This is a prospective, multicenter, analyst-blinded, randomized controlled trial. There will be one healthy control group and two parallel experiment arms in this study: (1) people without PCOS (health control group); (2) PCOS patients diagnosed based on clinical indexes (group 1); (3) PCOS patients diagnosed based on metabolomic indexes (group 2). A total of 276 eligible people will be recruited, including 60 people without PCOS in the healthy control group while the other 216 patients with PCOS will be randomly assigned to different diagnosis groups in a 1:1 ratio. After the group assignment, necessary medical detection, relative information, and biological samples of the healthy control group, as well as the two diagnosis groups, will be collected. Additionally, the blood samples for the participants in all of the groups will be tested using metabolomics for further studies, while the analysis results will be totally blinded to the participants in the groups and their research doctors. Based on our previous study ,we found there were two subgroups of PCOS patients; one included participants mainly with hormone metabolism disorders (subgroup 1), while the other included participants mainly with lipid metabolism disorders (subgroup 2).
As a result of this, patients in the two diagnosis groups will be automatically divided into the two different subgroups according to their characteristics. The main difference is the division of group 1 diagnosed based on indexes recommended by the statement, such as clinical symptoms, signs, and common clinical indexes (including ultrasonography), while group 2, will be selected mainly based on metabolomic indicators. All patients will receive corresponding treatment according to their subtypes, one being darin–35 and the other being metformin. The interventions for all experimental groups will last for 6 months and the results will be evaluated if three constant sessions( 3 months) of treatment is finished.
Outcome assessment will be conducted and data will be analyzed by someone who is blinded to the assignment of the participants. The design of study is briefly illustrated in the flow chart in Fig. 1, and the study timetable is presented in Figs. 2 and 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 the healthy control group and 108 participants in experiment group. Two other treatments centers—Wuxi Maternal and Child Health Centers Clinical Hospital and Jiangyin People’s Hospital—will enroll the other 108 participants.
Participants and recruitment
People without PCOS will mainly be enrolled in the healthy control group excluded other disorders may influence the sex related hormone and metabolism conditions. For the experiment group, all eligible participants will be diagnosed with PCOS according to the 2018 Chinese Endocrine Society clinical practice consensus on PCOS , which is based on the globally accepted criteria published by Rotterdam [22, 23]. Patients who are of reproductive age and can obey the protocol including strict contraception over the course of 6 months will be informed of this trial. If the potential participant 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 are eligible for enrollment in the trial if they are willing to provide written informed consent. We will publicize the trial to potential participants in two ways: 1) experimental 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 groups will be directly recruited by approaching volunteers in the health examination center of Wuxi People’s hospital.
Participants who meet all the following criteria will be enrolled in the experimental group:
- Diagnosed with PCOS according to the 2018 Chinese Endocrine Society clinical practice consensus. (Diagnosed with the presence of at least two of the following three conditions, and the exclusion of other etiologies must be followed: 1. Clinical or biochemical hyperandrogenism; 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 evaluations.
- Provides written informed consent prior to being enrolled.
Participants will be excluded if they meet any of the following criteria:
- Currently receiving treatment in another experimentally study, or just finished another trial less than 30 days ago.
- Treated with daine–35, metformin, or other forms of estrogen, progesterone, lipid-regulating, and hypoglycemic drugs (such as glucocorticoids, spironolactone, antibiotics, bacteria regulators, and anti-inflammatory drugs) within 12 weeks.
- An allergy or intolerance to daine–35, metformin, or any of its components.
- Suffers from congenital adrenal cortical hyperplasia, hypercorticosis, androgen-secreting tumors, Cushing’s syndrome, thyroid dysfunction, hypogonadotropin deficiency, hyperprolactinemia disease, premature ovarian insufficiency, functional hypothalamic amenorrhea, or diabetes.
- A medical history of malignant tumors, in particular a gynecological malignant tumor history of surgery, radiation, and chemotherapy.
- Suffers from liver function damage (ALT and AST above 1.5 times of the normal limits of the laboratory index) and chronic liver disease.
