2.7.1 Screening visit

(1) Obtain signed informed consent and detailed medical history collection;

(2) Semen analysis: semen analysis after 2–5 days of sexual abstinence included standard measurements, such as volume, pH, count, and motility. Semen samples will be assessed in the hospital laboratory using WHO 5.0 criteria (31) for sperm morphology. In addition, DFI will be evaluated using Sperm Chromatin Structure Analysis (SCSA) test (32);

(3) Laboratory examination: including reproductive hormone, blood routine test (BRT), thyroid function, liver function, kidney function, chromosome examination of peripheral blood.

2.7.2 Baseline visit

After the screening visit, couples who meet the inclusion criteria will be assigned to the baseline visit and randomly allocated. Complete anthropometric (height, weight, hip circumference, waist circumference), vital signs measurement.

2.7.3 Treatment visit

During the treatment period, visits were conducted at the end of each month, including the records of anthropometric, vital signs, compliance, adverse events and concomitant medication. At the end of the third month, the male partners in both groups will measure the following parameters within 1 week:

(1) Anthropometric: height, weight, hip circumference, waist circumference;

(2) Semen analysis, DFI;

(3) Laboratory examination: reproductive hormone, BRT, liver function, kidney function.

2.7.4 Follow-up visit

Following the end of the final treatment, couples need to be followed up for another 12 months to observe the long-term effect of CHM on male factor infertility. The follow-up visit includes:

(1) Follow up whether the female partner is pregnant;

(2) At the end of the follow-up, the semen analysis and DFI of male partners will be rechecked.

2.7.5 Pregnancy visit (only with conception)

Female partners who become pregnant within 15 months after randomization will have pregnancy visit, which will last until the termination of pregnancy (pregnancy loss or delivery). During the pregnancy visit, the following procedures will be completed.

(1) Blood human chorionic gonadotropin (HCG) test: pregnancy confirmation;

(2) Ultrasound examination (Conducted at 6–8 weeks of pregnancy): to access the quantity and of gestational sacs and the placement, as well as the dimensions, presence and size of fetal structures and to record the visualization of fetal heart motion and any pregnancy-related abnormalities;

(3) Record pregnancy loss: record biochemical pregnancy, ectopic pregnancy and all other intrauterine pregnancy loss (including spontaneous abortion, missed abortion, selective abortion and stillbirth);

(4) Record the confirmed pregnancy complications;

(5) After the female partner has given birth, the researcher will obtain a copy of the check list during pregnancy from the obstetrician to determine the pregnancy and delivery process of the female partner and the information of the newborn (whether it is a live birth, gender, weight, length, whether there is a congenital defect, etc.).

2.8 Adverse events

Participants involved in this study will not encounter major risks. The minor side effects of CHM may be dizziness, vomiting and diarrhea. The majority of these side effects are temporary and mild, which will disappear in a few days. During the study, the subjects will be asked about their drug tolerance, adverse events and serious adverse events will be recorded.

2.9 Outcome measures

2.9.1 Primary outcome

Live birth rate: defined as a delivery taking place after gestation of ≥20 weeks.

2.9.2 Secondary outcomes

(1) Semen parameters

1) sperm concentration: quantity of spermatozoa per unit of volume of semen, 10⁶/mL;

2) morphology: percentage of normal morphology;

3) motility: percentage of motile (including nonprogressive motile and progressive motile);

4) DFI: evaluate using SCSA test.

(2) Conception rate

Conception will be determined by a positive serum β-hCG measurement (β-hCG >10 mIU/mL).

(3) Pregnancy rate

Pregnancy will be characterized as an intrauterine pregnancy with confirmed fetal heart motion via transvaginal ultrasound.

(4) Pregnancy loss rate

Pregnancy loss will be refers to the intrauterine pregnancy lost before 20 completed weeks of gestation.

(5) Pregnancy complications

Including pregnancy diabetes, pregnancy induced hypertension, preeclampsia/preeclampsia, premature birth, congenital malformations, perinatal death, etc.

(6) Reproductive hormone

Including total testosterone, follicle stimulating hormone, luteinizing hormone, estradiol, progesterone, and prolactin.

(7) Adverse events.

2.10 Interim analysis

We propose abstain from conducting an interim analysis, and the final data analysis will be carried out after all live births in the trial.

2.11 Power calculation

Previous studies have shown that (33), among infertile men with abnormal semen, after taking placebo for 5 months, the live birth rate of spontaneous pregnancy of their female partners is 2.4%. It is assumed that in this study, the live birth rate of spontaneous pregnancy of couples in the waitlist-control group within 6 months after randomization is 2.5%, and 12.5% in the treatment group according to our clinical experience. Hence, the difference amounts to 10%, with two-sided α assigned to be 5% and β at 20% as the upper limit, while maintaining an 80% power and estimating a 15% dropout rate. Consequently, the sample size has been inflated from 105 to 125 per group, resulting in a total of 250 cases for the study.

2.12 Imputation procedure for missing data

We will document reasons for withdrawal in both randomization groups and qualitatively compare these reasons. The impact of any missing data on the results will be evaluated through sensitivity analysis using augmented data sets. To evaluate the effect of missing baseline data, we will employ the multiple imputation method with the missing-at-random assumption.

2.13 Data management

Upon enrollment in the study, each participant will be assigned a unique identifier, which will be utilized across all data records to maintain participant confidentiality. Participant data will be managed using paper case record forms (CRF) and a web-based electronic database (ResMan). Following the collection of original participant documents, they will be transferred to the CRF. Subsequently, all data will be stored in the ResMan. Quality control of the data will be managed at two distinct levels: the investigators will be responsible for ensuring data accuracy as the initial level of control during record input into the CRF. The second level will involve regular data monitoring and validation via ResMan by DCC. Any changes to the current study protocol will be presented to the institutional review board, all trial participants, and trial researchers. Ultimately, individual participant data, excluding private information, will be available to the public within 6 months after the trial’s completion.

