Study design and participants
This is a randomised, double-blinded, placebo-controlled, four-center clinical trial protocol for studying the effects of BSHX on women with POI. There are four hospitals (table 2)that have participated in this project. The clinical trials was conducted according to the principles of the World Medical Association Declaration of Helsinki and the guidelines of Good Clinical Practice of the International Conference on Harmonisation (ICH-GCP). The trial was registered as ChiCTR1900028451 at the Chinese Clinical Trial Registry (www.chictr.org) on 22 December 2019. This study has been approved by each hospital's ethics committee and the responsible regulatory authorities. All participants will provide written informed consent before randomization. Qualification screening and information on demographic and clinical characteristics will also be collected. After passing the qualification screening, participants will undergo a number of medical examination to obtain baseline data. These data are the minimum eligibility criteria for each participant. Meanwhile, it is possible to screen out respondents who may not follow the research procedures. All study participants will be randomly divided into two parallel groups, the BSHX group, and the placebo group, respectively. The trial consisted of 3 months of treatment with 2 weekly interviews, and followed-up for another 3-month with 4 weekly interviews. Based on previous clinical trial studies, we will use a three-month drug observation period to monitor the safety and efficacy of the treatment.18-21 The study design flow chart is shown in figure 1.
Table 2 The hospitals participating in this study
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Code
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Participating hospitals
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01
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Jiangsu Province Hospital on Integration of Chinese and Western Medicine
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02
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Suzhou Hospital of Traditional Chinese Medicine
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03
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Taizhou Integrated and Western Medicine Hospital
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04
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Wuxi City Traditional Chinese Medicine Hospital.
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Sample size calculation
Sample size estimation refers to the calculation of the required sample size in order to meet the accuracy and reliability of statistics (the control of type I errors and the guarantee of inspection efficiency).22 The sample size of this study was based on the relevant literature reviews and the validity of hypotheses. The main indicator of this study is to detect the difference of the total score (CMS) between the two groups before and after treatment. There is one randomised clinical trial for BSHX in the treatment of POI14. However, there is no placebo control group in the clinical trial and the primary outcome measurements were different. Thus, the parameters are not suitable to estimate the sample size of the trial. We referred to other clinical trials of traditional Chinese medicine in the treatment of POI patients 23. We assumed that BSHX administration would reduce CMS score by 2.9 points and the SD is 7.42 in the study. If a type I error rate of α = 0.05, a power of 90 % (type II error rate of β = 0.1), then u1−α =1.64, u1−β =1.28. n1=n2≈70. During the 3-month treatment period, taking into account the possible protocol violations, and the maximum possible drop-out rate of 15%, We need 148 participants in total to achieve the number of people required for the efficacy analysis. 150 participants will be recruited for this study, and the following formula was used to estimate the sample size:
Patient and public involvement
Patients and the public were not involved in the trial design, and they will not participate in the guidance, or implementation of this study. Once the trial is completed, the results will be disseminated through social media, academic conferences or research publications.
Participants and recruitment
There were two ways to recruit patients. One was to display recruitment posters of the study in all participating hospitals. The posters will contain brief descriptions of inclusion and exclusion criteria, the medicines, the ways of participation, and study purposes. Another way is to publish announcements on the hospital’s website. Prior to the start of the trial, all researchers are clinical obstetricians and received formal professional training to do diagnostic interviews. If there were people who were not eligible or refused to participate, we recorded the detailed reasons.
Inclusion criteria
Diagnostic criteria are based on the guidelines of the European Society of Human Reproduction and Embryology 2016. If they meet these criteria, the subjects will be enrolled in this study, 1) Chinese women aged less than 40 years, 2) Oligomenorrhea/amenorrhea for more than 4 months, 3) At least 2 basal FSH level more than 25 IU/L, on two occasions more than 4 weeks apart, and 4) Sign the informed consent.
Exclusion criteria
Excluded individuals who has met either one of the following criteria, 1) Patients with serious diseases, such as malignant tumour , thrombosis, 2) Patients with severe mental disorders, 3) Congenital abnormal development of reproductive organs, or amenorrhea caused by acquired organic lesions and injuries, 4) People with chronic diseases that requires hormone therapy, 5) Pregnant or breast feeding women, 6) Patients are taking drugs or acupuncture that could affect menstrual cycle or ovarian function, or had participated in another clinical trial in the previous three months that could affect the reproductive system, 7) The patient is allergic constitution or allergic to the study drugs, 8) Drink or use drugs, 9) The patient is not able to provide written informed consent or is likely to be lost to follow-up.
