Efficacy and safety of Ding-Kun-Dan for female infertility patients with predicted poor ovarian response undergoing in vitro fertilization/intracytoplasmic sperm injection: study protocol for a randomized controlled trial

Potential participants will be recruited from Tianjin Medical University General Hospital (Tianjin, China), Tianjin First Center Hospital (Tianjin, China) and The Second Hospital of Hebei Medical University (Hebei, China). The participating center must have an IVF program and be able to participate fully in the study. In China, IVF centers are regulated and typically located in level-3 (the highest level) hospitals.

Our study will be propagated via the Internet, bulletin boards, and posters in communities and hospitals, with a hotline provided for potential volunteers to call. Potential participants will be asked and talk face to face with researchers, and will be given detailed information about the study. If patients are eligible and interested in participating, they will be asked to sign an informed consent. An overview of specific measurements and time points of data collection can be found in the SPIRIT Figure (Fig. 2).

Eligible participants will be randomly assigned to the experimental group (DKD for 3 months, then IVF/ICSI) or the control group (immediate IVF/ICSI) in a 1:1 ratio by central randomization performed by an independent statistician from Tianjin CLINDA Medical Technology Co., Ltd. Random numbers will be generated by using dynamic randomization on an online computer, which will generate a randomization schedule. Recruitment, allocation, instructions how to take DKD, and study investigations will be carried out by the study investigators. The IVF/ICSI is carried out by the treating team.

The sample size calculation is based on the ongoing pregnancy rate. Studies indicated that ongoing pregnancy rate of female infertility with POR undergoing IVF/ICSI cycles was 12.5% to 15.8%, with an average of 13% [23‐27]. We hypothesized that a supplementation of DKD can increase the ongoing pregnancy rate to 26%.

According to the sample size of the estimation formula [28]:where n₁ = n₂ = n/(1–20%), Z_α = Z_0.05 = 1.64, Z_β = Z_0.8 = 0.84, P₁is the ongoing pregnancy rate of the treatment group = 0.26, P₂ is the ongoing pregnancy rate of the control group = 0.13, and ε is the difference of P₁ and P₂. Assume the superiority margin is 0 (δ = 0).

For 80% power to detect a difference between the experimental arm and the control arm at a one-sided 5% type I error rate, we need 112 per arm. This assumes standard sample size formulae for binary outcomes using a normal approximation [28]. To account for a 20% dropout rate, we will recruit 139 patients per arm. Considering the number of outpatients in each centre will be fluctuating during the trial; therefore, we use a dynamic randomization method to generate random numbers, and so there is no strict restriction on the number of participants in each centre.

Patients are eligible to be included if they meet the following criteria: (1) are female, aged between 25 and 38 years, (2) have regular menstrual cycles of between 25 to 35 days, (3) are diagnosed as likely to have a POR where at least two of the following three features must be present: at least five oocytes in a previous IVF/ICSI treatment cycle with a conventional stimulation protocol; combined antral follicle count of less than seven; or anti-Müllerian hormone (AMH) < 1.1 ng/ml, (4) diagnosed with infertility (aged under 35 years, the duration of infertility is more than 12 months and, for those who over 35 years, their duration of infertility can be shortened to 6 months or any other factors which are likely to cause infertility), (5) planning to do IVF/ICSI, and (6) competent and able to give informed consent.

Patients who present with any of the following conditions will be excluded: (1) other endocrine disease or autoimmune disease, e.g., polycystic ovarian syndrome (PCOS), hyperprolactinemia, thyroid dysfunction, diabetes etc., (2) any other factors known to reduce pregnancy rates in IVF/ICSI cycles, e.g., endometriosis, intrauterine adhesions, sub-mucosal fibroids, significant uterine malformations, hydrosalpinx, etc., (3) suffering serious medical, surgical or mental illness, (4) a hereditary disease or other serious illness which would be a contraindication to pregnancy, (5) allergy to any ingredient in DKD, (6) use of hormones or TCM (contraceptive drugs, ovulation-stimulating medicine, androgens, and glucocorticoid, etc.) within 3 months prior to study entry.

