Efficacy and safety of recombinant human granulocyte colony-stimulating factor in patients with poor ovarian response: protocol for a multicentre, randomised, controlled trial

Trial design and setting

The trial will be a multicentre triple-blind RCT with balanced randomisation (1:1) to examine the clinical efficacy of G-CFS at improving ovum quality and maturity among patients with POR undergoing IVF/ICSI. Included participants with POR will meet one of the three following conditions and will be randomly assigned to an intervention or control group: (1) anti-Müllerian hormone (AMH)≤2.0 ng/mL, (2) 10 IU/L≤basic follicle-stimulating hormone (bFSH)≤20 IU/L and (3) antral follicles≤7. (According to the Bologna criteria and the population admitted to each centre, the criteria for our study were formulated.23) The participants will be recruited continuously by reproductive doctors from four reproductive centres in different cities: Lanzhou University Second Hospital, Hebei Institute of Family Planning Science and Technology, Qinghai Province People’s Hospital and Qinghai Red Cross Hospital. The trial is scheduled to begin on 1 April 2023 and end in May 2024. The flow diagram of the study for the intervention and control groups is illustrated in figure 1.

Figure 1

Study flow chart. G-CSF, granulocyte colony-stimulating factor; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilisation.

Patient and public involvement

The study is triple-blind and has no priority for patients. Development of the research question and outcome measures are not informed by patients’ priorities. Before the trial, patients’ opinions will be considered according to the test procedure to optimise the procedure. Patients were involved in the recruitment and the process of the study. The process of patient participation in this study was designed and implemented after discussion with the principals of each centre. (Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.) After the study is completed, patients will be informed of its results after unblinding. The burden of intervention was assessed by both the investigator and patient. We would like to express our appreciation for patients’ consultation.

Participants

Randomisation and blinding

Eligible subjects will be randomised in a 1:1 fashion to one of two treatments:

• G-CSF, 125 ug every other day from 1 to 3 days of menstruation of IVF/ICSI (Gn (gonadotropic hormone) administration) to ovum pick-up day.

• Normal placebo, 1 mL every other day from 1 to 3 days of menstruation of IVF/ICSI (Gn administration) to ovum pick-up day.

The subjects will be randomised into two groups by block randomisation method with an allocation ratio of 1:1. The random number table is produced by professional medical statisticians using the SAS V.9.2 statistical software package. The random number table, the selected block length and the random number initial value seed parameters are sealed in blinding codes as confidential data and stored in sequentially coded, sealed and stamped radiopaque envelopes. The blinding codes are in duplicate and stored at the drug clinical study site of the sponsor and the principal investigator. The packaging of G-CSF and placebo is in the same appearance and cannot be distinguished. The randomisation will be performed prior to the first dose of G-CSF or placebo. After providing informed consent, the patients will be randomly allocated to the study group (subcutaneous injection of G-CSF) or the control group (subcutaneous injection of a placebo).

Because this is a triple-blind clinical trial, the randomisation scheme will be kept strictly confidential by the professional medical statisticians and will not be disclosed to any patient, investigator or outcome evaluator, including the protocol leader. As the appearance of G-CSF and placebo is identical and indistinguishable in packaging, the participants, research staff and outcome evaluators will not know the allocation during the study.

Intervention

All participants assigned to the intervention group will receive G-CSF treatment based on conventional clinical treatment protocols for patients with POR. Considering the influence of G-CSF on ovum maturity, embryo quality and endometrium, a subcutaneous injection of G-CSF (125 μg every other day) will be administered from 1 to 3 days of menstruation of IVF/ICSI (Gn administration) to ovum pick-up day. All subjects assigned to the control group will receive conventional clinical treatment and subcutaneous injections of placebo every other day. Normal placebo (1 mL) will be subcutaneously injected into the control group in the same manner as the study group. With the exception of G-CSF, the intervention and control groups will undergo the same procedures throughout the study. The detailed research process is shown in online supplemental document 1.

