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.
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.
-Male infertility due to one testicle had been removed, history of undescended testis,
previous chemotherapy, testicular torsion or any other known abnormalities on reproductive
Patients with azoospermia (indicating structural abnormalities or chromosomal abnormalities);
Patients with known chromosomal abnormalities;
Patients with normal WHO (Version 5) in all parameters;
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).
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.
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.
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.
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.
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 as well as the safety assessment on the TCM formula.
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.
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.
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 assess the safety on both TCM formula and
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.
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.
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
receives ART in our hospital. If the patient receives ART elsewhere, the outcomes
will be recorded by our team research nurse for 2-year follow up
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, remind the participants the upcoming doctor appointment/data collection
and record the presence of adverse drug reaction.
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.
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  and CONSORT for TCM for reporting our parallel group randomised
trials . 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.
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 the adverse event and cases with abnormal liver or renal
function results after the intervention, reviewing core trial processes and documents,
and discussing any amendments to the main study protocol.
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
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.
For the baseline comparison, the independent Student t-test or the Mann-Whitney U
test is used to evaluate the mean or median differences between placebo and intervention
group for the continuous data depending on whether the data is normally distributed,
and the Chi-squared test is used for frequencies comparison. For the longitudinal
data with a baseline, we will use repeated measures Analysis of Covariance (ANCOVA)
to evaluate the mean difference between and within groups All the tests are two-tailed,
and the significance level was defined as value of <0.05.
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.
We have included liver and renal function tests for each patient at the baseline and
at the completion of the WZYZ treatment to assess the safety of the TCM remedy. Blood
sample taking will cause extremely mild irritation on skin and the infection risk
This study is compliance with the Declaration of Helsinki and Guideline for Good Clinical
Practice of the International Conference on Harmonisation.