- Suffers from hematopenia or thrombotic disease.
- Is pregnant or expects to be pregnant during the study.
- In an unstable medical, physical, or mental state.
- Has a medical history of symptomatic ventricular arrhythmias with torsion ventricular tachycardia.
- Unable to complete the procedures outlined in the study protocol.
- Any other situation that would interfere with the study evaluation, procedures, or completion.
The dropout criteria are defined as follows:
- The subject quits.
- Safety issues (e.g. adverse events, failure of contraceptive measures, and accidents).
- Lost to follow-up.
4) Researchers remove the participant from the study (e.g. poor compliance, such as severe liver dysfunction,complication considerations, or serious adverse events.).
The comprehensive suspension criteria will be as follows:
1) A significant safety problem is found, such as the liver function of the patient is seriously impaired or sudden onset of other life-threatening illness that be unble to continue the study.
2) The diagnostic and therapeutic effect is poor, such as menstrual/metabolic disorders did not improve (we will evaluate this in the 3rd month of therapy).
3) A major mistake ( such as patients taking the wrong doses, taking or using medicine without following doctor’s advice) is made that will affect the results of the study.
4) There is a huge problem in funding or management, such as withdrawal of funding.
The Department of Good clinical practice of Wuxi People’s Hospital will lead the randomization process by using a random number generator in the Statistical Package for Social Sciences (SPSS) version 21.0 (SPSS Inc., Chicago, IL, USA).
Random sequences will be placed in opaque envelopes, numbered in order, and then will be sent to a clinical researcher and doctors.
The envelopes will be opened sequentially to decide upon the allocation for participants. In this trial, the doctors and the trial participants will be blinded to the group assignment.
In the experimental groups, participants will not be informed of the group assignment, the type of diagnosis, or the treatment that they will receive. Data managers, and the statistician will be blinded in this trial. But the trial administrator ( The steering committe, the Department of Good Clinical Practice in Wuxi People’s Hospital) will be not blinded in order to be responsible for the assignment of patients in different subgroups and monitor the whole study process. Besides, the clinical research doctors will be blinded to the assignment of group 1 whose treatment will be decided based on their clinical practice, while they have access to the diagnosis and treatment allocation for group 2. The clinical doctors will be responsible for learning how to use the blinding method to communicate with participants to ensure the diagnosis and treatment blinding. The blinding procedure will be conducted until the data are locked and the trial is completed. Additionally, unblinding will only be permitted in the case of a medical emergency or the participants quits the trial. All cases of unblinding will be documented.
Healthy control group
Participants will be recruited if they do not have PCOS, metabolism conditions, or any other disorders which may influence their sex related hormones and if they are not using any drugs which could also influence sex hormones. Necessary medical physical diagnosis will be conducted to confirm the qualification. Then, the trial team will collect information regarding the medical history, will conduct a physical examination, and collect blood samples from all qualified participants for further investigation.
Clinical indexes group
For the 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 on PCOS in 2018, the participants will be divided into two subgroups; one in which participants have hormone metabolism disorders (subgroup 1) and the other in which participants mainly have lipid metabolism disorders (subgroup 2). Based on several guidelines and clinical experience, subgroup 1 will receive oral Daine–35 once a day for 21days; thereafter this will be paused for 7 days. Subgroup 2 will receive oral metformin (2000–2500mg) once a day based on their clinical conditions. The whole treatment session will last for at least 6 months. During the trial, blood samples will be collected three times (the week 0, Week 15±1, and Week 26±1) in order to conduct metabolomic detection to assess the changes. However, the detection information will be blinded to all the participants and the clinical research doctors in case it influences the results. All clinical research doctors will be required to have majored in endocrinology and metabolism or gynecological endocrinology for at least 10 years. They also must be employed as an attending doctor for more than 5 years. Finally, they must receive professional training on clinical trials and pass a test to ensure their consistency performance in study methods.
