A randomized double-blind, placebo-controlled, parallel multicenter phase III study to evaluate the effectiveness and safety of Probiotic IND 2-A and IND 2-B Blends of strains VHBSUB, VHBCSI, VHBAX on the clinical and biological parameters of reproductive-aged PCOS women

Protocol Full Title:A randomized double-blind, placebo-controlled, parallel multi-center phase III study to evaluate the effectiveness and safety of Probiotic on the clinical and biological parameters of reproductive-aged PCOS women.

Phase of Development :Phase III Name of the Investigational product:Probiotic

STUDY OBJECTIVE(S) :Primary Objective: To evaluate effectiveness of Probiotic on the clinical and biological parameters of reproductive-aged PCOS women Secondary Objective : To evaluate safety of Probiotic  on the clinical and biological parameters of reproductive-aged PCOS women.

STUDY DESIGN:A randomized double-blind, placebo-controlled parallel multi-center phase 3 clinical trial.

STUDYCENTER(S):Multicenter

SAMPLE SIZE:The sample size required for this study was estimated to be 60 patients (30 patients per treatment group). This number was based on the results in an earlier unpublished study. The assumed study parameters would provide 80% power for a 2-sided t test at a significance level of 0.05.

STUDY DURATION:06 month

PLANNED DURATION OF TREATMENT: 90 days

STUDY ENROLLMENT AND WITHDRAWAL CRITERIA:Inclusion criteria: Patients with meeting all of the following criteria will be considered for enrollment in the study: • Women between 18-70 years of age. •Diagnosed PCOS defined as 2003 Rotterdam diagnostic criteria (the presence of antral follicle count (AFC) of 12 or more and/or an ovarian volume greater than 10 mL) •Voluntarily participating in the clinical study; fully understanding and being fully informed of the study and having signed the Informed Consent Form (ICF) •Willingness and capability to complete all the study procedures. Exclusion criteria: Patients with history or significant presence of the following will be excluded from participation/enrollment in the study trial: • During the pregnancy and lactation period. • Significant impaired liver function, impaired renal function, mental disease, severe infection, severe anemia, severe heart disease and neutropenia disease. •Use of antibiotics within 3 months. •Symptoms of any infection at screening. •Immunodeficient or use of immunosuppressive drugs. • Use of products containing prebiotics or probiotics within the last 3 months. • Previous history of gastrointestinal surgery or disease (such as peptic ulcer, irritable bowel syndrome, inflammatory bowel disease or other gastrointestinal disorder). • Medical conditions or diseases that may affect subject safety or confound study results in the opinion of the investigator

DOSE AND MODE OF ADMINISTRATION (Need to be provided by  sponsor) : •The investigational product (IP) is a Probiotic which will be supplied by Sponsor. • The placebo contained only excipient, maltodextrin (1.00 g). The test group –(n = 30) will Probiotic (______)billion colony-forming unit activity powder (carrier maltodextrin) thrice a day.  (details to be provide by sponsor) Control (placebo) group – (n = 30) will receive maltodextrin with similar dosing schedule. • The packaging and labeling for both the products were same except the coded batch numbers used for differentiation.

EVALUATION SCHEDULE:The patients will be followed up during the Day 1, Day 45 and Day 900. All patients were required to log their compliance in the provided forms given to them throughout the study. Improvement in menstrual cycle Patients will be required to maintain a menstrual calendar and record their basal body temperature for the duration of the study and asked to fill a questionnaire before and after study. The questionnaire will cover menstrual cycle dates, duration and amount. Menstrual Symptom Questionnaire Menstrual symptoms were measured using the MSQ (Chesney & Tasto, 1975). The MSQ is a 24 item self-report measure which assesses menstrual pain and symptoms (e.g. ‘I have cramps that begin on the first day of my period’; ‘I feel depressed for several days before my period’). The score on each item ranges from 1 (never) to 5 (always) with a higher composite score indicating more symptoms. 12 weeks Improvement in hirsutism Modified Ferriman and Gallwey score (m-FG scores) will be measured before and after study. M-FG score > 8 is considered hirsutism and when the score is higher, the symptom is more serious. 12 weeks Improvement in acne score Visual assessment of acne using "mild/moderate/severe" grades will be assessed before and after study. 12 weeks Hematological and hepatic biomarkers were analyzed following the standard medical test protocols at screening (baseline) and end of treatment (Day 90) • Fasting Blood sugar level • HbA1c • Lipid profile • FSH • LH • Serum estradiol levels •Serum testosterone levels • C-reactive protein To assess effectiveness on polycystic ovaries using ultrasound (USG) will be used. Vital signs measurement and physical examination will be carried out at Screening, Day1, Day 45 and Day 60. All adverse events will be reported during the entire duration of study.