2.14 Data monitoring and access

To ensure the safety of the study subjects, Data and Safety Monitoring Board (DSMB) will be established independently to review and interpret the data produced in this study. DSMB will oversee the study’s safety of and the integrity of data through routine video conferences or on-site inspections, offering recommendations on study conduct. In addition, DSMB will assess the advancement of the study, adjudicate adverse events, and determine on any potential premature closure of the study.

2.15 Auditing

Audit trail is another measure to ensure data quality and integrity. Authorization is required for any additions, deletions, or modifications of information in the electronic system record. This study will use computer-generated audit trail with time stamp to track changes in electronic source files. In addition, controls will be implemented to verify the accuracy of the date and time in the electronic system, with only authorized personnel permitted to make adjustments to the date or time. Once any abnormal system date or time is detected, they will be immediately notified. Internal security measures will be established in the computerized system. The data will be stored at the servers located at Foshan Fosun Chancheng Hospital, under the supervision and access of DCC. To prevent data loss, regular backup records will be conducted.

2.16 Data analysis

The outcomes were analyzed according to the intention-to-treat principle. The Kolmogorov–Smirnov test was used to test the normal distribution of continuous variables. Between-group comparisons were carried out by either a chi-squared test or Fisher’s exact test for categorical variables and by either Student’s t-test or Mann–Whitney U test for continuous variables. Comparisons of variables such as live birth rate, conception rate, pregnancy rate and pregnancy loss rate will include relative risk (RR) and 95% CIs in addition to the chi-squared test. A P-value <0.05 was considered to be statistically significant. All statistical analyses were performed using SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA).

3 Discussion

The significant decrease in male fertility is closely related to factors such as unhealthy lifestyles and environmental pollution (34). The effectiveness of commonly used drugs for treating male infertility is still uncertain. Hormone therapy, antioxidant, antibiotics, corticosteroids, methylxanthine, vitamins, minerals and amino acids, and angiotensin converting enzyme inhibitors are the common drugs for treating male infertility (35). However, owing to inadequate controlled studies on potential therapeutic drugs or the absence of substantial enhancements in fertility, the United States Food and Drug Administration has not yet approved any drugs for treating male infertility. An analysis by Cochrane comprising 61 studies on male infertility indicated that antioxidant therapy might enhance the live birth rate, but the quality of evidence is inadequate and upon exclusion of studies at high risk of bias, the observed increase in the live birth rate was no longer apparent (36). A large multicenter randomized controlled trial showed that, compared with placebo, folic acid and zinc supplementation did not significantly improve sperm quality and live birth rate, and adverse reactions such as gastrointestinal tract increased (37). Similarly, another multicenter placebo-controlled randomized controlled trial also showed that antioxidants could not improve semen quality and partner pregnancy rate or live birth rate of male infertile patients (29). In addition, the application of hormone stimulation therapy in male infertility is still controversial due to its potential side effects (38). Therefore, it is crucial to identify a safe and effective treatment for male infertility.

Unlike the direct supplementation of certain hormone in Western medicine treatment, the CHM therapy emphasizes overall balance by enhancing the physical condition and regulating testicular function. TCM believes that the etiology of male infertility is multifactorial, and its pathogenesis is related to kidney, liver and spleen disorders. Therefore, the formulation principles of CHM in this study mainly include tonifying the kidney, soothing the liver and strengthening the spleen. Although studies have shown that traditional CHM can improve male semen quality, few studies include data on pregnancy outcomes, especially the lack of live birth rate, which is a key outcome of male factor infertility. Thus, the findings of this study could offer supportive evidence for the use of CHM in future clinical practice in this field.

Trial status

The recruitment of participants has started in November 2022 and is going. The first version of the protocol was finished on June 30, 2022. After numerous discussions and amendments by the authors, there are 3 versions of the protocol. The enrollment of the current study will be conducted till December 2024.

Data availability statement

The datasets used and/or analyzed after completing the current study will be available from the corresponding author by reasonable request.

Ethics statement

The trial received ethics committee approval from Foshan Fosun Chancheng Hospital, China (ethics approval no. CYIRB-LCYJ-2022113). It has been registered at Chinese Clinical Trial Registry (ChiCTR2200064416). Written informed consent will be obtained from all participants. All procedures in the study are in accordance with the Declaration of Helsinki. The findings of this trial will be disseminated in peer-reviewed journals and presented at conference.

Author contributions

QW: Writing – original draft, Writing – review & editing. HX: Writing – original draft, Writing – review & editing. HZ: Data curation, Investigation, Methodology, Writing – original draft. PW: Methodology, Writing – original draft. XX: Methodology, Writing – original draft. YiC: Data curation, Writing – original draft. QZ: Data curation, Writing – original draft. YuC: Data curation, Writing – original draft. MT: Data curation, Writing – original draft. MZ: Formal analysis, Writing – original draft. TP: Validation, Writing – original draft. YaC: Validation, Writing – original draft. YW: Validation, Writing – original draft. SW: Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Traditional Chinese Medicine Bureau of Guangdong Province, China (grant number 20221371), Guangdong TCM anti-aging Engineering Technology Research Center (No. 2022E040), Guangdong Famous Traditional Chinese Medicine Inheritance Studio Construction Program, and Foshan Anti-aging Engineering Technology Research Center.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fendo.2024.1418936/full#supplementary-material

References