Removal, dropout, suspension and termination criteria
During the clinical trial, participants who violate the protocol operation will be removed their participation, for instance, failed to take the drug in accordance with the regulations (medication compliance less than 80%) that affect the judgment of drug efficacy because incomplete data will affect the judgment of efficacy and safety. Participants can voluntarily drop out at any time during the trial. Emergency unblinding or loss of eligible subjects to follow-up will be considered dropping out. If the participant drops out, the last recorded data will be recorded and analyzed. These will be recorded in detail in electronic medical records. If participants have any of the following circumstances, the drug treatment discontinues immediately, while safety or post-treatment visits may continue. These circumstances were 1) a serious adverse event occurred, 2) pregnancy, 3) use of medication that can affect menstrual cycle or ovarian function, 5) the participant withdrew the informed consent form, and 6) the subjects showed hypersensitivity reactions to BSHX, such as stomach pain, diarrhea, etc. Once the blindness is made public, or the opening rate of emergency letters exceeds 20% of the sample size, it means that the double-blind experiment will be invalidated and the entire study will be terminated.
Randomisation and blinding
Participants will be randomly assigned (1:1) using a computer-generated randomisation sequence to receive oral BSHX or BSHX placebo. The statistician who is not involved in recruiting will prepare the randomisation list, and the list will not be available to anyone in any center. The randomisation distribution sequence is hidden in sealed, opaque, and sequentially numbered envelopes, which are properly kept until the end of the trial. Researchers who do not participate in the recruitment label the envelopes. Meanwhile the size, color, taste, shape of the placebo are consistent with the corresponding drugs, and all study groups assigned by different numbers will use the same packages of medicines. Placebo consists of starch and no active ingredients. Packages of BSHX and placebo were provided to randomly assigned two groups of women in each hospital's centers. This is a double-blind trial, in which all researchers and subjects will be blinded for the treatment allocation until the trial is completed. If there is a serious adverse event or an emergency unblinding event, the blind will be immediately broken.
Intervention
All clinics followed the same study protocol. BSHX granules and placebo granules were assigned to different groups for a period of three months of treatment. The BSHX and placebo were manufactured by Jiangyin Tianjiang Pharmaceutical Co. Ltd. (Jiangyin, Jiangsu Province, China) in compliance with China Good Manufacturing Practice standards. Both BSHX and placebo were approved and regulated by the China Food and Drug Administration (FDA) and can be valid for 2 years. The medicines have been widely used in our hospital for many years, and the clinical efficacies were noticeable. Different from other traditional herbs prepared by boiling, it is easier and quicker by dissolving BSHX or placebo (one bag three times a day, 30 g/bag) in water for 30 minutes. Treatment was taking these medicines after meals three times a day for 3 months. After treatments, medicine packages will be required to return, and compliance will be calculated based on the residual medication. Dosages of Chinese medicines were in accordance with the latest standards of the Chinese Pharmacopoeia (2015 edition). Trial records were confidential and secure.
Follow-up
All included patients were re-evaluated in 2, 4, 6, 8, 10, 12, 16, 20 and, 24 weeks follow-ups. Wherever in-person follow-up was not possible, video conferencing or telephone follow-up was carried out.
Safety assessments
Studied medicine safety testing includes liver and renal function test, blood routine examination, urine routine examination, stool routine examination, and electrocardiogram. These parameters will be obtained both at screening and after treatment, and all samples were tested by the Jiangsu Province Hospital on Integration of Chinese and Western Medicine in China. All adverse events during treatment and follow-up will be recorded and analyzed in detail. If a participant has a serious adverse event and was not safe to continue the trial, the participants were requested to withdraw from the trial. Immediate relevant treatments were provided to those participants, and following up continued until the response was terminated.
Outcome measures
Primary outcome
The primary outcome measurement was the CMS questionnaires. The CMS was translated by Yang Hongyan and others24. In clinical practice, the Chinese version of the MENQOL Questionnaire (table 3)(supplementary material)has been proven to be highly reliable, effective, and responsive. It is a reasonable and effective tool to comprehensively evaluate the current physical and mental health problems of menopausal women in China 25. CMS has a total of 29 items, which are divided into 4 dimensions, including vasomotor symptoms (3 items), psychosocial symptoms (7 items), physiological health (16 items), sexual health-related questions (3 items). Differences in this questionnaire are that MENQOL appears not only as symptoms based on the frequency, it also includes the troubles caused by various severity of symptoms degree. Participants will complete the questionnaires before, after the intervention, and follow-up. Items measured and data collection schedule are shown in table 4.
