Aim
The aim of this study is to investigate the therapeutic effect of Wuzi Yanzong Pill on semen quality, sperm functions, and natural conception rate.
Trial design and setting
This is a prospective, randomized, placebo-controlled, double-blinded, superiority trial with 1:1 allocation ratio. It is conducted at Assisted Reproductive Technology Unit, Department of Obstetrics and Gynaecology, Faculty of Medicine, The Chinese University of Hong Kong. Our department conducted 892 semen analyses in 2017, in which 273 were diagnosed with oligoathenospermia under the WHO criteria.
Eligibility criteria
Inclusion criteria
- Patients with less than 20 million total progressive motile sperm will be recruited in this study. Since the TMC is so far the most predictive indicators for achieving pregnancy [4, 5]. Other Seminal parameters each as morphology and other physical properties are not included in the criteria.
Exclusion criteria
- Male infertility due to one testicle had been removed, history of undescended testis, previous chemotherapy, testicular torsion or any other known abnormalities on reproductive organs;
- Patients with azoospermia (indicating structural abnormalities or chromosomal abnormalities);
- Patients with known chromosomal abnormalities;
- Patients with normal WHO (Version 5) in all parameters;
Interventions
TCM WZYZ formula preparations
Traditionally and clinically most Chinese herbal medicines are prepared by decoction in boiling water for hours. In this study, the individual concentrated herbal medicine granules prepared from standardized decoction methods using state-of-art concentration technologies in Good manufacturing practices (GMP) standard will be obtained from PuraPharm® International (H.K.) Ltd. The individual concentrated herbal medicine granules will be prepared in sachet form. The modified Wuzi Yanzong Pill will be composed of Lycii Fructus 2.4g, Rubi Fructus 3g, Cuscutae Chinensis Semen 2.4g, Plantaginis Semen 2.4g, and Schizandrae fructus 2.4g, Rehmanniae Glutinosae Conquitae Radix 3g, Polygonati Rhizoma 3g, Cistanches Deserticolae Herba 2g, Epimedii Herba 2g, and Cornus Cervi Colla 0.6g, totally 23.2g divide into two doses per day. For oral administration, the sachets at designated daily dose are mixed to combine each Chinese medicine. The Certificate for Clinical Trial is not required as our formula dose package has satisfied the Chinese Medicine Ordinance (Cap. 549 Laws of Hong Kong).
Placebo preparation
The placebo is produced by PurePharm International (H.K.) Ltd. newly established technology that can mimic the formula’s outlook, colour and smell however without any active ingredients thus no therapeutic effect. The placebo herbs are made indistinguishable in appearance and favour from the treatment formula. The daily dose of single form of herb is packed in the individual sachet for easy consumption under the GMP licence. This new technology, on one hand, can prevent participants from recognizing being allocated in the placebo group may result in lower compliance rate, on the other hand, can prevent the potential beneficial effect from using vitamin or clomiphene as placebo. Patient in the placebo group will consume the same amount of placebo herb as the treatment group.
Study process
The semen analysis will be performed for patients in our andrology unit. If the TMS count is less than 20 million per ejaculate, our reproductive medicine practitioner will exam the inclusion and exclusion criteria and consent the patient. After recruitment, the patients are assigned randomly by the TCM team and are divided into the control group taking placebo and treatment group taking WZYZ formula twice a day regularly in a period of three months. Semen samples will be taken on the baseline (week 0), 6 weeks and 12 weeks to determine if the semen quality has been improved by the formula, an additional semen analysis will be performed three months after completion of the treatment. Natural conception and pregnancy outcome will be followed up by our clinical nurse for another six months. Compliance will be recorded in the patient diary and research nurse by weekly phone calls (90 days treatment, i.e. less than 63 days of treatment intake will be excluded, the incomplete intake will be considered as fail of compliance too). If the total compliance is less than 70%, then the patient will be considered to drop out of the study. The flow chart and study period are shown in Figure 1 and Table 1. Part of the semen sample will be cryopreserved and separately stored in -80℃ freezer for DNA fragmentation test and other sperm function tests, which will be explained in the outcome measurement sections. The blood sample will as well be taken concurrently with the semen samples for hormonal level examination, including routine follicle-stimulating hormone (FSH) and free androgen concentrations.