Metabolomic indexes group
For the metabolomic indexes group, the majority of the interventions will be similar to those from the clinical indexes group. The main difference is that the division of the subgroup will be based on metabolomic indexes. The doctors in this group will be informed of the allocation of the subgroup and give the corresponding treatment scheme to all of the participants. It should be mentioned that there will be no chance for doctors to change the scheme during the trial and they will still be blinded to the metabolomic results to minimize the possibility of any influence on treatment decisions.
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 that might influence the results. Participants may receive any treatment that is not related to PCOS and should be instructed to use condoms when having sexual intercourse. Any change in concurrent treatment will be recorded at every visit.
For the healthy control group, they will remain enrolled in the trial until all their samples are collected. For the experimental groups, the primary outcome will be the changes in their relative PCOS condition evaluated using clinical indexes. The main clinical index that will be evaluated will be the LH/FSH ratio.
The secondary outcomes will be assessed using the other indexes, signs, and symptoms as described in the consensus of the Chinese Endocrine Society in 2018,such as 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 and hsCRP); endocrine-related indicators (GLU [0h,30min,120min], INS [0h,30min,120min], and BUA). There are also three metabolic markers will help us to evaluate the outcomes, which contains palmitoyl sphingomyelin, cGMP, and DHEAS. Ultrasonography examination is also included; the number and diameters of the follicles of the bilateral ovary will be measured.
The safety outcome will be any severe impairment in liver function or the inability to tolerate (such as nausea and vomiting which caused by metformine) related therapeutic drugs. It will be monitored after each treatment session(one month) via the hepatic function test. The exploratory outcome will be the entire recovery of PCOS after treatment within 26 weeks.
The occurrence of adverse events (AEs) will be evaluated at each visit; this will include every unexpected or unfavorable response that occurred during or after treatment. These events may not have a causal relationship with this study. However, the investigators need to ensure that all AEs are reported and recorded in the subject’s medical records. In this trial, AEs are defined as (1) disorders that will hinder one’s ability to work or be life threating (especially liver dysfunction) (2) lead to hospitalization or prolong a department or hospital stay. Remedial treatment should be given immediately to resolve the observed AE, and all AEs will be reported to the responsible units, ethical committees, and to the trial administrator to determine whether the participant ought to remain in or drop out of the trial. No matter what is decided, all participants with an AE will be followed up until the event has been resolved or the condition has become chronic or stable.
The study will not add the times of collection of blood samples and the drugs of treatment in this trial is suggested by Consensus of Chinese endocrinologists for the diagnosis and treatment of polycystic ovary syndrome. In this situation, our study do not add any extra harms to participants. However, once serious damage related to the study is happened, we will take the relevant expenses (including the diagnosis and treatment) and follow up the situation of the participants until recovery.
To evaluate the accuracy and efficacy of diagnosis, the safety, and the superior effects of individual interventions, a 6 month telephonic follow-up (or via ace-to-face, emails, text messages, or WeChat) will be conducted after the trial. During the follow-up 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 call participants to investigate the PCOS condition, asking mainly about menstrual cycle length and frequency, the appearance of hyperandrogenism (such as weight loss, acne, excessive facial and body hair), and pregnancy. Participants are welcomed to inform the assessors of their clinical symptoms and AEs via face-to-face, emails, phone,text messages, or WeChat at the relevant time points.
Blinding and credibility tests
The findings from metabolomic 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 the diagnosis methods and different types of treatments will be estimated using a credibility test at week 15, 26, and at week 41 during the follow-up period.
Data collection and monitoring
Data on and basic personal medical characteristics will be collected and recorded by screeners when participants are recruited. Clinical signs and symptoms, physical examination findings, metabolomic detection results, short- and long-term outcomes, assessment of diagnosis accuracy and treatment efficiency, and details of the AEs will be recorded by trial assessors and clinical researchers in case report forms (CRFs).
Completed CRFs will be 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. Committees are completely independent from the research team and constantly blinded to the group allocation.
