. Given the close association of PCOS with mood disorders, inflammation, and oxidative stress, probiotics may offer benefits for managing PCOS symptoms by restoring gut balance and improving antioxidant, inflammatory, and hormonal status [ 25 ]. To our knowledge, no clinical trial has evaluated the combined effects of Lactobacillus helveticus and Bifidobacterium longum on biochemical markers and clinical symptoms in women with PCOS. Therefore, this randomized controlled trial (RCT) was designed to assess the effects of supplementation with these two probiotic strains on hormonal status, oxidative stress, and clinical symptoms in women with PCOS.

Supplementary Material 1. Supplementary Material 2. Supplementary Material 1. Supplementary Material 2.