Adherence
To enhance the validity of data, multiple methods will be used to assess medication adherence including TCM package count; phone call follow-up every week asking whether the participant takes drug accordingly, reasons for non-compliance. Participants will return the unused drugs after 90 days. Unused drugs will be counted and recorded.
Concomitant care
The administration of other TCM, as well as the nutritional supplements for enhancing sperm parameters, are not permitted for all participants during the first 90 days as these drugs/supplement may confound the effect of TCM WZYZ formula.
Outcome measurement
The primary outcome is to compare the total motile sperm count, natural conception rate, pregnancy outcome, and live birth rate. Secondary objects are sperm functions and assisted reproductive technology (ART) outcome.
Natural conceptions and pregnancy outcome
Natural pregnancy, ongoing pregnancy, and live birth will be recorded by our team research nurse for 2-year follow up after the participant finish 90 days of drug taking.
Semen analyses
A semen analysis will be performed using both manual analyses (by andrologist) and using Computer-Aided Sperm Analyzer under WHO criteria. The clinical andrologist has passed the quality assurance test (UK, NEQAS) to make sure up to standard. Semen parameters include volume, motility, viability, concentration, morphology, total sperm count, and another physical status will be measured. A repeat semen analysis will be performed at 3 and 6 months after the treatment to assess the response duration.
Blood tests
Hormone FSH and testosterone will be tested because the levels of the two hormones are closely related to the testis function, complete blood picture; liver and renal functions test will also be included to demonstrate both TCM formula and placebo do not have adverse effects to the patients.
Sperm DNA fragmentation
DNA integrity has been shown strongly associated with reduced fertility in males. The increment of DNA fragmentation level reduces fertility in men and affects subsequent embryo development. The level of sperm DNA fragmentation will be analyzed by DNA fragmentation assay to distinguish the effect of the TCM.
Sperm cryo-tolerance
Cryopreservation of sperm is a common clinical procedure for ART treatment; some of the patient’s samples show better cryo-tolerance sperm than others. Cryo-tolerance is a test to demonstrate sperm are tolerable to the freeze-thaw procedure, the value is useful to predict the percentage of sperm survive from cryopreservation for subsequent ART treatment. This test also provides information on sperm robustness indirectly.
Endocrine-disrupting chemicals test
Estradiol like substances in the formula or placebo can possibly reduce the therapeutic effect in males [10, 11]. We will use a patented estrogenic testing method to test the analogs of WZYZ formula and placebo.
DNA maturity assay
Sperm DNA maturity will be tested using aniline blue solution staining, the solution stain histone protein, which is replaced by protamine during spermatogenesis for the dense sperm head compaction. The high histone content indicates poor embryo development and pregnancy outcome.
ART outcomes
The ART outcomes include fertilization rate, embryo quality, blastocyst rate, implantation rate and pregnancy rate. They will be recorded by the treatment record if the patient receive ART in our hospital. If the patient receive ART elsewhere, the outcomes will be recorded by our team research nurse for 2-year follow up
Retention
Once a participant is enrolled, our study investigators and staff will provide the written feedback of their semen analysis and liver and kidney function tests. Our research assistant will maintain their interest in the study through weekly phone call follow-up and remind the participants the upcoming doctor appointment/data collection.
Sample size calculation
Our andrology unit conducted 892 semen analyses in 2017, in which 273 were diagnosed with oligoathenospermia, and the mean of the Total Motile Sperm count of all patients failed to fulfil WHO criteria (5th edition, <20 Million per ejaculate) is 8.08 ±6.19 million total motile sperm. To detect 100% (double of TMS) of improvement by WZYZ TCM formula and accepting a type I error of 0.05 and a type II error of 0.2, the number of subjects to be recruited into each arm to detect a significant difference is 115 on each arm (Medcalc®, Version 15.6.1). To make allowance for a dropout/non-compliance rate of 20%, the number of subjects required in each arm is therefore 181 and the total number of subjects required in the study is 286. Therefore, the whole recruitment approximately takes 12 to 18 months to finish.