STUDY ENDPOINTS

Primary End Point: •Change in the MSQ symptom score from baseline to Day 90 (end of study) Secondary End Point: •Change in the MSQ symptom score from baseline to Day 45 (end of study) •Change in the Modified Ferriman and Gallwey score from baseline to Day 45 and Day 90 (end of study) •Change in the Visual assessment scale score for acne from baseline to Day 45 and Day 90 (end of study) •Change in the Fasting Blood sugar level from baseline to Day 90 (end of study) • Change in the HbA1c level from baseline to Day 90 (end of study) •Change in the FSH from baseline to Day 90 (end of study) • Change in the LH  from baseline to Day 90 (end of study) • Change in the Serum estradiol levels from baseline to Day 90 (end of study) •Change in the Serum testosterone levels from baseline to Day 90 (end of study) •Change in the C-reactive protein (mg/L) levels from baseline to Day 90 (end of study) •Change in the lipid profile parameters [triglycerides (mmol/L), total cholesterol (mmol/L), low density lipoprotein cholesterol (mmol/L), and high density lipoprotein cholesterol (mmol/L)] from baseline to Day 90 (end of study) •Change in the antral follicle count from baseline to Day 90 (end of study) •Change in the ovarian volume from baseline to Day 90 (end of study) Safety endpoints • Number of participants who Experienced at least one Adverse Event during the study duration •Number of participants who discontinued study drug due to an Adverse Event during the study STATISTICAL ANALYSIS:The statistical evaluation will be performed using appropriate statistical tests. The details of statistical analysis to be performed will be described in Statistical Analysis Plan (SAP). Statistical analysis will be performed using the latest version of SAS® system software (SAS Institute Inc., USA). Symptoms considered were bloating and cramping, abdominal pain, diarrhoea and constipation, stomach rumbling, nausea, vomiting, headache, and anxiety. Intergroup mean difference [(Test) – (Placebo)] for all the symptoms was analyzed through ANOVA and 95% confidence interval (CI) estimation. Safety analyses were based on the safety population, which was defined as all randomized patients who received study treatment, and were summarized by the actual treatment received. Significant levels will be set at p<0.05. All analyses will be based on the intention-to-treat principle. Missing values will be handled by the modern imputation methods which will be accomplished using a set of repeated imputations created by predictive models based on the majority of participants with complete data. Demographic characteristics and other baseline values will be described using descriptive statistics for each group. Continuous variables with normal distribution will be expressed as the means with SD; for abnormally distributed variables, the data will be expressed as medians with a centile range (such as the 25th and 75th centiles). Numbers and proportions will be used to describe the categorical variables. Between-group differences in primary and secondary outcomes will be tested using repeated measures analyses of variance.

**ETHICAL CONSIDERATIONS:**The study will be conducted as per the National Ethical Guidelines for Biomedical and Health Research involving Human participants ICMR (2017), ICH (Step 5) ’Guidance on Good Clinical Practice’, New Drugs and Clinical Trials Rules 2019 G.S.R. 227(E) dated 19 Mar 2019, ’Good Laboratory Practice’, ‘Good Clinical Practices for Clinical Research in India’ Guidelines, Good Clinical Laboratory Practice (GCLP) and Declaration of Helsinki (Fortaleza, October 2013).