Table 3 Chinese Version of the Menopause-Specific Quality of Life Questionnaire
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In the last month, have you experienced the symptoms in the following questionnaire? If you have not experienced these symptoms, please tick “√” in the“□” above “No”; if you have experienced this symptom, tick “√” in the “□”above “Yes”, and select a level from "0~6" according to the degree of the symptom affecting you. "0" means not affected at all, and "6" means extremely affected; at 0 Between and 6, the closer to 0, the less affected, and the closer to 6, the greater the impact.
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1.Hot flushes
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□ □ → 0 1 2 3 4 5 6 No Yes
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2.Night sweats
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□ □ → 0 1 2 3 4 5 6 No Yes
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3.Sweating
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□ □ → 0 1 2 3 4 5 6 No Yes
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4.Dissatisfaction with my personal life
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□ □ → 0 1 2 3 4 5 6 No Yes
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5.Feeling anxious or nervous
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□ □ → 0 1 2 3 4 5 6 No Yes
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6.Poor memory
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□ □ → 0 1 2 3 4 5 6 No Yes
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7.Accomplishing less than I used to
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□ □ → 0 1 2 3 4 5 6 No Yes
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8.Feeling depressed, down or blue
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□ □ → 0 1 2 3 4 5 6 No Yes
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9.Being impatient with other people
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□ □ → 0 1 2 3 4 5 6 No Yes
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10.I always want to be alone
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□ □ → 0 1 2 3 4 5 6 No Yes
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11.Gastric flatulence or distending pain (with farting or belching)
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□ □ → 0 1 2 3 4 5 6 No Yes
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12.Aching in muscles and joints
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□ □ → 0 1 2 3 4 5 6 No Yes
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13.Feeling tired or worn out
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□ □ → 0 1 2 3 4 5 6 No Yes
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14.Difficulty sleeping
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□ □ → 0 1 2 3 4 5 6 No Yes
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15.Aches in back of the neck or head
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□ □ → 0 1 2 3 4 5 6 No Yes
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16.Decrease in physical strength
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□ □ → 0 1 2 3 4 5 6 No Yes
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17.Decrease in stamina
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□ □ → 0 1 2 3 4 5 6 No Yes
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18.Lack of energy
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□ □ → 0 1 2 3 4 5 6 No Yes
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19.Dry skin
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□ □ → 0 1 2 3 4 5 6 No Yes
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20.Weight gain
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□ □ → 0 1 2 3 4 5 6 No Yes
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21.Facial hair increased
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□ □ → 0 1 2 3 4 5 6 No Yes
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22.Changes in appearance, skin texture or complexion
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□ □ → 0 1 2 3 4 5 6 No Yes
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23.Feel uncomfortable with swelling
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□ □ → 0 1 2 3 4 5 6 No Yes
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24.Low backache
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□ □ → 0 1 2 3 4 5 6 No Yes
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25.Frequent urination
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□ □ → 0 1 2 3 4 5 6 No Yes
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26.Involuntary urination when laughing or coughing
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□ □ → 0 1 2 3 4 5 6 No Yes
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27.Decrease in my sexual desire
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□ □ → 0 1 2 3 4 5 6 No Yes
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28.Vaginal dryness
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□ □ → 0 1 2 3 4 5 6 No Yes
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29.Avoiding intimacy
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□ □ → 0 1 2 3 4 5 6 No Yes
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Table4 Schedule of data collection
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Items
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Screening period
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Treatment period (1-3 months)
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Follow-up period (4-6 months)
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Months
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√
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0
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1
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2
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3
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4
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5
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6
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Eligibility screening and signed informed consent
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√
|
|
|
|
|
|
|
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Inclusion/exclusion criteria
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√
|
|
|
|
|
|
|
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Demographic characteristics
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√
|
|
|
|
|
|
|
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Medical history, course of disease, treatment history
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√
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|
|
|
|
|
|
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Combined diseases
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√
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|
|
|
|
|
|
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Concomitant medications
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√
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√
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√
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√
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√
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|
|
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CMS
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√
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|
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√
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|
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√
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AFC, PSV
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√
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√
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|
|
|
|
|
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Serum sex hormone levels, AMH
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|
√
|
|
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√
|
|
|
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Vital signs
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√
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√
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√
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√
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√
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√
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√
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√
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Laboratory tests for safety assessment
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√
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|
|
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√
|
|
|
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Adverse events
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|
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√
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√
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√
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√
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√
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√
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1.CMS: Chinese version of the Menopause-Specific Quality of Life questionnaire. 2.AFC:antral follicle count. 3.AMH :Anti-mullerian hormone. 4.PSV: ovarian peak systolic velocity (;cm/s)5.Safety assessments: Liver and kidney function test, routine blood test, routine urine test, routine stool test and electrocardiogram. 6.Vital signs: temperature, heart rates, breathing and blood pressure.