Recruitment
It is estimated that four to five new semen analyses are conducted in our andrology unit per day. We will screen the semen analysis result for the potential eligibility following the TMS criteria. Once identified, our research assistant will contact them, explains the study and invite them to join it. If the patient is interested, the patient will have another semen analysis and blood taking for confirming his eligibility. Once the patient fulfils our criteria, our reproductive medicine practitioner will further inform him of our project and ask for his consent.
Randomization and allocation concealment
For those consented patients to join the study, they will be randomly assigned in two groups (1:1 ratio) using computer-generated random numbers manage by Integrative medicine team. The study medication (TCM WZYZ formula and placebo) will be prepared as similar looking medication not only in blisters but also in mimic herb’s outlook as well as their smell. Our clinicians providing care and patients will be masked from the type of treatments for the patients. To achieve high accuracy, the trial will follow the updated guidelines from Consolidated Standards of Reporting Trials (CONSORT) 2010 Statement [12] and CONSORT for TCM for reporting our parallel group randomised trials [13]. Unblinding should occur only in exceptional circumstances when problems cannot be solved with ongoing randomization. Unblinding should be performed by an authorized investigator. The investigator should report all the unblinding and their reasons on the corresponding case report form.
Quality assurance
A Data Monitoring Committee (DMC) has been established. The DMC is composed of one independent chairman from the Clinical Research Management Office of The Chinese University, one independent member from the Department of Obstetrics and Gynaecology, and one independent member from the School of Chinese Medicine. The responsibilities of the DMC are data monitoring, interim-analysis, assessing adverse event, reviewing core trial processes and documents, and discussing any amendments to the main study protocol.
Data management
All information the participant will be carefully recorded on the case report forms (CRF). All errors will be crossed out and corrected and signed by the corresponding investigator. The study data in the CRF will be entered and coded to a corresponding e-CRF by the double-entry method.
Hard copy CRFs are stored in a separated room, locked in filing cabinets and only authorized investigators are permitted to access this information. The e-CRFs are stored in a server encrypted using the Advanced Encryption Standard and only authorized investigators are permitted to access them. The researchers will ensure the confidentiality of sensitive data by minimizing the number of personnel who handles subject data.
All the data are reported as percent for nominal data or means ± SD for continuous data. Patients with less than 70% of compliance (90 days treatment, i.e. less than 63 days of treatment will be excluded) on taking WZYZ formula will be considered dropping out and will not be included in the final analysis but will include in the intention to treat analysis. The compliance will be checked by our research staff on the patient diary and every two weeks phone call. For noncompliance and missing outcomes, we will use two sets of analyses. The full analysis set (FAS) will include all patients who are randomized and will be conducted according to the intention to treat principle. The per protocol set will include all patients of the FAS complete follow-up and not have serious protocol violations.
An unpaired Student t-test is used to test for differences between placebo and intervention group, and a paired student t-test was used to test for differences within group from baseline to 12 weeks for variables that followed a normal (Gaussian) distribution, if normal distribution was not satisfied; variables were then transformed to approximate normal distribution by using the Mann-Whitney U test and Wilcoxon matched-pairs signed-rank test. The two-tailed significance level was defined as a P value of <0.05.
Ethical Consideration
Ethical approval of the protocol and informed consent forms has been granted from the Clinical Research Ethics Committee (CREC), CREC Reference number 2016.617-T (Appendix 1).
All subjects will be given a detailed explanation and their permission will be obtained before they are recruited into the study. A written consent form will be signed by the patient (Appendix 2, 3). Semen analysis is a direct and non-invasive method for semen quality evaluation. The TCM remedy is a mild formula with no observable side effect and no known toxicity. Blood sample taking will cause extremely mild irritation on skin and the infection risk is low.
This study is compliance with the Declaration of Helsinki and Guideline for Good Clinical Practice of the International Conference on Harmonisation.