This is a prospective, multicenter, analyst-blinded and randomized controlled trial. One healthy control group and two parallel experiment arms will be proposed in this study: (1) people without PCOS (health control group); (2) PCOS patients diagnosed based on clinical indexes (group one); (3) PCOS patients diagnosed based on metabolomics indexes (group two). A total of 276 eligible people will be recruited, which includes 60 people without PCOS in healthy control group while other 216 patients with PCOS will be randomly assigned to different diagnosis groups in a 1:1 ratio. After group assignment, necessary medical detection, relative information, biological samples of healthy control group will be collected. So the other two diagnosis groups as well. In addition, the blood sample of all groups will be detected by metabolomics for further study while the analysis results will be totally blinded to the groups and their research doctors. Based on our previous study ,we found there were two subgroups of PCOS patients, which one was mainly relevant to hormone metabolism disorders (subgroup one) while the other was mainly to lipid metabolism disorders (subgroup two).
Due to this, patients in two diagnosis groups will be automatically divided into two different subgroups according to their characteristics. The main difference is the division of group one diagnosed based on indexes recommended by the statements, such as clinical symptoms, signs and common clinical indexes (includes the ultrasonography), while the group two, further than that, mainly based on metabolomics indicators. All patients will receive corresponding treatment according to their subtypes, one is darin–35 and the other is metformin. The interventions for all experiment groups will last at least for 6 months and periodically evaluate in this trial.
Outcomes assessment and data will be analyzed periodically and by the one who is blinded to the assignment of participants. The design of study is briefly illustrated in the flow chart in Fig. 1, the timetable of the study is presented in Fig. 2 and Fig.3.
The main treatment center will be the Department of Endocrinology and Metabolism in Wuxi People’s Hospital affiliated with Nanjing Medical University, which will enroll 60 participants in healthy control group and 108 participants in experiment group. Two other treatments centers—Wuxi Maternal and Child Health Centers Clinical Hospital, Jiangyin People’s Hospital will enroll the other 108 participants.
Healthy control group mainly recruit the people without PCOS and other disorders may influence the sex related hormone and metabolism conditions. For the experiment group, all eligible participants are diagnosed as PCOS according to the 2018 Chinese Endocrine Society clinical practice consensus of PCOS , which based on worldly accepted criteria published by Rotterdam [22, 23]. Patients who are in reproductive age and can obey the strict contraception within six months will be informed of this trial. If the recruiter expresses interest, a face-to-face interview about the whole trial process will be conducted in a reception room for clinical research subjects in the three hospitals. Patients who meet the inclusion criteria and except from who meet the exclusion criteria can join in the trial once the consent form is signed. We publicized the trial to potential participants in two ways: 1) experiment groups will be recruited by approaching patients with PCOS admitted to an outpatient department or inpatient ward in each center, the official microblog and WeChat platforms of each center will also be used; 2) healthy control group will be directly recruited by approaching volunteers in health examine center of Wuxi People’s hospital.
Participants who meet all the following criteria can be enrolled in experimental group:
1) Diagnosed as a PCOS according to the 2018 Chinese Endocrine Society clinical practice consensus.
(diagnosed with the presence of at least two of the following three, and the fourth must be followed:
2. oligo-anovulation (menstrual cycle length > 35 days and < 8 menstrual cycles per year);
3.polycystic ovaries morphology (having at least 25 small follicles (2–9 mm) in the whole ovary, and/or increased ovarian volume ≥ 10 ml;
2) Aged 18–45 years, reproductive female.
3) Able and willing to comply with the intervention and follow-up evaluation.
4) Signed the consent before the study.
1) Currently receive treatment in another experimental study, or just finished another experiment less than 30 days.
2) Treated with daine–35, metformin or other estrogen, progesterone, lipid-regulating and hypoglycemic drugs (such as glucocorticoids, spironolactone, antibiotics, bacteria regulators and anti-inflammatory drugs) within 12 weeks.
3) Allergy or intolerance to daine–35, metformin or any of its components.
4) Suffers from congenital adrenal cortical hyperplasia/ hypercorticosis/ androgen-secreting tumor/ Cushing’s syndrome/thyroid dysfunction/ hypogonadotropin deficiency/ hyperprolactinemia disease/ premature ovarian insufficiency/ functional hypothalamic amenorrhea/ diabetes.
5) A medical history of malignant tumors, especially gynecological malignant tumor history of surgery, radiation and chemotherapy.
6) Suffer from liver function damage (ALT, AST upper 1.5 times of the limits of laboratory index) and chronic liver disease.
7) Suffer from hematopenia/ thrombotic disease.
8) In pregnant or expect to be pregnant during the study.
9) In an unstable medical physical and mentality condition.
10) Medical history of symptomatic ventricular arrhythmias with torsion ventricular tachycardia.
11) Unable to complete the procedures of the study protocol.
12) Any other situation that would interfere with the study evaluation, procedures, or completion.
1) The subject quits.