Secondary outcomes
The secondary measurements included serum sex hormone levels, AMH, AFC,PSV; cm/s. The regulatory effect of tested medicine on hormone balance was monitored by the level of serum sex hormones (FSH, LH, E2). The blood samples of all subjects were taken in the early antral follicular phase before treatment and after the withdrawal of the drug. As an ovario-specific growth factor, AMH is an effective and reliable indicator to predict ovarian reserve function26,and it has no cycle-dependent 27. AFC is helpful to evaluate the relative accuracy of ovarian reserve function when used in combination with sex hormone detection, and can also provide a reference for the formulation of the treatment plan 28. Transvaginal ultrasound is the best way to observe AFC indicators, which can be accurately evaluated and calculated the number of follicles. Generally, the AFC is measured at the beginning with the early antral follicular stage. Transvaginal ultrasound is performed by an experienced ultrasound expert, and the follicle count of both ovaries with a diameter of 2-10mm (measured by clinicians rather than automatically calculated) is used to calculate AFC. All subjects underwent vaginal ultrasound examination at the follicular stage before treatment and after drug withdrawal.
Quality control
The multicenter clinical trial is a complex process, and quality control is a reliable guarantee for this trial. Effective quality control measures were carried out to ensure the quality of the trial and the accuracy of results, completeness, and authenticity of the information. At the same time, the rights and welfare of participants should be guaranteed. Being the authority among research centers, Jiangsu Province Hospital on Integration of Chinese and Western Medicine is in charge of formulating the standard operating procedures (SOPs) training, and visiting each site regularly to guidance the trial, which strictly followed the protocol. The data were collected timely, directly, accurately, and clearly. The recordings were regularly self-checked its accuracy and completeness. Any errors were corrected in accordance with the prescribed methods. Data from case reports were published on the public clinical trial management platform (www.medresman.org) within 6 months after the end of the trial. An independent data safety monitoring committee, which is independent of the sponsor without competing interests, was in charge of data validation.
Statistical analysis
Data will be analyzed with SPSS V.21 (IBM) by statisticians independent from involved researchers in this study. Efficacy and safety will be evaluated by Intention-to-treat (ITT) analysis within the full analysis set (FAS) according to the ITT principle. At the same time, per-protocol (PP) analysis will apply to evaluate data collected from subjects who have completed all steps of the trial protocol. Results of the full analysis set and the per-protocol set will be compared. All prespecified outcomes will analyze in the ITT population because ITT analysis often underestimates the efficacy and was usually conservative. On the contrary, PP analysis overestimates the efficacy and the differences between the two groups. Therefore, this study mainly uses ITT analysis, and PP analysis will be applied if there was a statistical significance. Various assignment methods were used to detect whether the results of different assumptions of missing data are robust. The safety evaluation will be conducted by ITT analysis. We use mean± SD for continuous variables and percentages for categorical variables. The incidence of binary outcomes will be estimated for each treatment group and the differences between groups will be presented using the χ2 test. Estimates of the adjusted differences in risks are presented with 95% confidence intervals (CIs) of the difference. The study will set an α level of 0.05 two-sided for all statistical tests. Demographic data and clinical characteristics (e.g., menstrual history, pregnancy history, marital status, smoking and alcohol abuse, and employment status) are analyzed by descriptive analysis. An independent t-test was conducted to compare the differences between the BSHX and placebo groups. In all tests, a value of p<0.05 will be considered statistically significant.
Ethics and dissemination
The protocol has been approved by the Ethics Committee of Jiangsu Province Hospital on Integration of Chinese and Western Medicine. (No. 2019LWKY014). It has been registered in the Chinese Clinical Trial Registry (ChiCTR1900028451). The results of this study provide a better understanding of the efficacy and safety of BSHX in treating patients with POI. Results from this study will be published to the public through academic conferences and peer-reviewed journals.