Reasons for discontinuation of treatment may include, but are not limited to, the following: (1) participants become pregnant during the trial, (2) participants who have experienced some complications or serious side effects), (3) participants who do not comply with the DKD regimen, as defined by less than 80% or more than 120% of the prescribed amount; using prohibited drugs as described in the protocol, such as contraceptive drugs, ovulation-stimulating medicine, glucocorticoids etc., (4) participants who request to withdraw from the trial.

The study will be conducted in four stages in the DKD group. After a recruitment period prior to baseline assessment and randomization, DKD (water-honeyed pill) will be given to patients in the experimental group 7 g twice daily orally from the first day of their menstrual period, for 3 months. If the participant has side effects on this dose, it is permissible to reduce the dose to 3.5 g twice daily. After that, participants will undergo their IVF/ICSI cycle. Finally, a 12-week follow-up will be included.

COH prior to IVF/ICSI will be initiated on day 3 of the menstrual cycle. All participants will receive a fixed initial daily follicle-stimulating hormone (FSH) (Gonal F, Merck Serono SA, Switzerland) dose of 300 IU subcutaneously for the first 5 days [29]. Thereafter, the daily dose will be adjusted up or down according to follicular response and serum estradiol (E₂) level, with 450 IU as the maximum daily dose allowed. Participants will receive a gonadotropin-releasing hormone (GnRH) antagonist (Cetrorelix Ac, Merck Serono, Darmstadt, Germany, or ORGALUTRAN, Merck, Kenilworth, NJ, USA) when the dominant follicle reaches 14 mm in diameter or serum E₂ level higher than 350 pg/ml, at a dose of 0.25 mg daily subcutaneously throughout the remainder of the stimulation period. Triggering of final follicular maturation is performed as soon as one or more follicles are ≥ 18 mm in diameter with 250 μg recombinant hCG (choriogonadotropin-α, Ovitrelle, EMD Serono, Darmstadt, Germany).

Transvaginal ultrasound-guided oocyte retrieval will take place 36 ± 2 h after triggering of final follicular maturation. Oocytes will be inseminated using conventional IVF or ICSI, according to the clinical indication. Embryo transfer will be performed on days 2–3 after oocyte retrieval and a maximum of two embryos will be transferred. Surplus embryos will be cultured until the blastocyst stage and subsequently cryopreserved, for use after trial completion.

Vaginal progesterone gel (Crinone gel, 8%, Merck Serono, Switzerland) 90 mg once daily or dydrogesterone tablet (Duphaston, Abbott, Netherlands) 10 mg twice daily orally will be used for luteal-phase support from the day after oocyte retrieval for 13–15 days. If serum hCG positive, progesterone is continued until 10–12 weeks’ gestation. If hCG is negative, or if the pregnancy fails, P support is stopped. Ultrasound will be performed at 4–6 weeks and 10–12 weeks after embryo transfer to confirm clinical and ongoing viable pregnancy, respectively.

The study will be conducted in three stages in the control group. After a recruitment period prior to baseline assessment and randomization, patients in the control group will be arranged to accept IVF/ICSI directly without any other intervention. The participants in this group will receive the GnRH antagonist protocol for COH that is the same as the DKD group. Finally, a 12-week follow-up will be included.

All participants in both groups will be advised to maintain a healthy lifestyle during the study, such as no smoking, no alcohol, low-fat high-protein diets, regular exercise, and to get plenty of sleep.

All the DKD (water-honeyed pill) will be provided free of charge by the Shanxi Guangyuyuan TCM Co., Ltd. It will be stored at room temperature and dispensed locally at the treating hospital. Patients will take DKD 7 mg twice daily orally from the first day of their menstrual period for 3 months. If the patient has side effects on this dose, it is permissible to reduce the dose to 3.5 mg twice daily. If on completion of the trial, or in case of withdrawal from the study, any unused DKD will be returned to the pharmacy. As DKD is the standard of care within TCM, no special drug accountability is required. Patients will be given a diary and asked to keep a record of their daily medication usage, which is returned to the investigator on completion of the course of DKD treatment. A complete listing of all concomitant medication received during the treatment phase must be recorded in the relevant Case Report Form (CRF).