2) Safety issues (e.g. adverse events, failure of contraceptive measures and accidents).
3) lost to follow-up.
4) Researchers remove (e.g. poor compliance, complications considerations or serious adverse events).
Comprehensive suspension criteria:
1) A significant safety problem is found.
2) The diagnostic and therapeutic effect is poor (we will evaluate the process in the 3rd month of therapy).
3) A major mistake which will affect the results in the plan.
4) A huge problem in funding or management.
The Department of Good clinical practice of Wuxi People’s Hospital will generate the randomization process through a random number generator (SPSS 21.0, SPSS Inc., Chicago, IL, USA).
Random sequences will be placed in opaque envelopes, numbered orderly and sent to a clinical researcher and therapist.
The envelopes will be opened sequentially to decide the allocation for participants. In this trial, the therapist and the trial participants are blinded to the group assignment while the trial administrator will monitor the whole study process.
In the experiment groups, participants will not be informed of the group assignment, the type of diagnosis and treatment that they will receive. Study assessors, data managers and the statistician will be blinded in this trial. In this trial, the clinical therapists will be blinded to the assignment of group one which would decide only by their clinical practice, while they have an access to the diagnosis and treatment allocation of group two. It should be mentioned that the clinical therapists must learn how to use the blinding method to communicate with participants to ensure the diagnosis and treatment blinding. The blinding procedure will be operated until the data are locked and the trial is finished.
Healthy control group
Participants without PCOS and exclude any other disorders which may influence the sex related hormone and metabolism conditions, including the usage of drugs, will be recruited. Necessary medical physical detection will be made to confirm the qualification. Then, our group will collect relative medical history, physical examination and the blood samples of all qualified participants for further study.
Clinical indexes group
For clinical indexes group, blood samples and medical information of all qualified participants will be collected. According to the clinical indexes and the Chinese Endocrine Society clinical practice consensus of PCOS in 2018, the participants will be divided into two subgroups, which one was mainly relevant to hormone metabolism disorders (subgroup one) and the other was mainly to lipid metabolism disorders (subgroup two). Participants in different subgroup will receive corresponding treatment. Based on several statements and clinical experience, subgroup one will receive oral Daine–35 once a day for 21days and pause for 7 days. Subgroup two will receive oral metformin (2000–2500mg) once a day based on their conditions. The whole treatment session will last at least for 6 months. During the trial, blood samples will be collected three times (baseline, Week 15±1,Week 26±1) for metabolomics detection to assess the changes. However, the detection information will be blinded to all the participants and clinical research doctors in case influences the results. It should be mentioned that there is one and only one chance on the 3rd month of the treatment to let the clinical research doctors to change the scheme of participants according to their condition. All clinical research doctors will be who has majored at endocrinology and metabolism or gynecological endocrinology for at least 10 years. They also must have become an attending doctor for more than 5 years. In addition, they must receive professional training of clinical trials and pass the test to ensure their consistency of study methods.
Metabolomics indexes group
For metabolomics indexes group, majority part of protocol is similar with the clinical indexes group. The main difference is that the division of the subgroup will be based on metabolomics indexes. The therapists of this group will be informed of the allocation of subgroup and give corresponding treatment scheme to all participants. It should be mentioned that there will be no chance for them to change the scheme during the trial and they will still be blinded to the metabolomics results to free of any possible influence.
Allowance of concurrent treatment of patients
All other treatments for PCOS are banned during the trial, including oral contraceptives, the broad-spectrum lipid drug and any other drugs will influence the results. Participants may receive any treatment that is not related to PCOS and use the condom. Any change in concurrent treatment will be recorded at every visit.
For healthy control group, they will complete the trial until their samples are collected. For experiment groups, the primary outcome will be the changes of their PCOS relative condition evaluated by the metabolomic indexes. The secondary outcomes will be assessed by the clinical indexes, signs, symptoms advised by the consensus of Chinese Endocrine Society in 2018.
There are three metabolic markers which is palmitoyl sphingomyelin, cGMP and DHEAS. There are twenty-five clinical indexes, which includes eight sex hormone-related indexes: FSH, LH, PRL, E2, P, Ts, AMH and VitD; nine blood lipid related indicators: TC, TG, HDL, LDL, ApoA1, Apo B, Lp(a), FFA, hsCRP; seven endocrine-related indicators: GLU (0h,30min,120min), INS (0h,30min,120min), BUA. Ultrasonography examination is also included, especially the number and diameters of follicles of bilateral ovary. The main evaluation clinical index was LH/FSH ratio.
The safety outcome will be any severe impairment in liver function or be unable to tolerate related therapeutic drugs and it will be monitored after each treatment session. The exploratory outcome will be the entire recovery of PCOS after treatment within 26 weeks.