All data entry and management will be conducted in an OpenClinica System (version 3.12) database. The committee members are qualified in data analysis and will have been trained uniformly.
To ensure the accuracy of the data, two data administrators will independently enter and validate data. If there are issues with the information in the CRF, the data administrators will bring this to the attention of the steering committee. Any revisions will be modified by the administrators according to the feedback provided by the committee. Once the accuracy of data is confirmed, the electronic database will be locked while the real-time tracking and monitoring will still be opened.
The steering committee and the department of Good Clinical Practice in Wuxi People’s Hospital will be in charge of monitoring the whole trial processes (e.g., checking the progress of recruitment, participant data, including CRFs, the protocols for researchers, informed consent forms, and any other study specific files.)and are going to audit trial conduct at least one time per month.
All these information will be made available to investigators whose proposed use of the data has been approved by the committee, for up to 15 years following publication. Findings will be presented at conferences and published in peer-reviewed journals. A summary of the findings will be provided to participants if they request via phone.
Smaple Collection and Handling
The actual dates and times of sample collection must be recorded in the laboratory requisition form. Instructions for the collection, handling, storageof samples are found in the laboratory manual that will be provided.
Standard operation of plasma separation for clinical patients:
(1) fresh anticoagulant peripheral blood was collected with the EDTA anticoagulant tube.
(2) the fresh peripheral blood was centrifuged at 4°C at 1700G for 10min, and the upper plasma was carefully collected to avoid touching the lower layers of red blood cells and white blood cells.
(3) the collected plasma was centrifuged at 4°C at 2000G for 10min and the supernatant was taken.
(4) divide the plasma and freeze it in ‐80℃ refrigerator for later use.
Precautions for plasma separation:
(1) the plasma separation process is performed on the ice to maintain the integrity of the blood cells.
(2) the whole separation operation shall be completed within 4 hours.
(3) the process of blood collection and plasma separation should be handled carefully to avoid the risk of hemolysis.
After the study, all the samples will be destruced.
The dataset will include a safety set, a full analysis set (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 conducted; however, individuals who miss the primary outcome evaluation will be excluded. In the per protocol dataset, participants will be included if they received at least three constant sessions of treatment.
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.
A sensitivity analysis will then be used to compare the results from the per protocol analysis and the different intervention analyses to evaluate the impact of the missing data on the trial results.
Descriptive statistics about the quantitative indicators, such as means, standard deviations (SD), medians, minimum and maximum., will be presented. Variance analysis will be used to compare the quantitative indexes among different groups (health control group; group 1 and group 2)，Student-Newman-Keuls will be used in analysis of two subgroups.
Classification indicators are described by the number and percentages of each category. The chi-square test or exact probability method was used to compare the qualitative indexes among the three groups, and the chi-square segmentation method will be used for the pair-wise two subgroups comparison.
SPSS version 21.0 (SPSS Inc., Chicago, IL, USA) will be used to analyze all trial data. All statistical tests were double-sided.Statistical significance will be considered when the p value is≤0.05.
Sample size calculation
Based on the literature and the findings from a previous small pilot study which used metabolomic techniques, the sensitivity and specificity needed to classify the control and PCOS groups are 100% and 86%. The sensitivity and specificity between the control group and subgroup 1 is 96.7% and 100%, while the rate between the control group and subgroup 2 is 100% and 86.2%. The sensitivity and specificity to divide PCOS into two different subtypes are 90.9% and 87.5%. In order to meet the lower Statistical limit, each subgroup should include at least 30 people.
According to a presumptive maximum dropout tolerance of 20%, with a significance level of 0.05 and power of 0.80, we calculated that the required sample size in the experimental groups were 216. In this trial, there are 60 people in healthy control group and 108 people in each experimental group across the three centers.
During the trial, quality control will be carried out by the steering committee. All researchers are required to attend training on trial methods, techniques, and protocol regulations in order to maintain the consistency and validity of the data. Any modifications or corrections required should be discussed, decided upon and submitted to the steering and ethics committees. In addition, details should be kept through the trial.