DKD is the standard of care within TCM. The TCM hospital in which the DKD is prescribed, and the hospital in which the IVF/ICSI treatment is carried out, provide indemnity for approved clinical research in their institution.

As DKD is the standard of care, no special arrangements need to be put in place in case of adverse events.

In case of emergency, the patient will be provided with a 24-h contact number to call.

The primary outcome of the study is ongoing pregnancy rate, defined by at least one intrauterine viable fetal heart beat at 12 weeks of gestation.

The secondary outcomes include total gonadotropin dosage; duration of stimulation; cycle cancellation rate; E₂ and progesterone (P) levels on the hCG trigger day; retrieved oocyte number; high-quality embryo development rate; biochemical pregnancy; as well as some specific endpoints indicative of the ovarian response, such as the change of serum AMH, FSH, E₂ level. All side effects, safety outcomes and adverse events will also be recorded. All data will be collected using an electronic Case Report Form (eCRF) and will be supervised by an independent statistician from Tianjin CLINDA Medical Technology Co., Ltd.

The standard of high-quality embryo development rate is according to the Istanbul criteria [30] as following: Grade 1 – good rating: 10% fragmentation, stage-specific cell size, no multinucleation. Grade 2 – fair rating: 10–25% fragmentation, stage-specific cell size for majority of cells, no evidence of multinucleation. Grade 3 – poor rating: severe fragmentation (> 25%), cell size not stage specific, evidence of multinucleation.

During the study, all the details including all adverse events, such as swelling and aching of the gums, oral ulceration, constipation, and ovarian hyperstimulation (OHSS) will be recorded in detail. Serious adverse events will be immediately reported to the principal investigator, and appropriate measures will be initiated instantly.

The Ethics Committee will determine whether the adverse event is likely to have been associated with the experimental drug, and the sponsor will provide the cost of the treatment associated with the study and the corresponding financial compensation.

All data will be collected using an eCRF and will be supervised by an independent statistician from Tianjin CLINDA Medical Technology Co., Ltd., and will be completed and recorded on the paper and eCRF. Paper files will be kept in a locked filing cabinet in the treating hospital. Electronic documents will be stored in a password-protected computer, with access restricted to the principal investigator. All research documents will be preserved for at least 5 years after publication.

All clinicians involved with the trial will be fully trained in the trial methodology. After the clinician has recruited the first patient the CRF will be reviewed by the researcher to ensure that it has been correctly completed.

To ensure trial quality and reliability, a clinical trial monitor from Tianjin Taicheng Yaozhong Biomedical Technology Co., Ltd. will verify all of the process details at when the experiment is half done and completed. Moreover, the monitor will check the authenticity, accuracy, and integrity of the data.

At the beginning of the study, blood will be taken for baseline FSH, E_2, and AMH, and an ultrasound will be performed to measure the antral follicle (AFC) in both groups. After 3 months of DKD treatment, and before commencing IVF/ICSI, the same data will be collected in the DKD group. These blood tests will be assayed within each participating center. The AFC will be performed by the treating team.

After the IVF/ICSI treatment cycle has been completed, the total gonadotropin dosage, duration of stimulation, E₂ and P  levels on hCG trigger day, cycle cancellation rate, retrieved oocyte number, high-quality embryo rate, and biochemical pregnancy will be recorded.

If the patient is pregnant, the presence or absence of a fetal heart will be noted 4–6 weeks after the embryo transfer, and the ongoing pregnancy rate at 12 weeks of gestation, and the miscarriage rate will be recorded during the follow-up.

Trained nurses will take the blood samples, and ultrasound physicians, independent of the researchers, will perform the ultrasonography.

The trial protocol is in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of the First Teaching Hospital of Tianjin University of TCM (approval no. TYLL2017[K] 004). This trial was registered at the Chinese Clinical Trail Registry, (ID: ChiCTR-IOR-17011697). Each participant will be fully informed regarding the study protocol. Written informed consent will be obtained from every participant.