Researchers will evaluate at each visit about adverse events (AEs), which include every unexpected or unfavorable responses occurred during or after treatment. These events may do not have a causal relationship with the study. However, the investigators have to ensure that all AEs should be reported and recorded in the subject’s medical records. In this trial, AEs are defined as (1)cause a disorders that will hinder ability to work or be a threat to life(especially the liver dysfunction) (2) lead to hospitalization or prolong a department or hospital stay. Remedial treatment should be given immediately and all AEs will be reported to the responsible units, ethical committees and trial administrator to determine whether the participant ought to stay in or have to drop out of the trial. No matter the decision is, all participants with an AE will be followed up until the event has been resolved or the condition has become chronic or stable.
To evaluate the accuracy and efficacy of diagnosis, the safety and superior effects of individual interventions, a six month telephone follow-up will be given after the trial. During the period, no participants will undergo special therapy with the exception of routine cervical care. At weeks 30,34,38 and 41, the outcome assessor will telephone participants to investigate the condition of PCOS, which mainly about menstrual cycle length and frequency, the appearance of hyperandrogenism(such as weight loss, acne, excessive facial and body hair) and whether be pregnant or not. Participants are welcomed to inform the assessors of their clinical symptoms and AEs via face-to-face, email, text message, or WeChat at the relevant time points.
Blinding test and credibility test
The results of metabolomics detection on blinding will be completed at weeks 0,15 and 26. The implementation of the blinding strategy will be crucial for the trial. The credibility rating for both diagnosis methods and different types treatments will be estimated using a credibility test at week 15,26 and at the follow-up period week 41.
Data collecting and monitoring
Demographic, basic personal medical related characteristic data will be collected and recorded by screeners when participants are recruited. Clinical symptoms and signs, physical examinations and metabolomics detection results, short-term and long-term outcomes, assessment of diagnosis accuracy and treatment efficiency, details of AEs will be recorded by trial assessors and clinical researchers in case report forms (CRFs).
Completed CRFs are checked and reviewed by a five-person steering committee, which is composed of two supervisors of the major trial center, a data statistician and two data administrators. Committee are totally independent from the research team and constantly blinded to group allocation.
All data entry and management will employ the Excel database. In addition, the committee members are qualified with data analysis and are trained uniformly.
To ensure the accuracy of the data, two data administrators independently enter and mutual proofread it. If there are issues with the information in the CRF, the data administrators will give it straightly to the steering committee. Any revision will be modified by the administrators according to committee. Once the accuracy of data be confirmed, the electronic database will be locked while the real-time tracking and monitoring will still be opened. The Department of Good Clinical Practice in Wuxi People’s Hospital, which is independent of the trial, will in charge of monitoring the entire data which includes the primary script of the CRFs, protocols for researchers, informed consents and other files.
The data set will include a safety set, a full analysis set of trial (FAS) and a per protocol set. The safety set will be designed for the participants who were randomly assigned and received at least one session of treatment. The FAS will include all medical data related to the trial which will indicate the intervention made while the one who misses the primary outcome evaluation will be excluded. In the per protocol set, participants at least receive three constant sessions of treatment (50%).
In this trial, the FAS will be used for the basic analysis. For missing data, we will take imputation adjustment approach and the last observation analysis will be selectively used to handle the missing data.
The sensitivity analysis will then be compared the results from the per protocol analysis and different intervention analysis to evaluate the impact of missing data on the trial results.
According to the homogeneity of data, various statistics methods, such as parametric statistics or non-parametric will be used for the analyses. Descriptive statistics about the demographic and baseline condition will mainly be presented at appropriate indexes, such as means± standard deviations (SD), medians and interquartile ranges (IQR), or percentages. The covariance analysis will be performed if it is necessary.
Efficacy will be measured at three time points. Therefore, a repeated measures analysis of variance will be used to investigate the efficacy of diagnosis and treatment between different intervention groups. Two-sided paired t tests, chi-squared test or a Fisher’s exact test will be performed in the analysis.
The Statistical Package for Social Sciences (SPSS, version 21.0, SPSS Inc., Chicago, IL, USA) will be performed to analyze all trial data. The confidence interval will be established at 95% and the significance level at 0.05.
Sample size calculation
According to the literature and previous small sample pilot research, via the metabolomics techiques, the sensitivity and specificity to classify the control and PCOS are 100% and 86%. The rate between the control and subgroup one is 96.7% and 100% while the rate between the control and subgroup two is 100% and 86.2%. The sensitivity and specificity to divide two subtypes of PCOS are 90.9% and 87.5%. In order to meet the lower Statistical limit, each subgroup should be at least 30 people.
According to a presumption maximum dropout tolerance of 20%, with a significance level of 0.05 and power of 0.80, we calculated required sample size in experiment groups were 216. In this trial, there are 60 people in health control group, 108 people for each experiment group across the three centers.
During the trial, quality control will be carried out by the steer committee. All researchers are required to train on trial methods, techniques and relative rules of the study which could keep the data consistent and stable. It should be mentioned that whatever the modifications or corrections to be performed in this trial, it all has to be discussed, decided by and submitted to the steer and ethics committee. In addition, details should be kept through the trial.