IVF/ICSI treatment

All participants will then undergo IVF treatment per local protocols, with agreed on standards. The standard IVF treatment for the study will be a PPOS protocol at all sites. Controlled ovarian stimulation will be performed based on female serum hormone levels, age and body mass index (BMI). Gonadotrophin stimulation with recombinant human follicle-stimulating hormone and menotropin (supplying 75 IU FSH and 75 IU LH activity per vial) will start at a daily dose depending on patient characteristics including age, early follicular phase FSH, AMH, antral follicle count and BMI. Dydrogesterone will be administered orally two pills daily from days 1–3 of menstruation to 10 days after ovum retrieval. Transvaginal ultrasonography to assess follicle growth and detect serum oestradiol, progesterone prog (P), luteinising hormone and FSH levels will be performed throughout the controlled ovarian stimulation cycle. When at least two leading follicles reach 18 mm in diameter, 6500–10 000 IU of recombinant human chorionic growth factor and triptorelin 0.2 mg will be injected, followed by transvaginal ultrasound-guided ovum pick-up 35.5–36 hours later. After 3–5 days of in vitro culture, we will be able to determine the number of D3 high-quality embryos and freeze all transferable embryos. We can then confirm the number of D3 high-quality embryos, ova retrieved, MII ova and transferable embryos according to embryo laboratory records. All the participants who are to be assigned to the control group will receive only conventional clinical treatments. With the exception of G-CSF, the intervention and control groups will undergo the same procedures throughout the study.

Initial screening, assessment and follow-up

After providing informed consent, the participants will be asked standardised questions regarding their demographic characteristics and medical history. Clinical data that are to be extracted from medical records will include assisted reproduction processes, surgical operation records, physical examinations, embryo laboratory records, laboratory tests, gynaecological examinations and imaging examinations (table 1).

All enrolled subjects will be followed up for 3 days after ovum retrieval. All outcomes will be assessed based on embryo laboratory records. Information related to laboratory information, clinical information, vaginal ultrasound and adverse events (AEs) will be collected and assessed. Baseline screening, evaluation and follow-up plans are presented in table 1.

Embryo cord (Istanbul embryo scoring criteria, 2011)24: Embryo quality will be evaluated according to blastomere homogeneity, morphology, cytoplasm uniformity, granulation, vacuolation and the amount of debris in the embryo.

The third day embryos will be divided into four phases.

Grade I: Blastomeres of equal size, regular morphology, uniform and clear cytoplasm, no granular phenomenon, no fragmentation or less than 10%.

Grade II: Blastomeres are slightly different, the morphology is not regular, the cytoplasm may have granular phenomenon and debris 10%–20%.

Grade III: The blastomeres are uneven in size and irregular in shape, and the cytoplasm may have an obvious granular phenomenon, with fragments of 20%–50%.

Grade IV: The size of blastomeres is seriously uneven, and the cytoplasm may have a serious particle phenomenon; fragmentation is over 50%.

Normal fertilised embryos on day 3 were divided into six to nine cells, grades I and II were good-quality embryos. Embryos with a cell count of less than six cells or grades III and IV were considered as non-high-quality embryos.

Currently, the maturity of ova is primarily assessed based on the morphological features of its polar body morphology.25 26

Study endpoints

The primary endpoint will be the total number of D3 high-quality embryos. All the outcomes will be assessed based on embryo laboratory findings according to the Istanbul embryo scoring criteria (2011). Three days after ova retrieval, efficacy is classified into one level. D3 high-quality embryos: observation on the third day after ova retrieval. Secondary endpoints will be: (1) the number of ova obtained: the total number of ova collected during ovum retrieval; (2) the total number of MII ova: the number of matured oocytes and (3) the number of transferable embryos.

Safety indicators and AEs

Safety indicators: (1) basic vital signs and general conditions: respiration, body temperature, blood pressure, rash, etc; (2) whole blood cell count, liver and kidney function determination and (3) evaluation of AEs.

Subjects will be tested for blood routine every 14 days after starting medication. If the subject’s white cell count is greater than 30×10⁹/L, the trial will be terminated and the investigator will be notified to urgently remove the blindness. The records, evaluations and reports of other adverse reactions and AEs can be found in online supplemental document 2.

Sample size consideration

The PASS software V.11.0 (NCSS, Kaysville, Utah, USA) will be used to calculate the sample size for both groups. Currently, there is no clinical data on G-CSF for POR. Two recent studies conducted in China reported that the clinical pregnancy rates of patients with POR receiving DKP treatment and those who are undergoing conventional IVF or ICSI based on a microstimulation protocol are 39.62% and 20.93%, respectively.27 28 In the present study, the clinical pregnancy rate in the DKP group is hypothesised to be 0.4 based on the null hypothesis and 0.25 based on the alternative hypothesis. The clinical pregnancy rate in the placebo group is hypothesised to be 0.25. A two-sided Z test with pooled variance will be used as the test statistic. The significance level of the test target will be set at p<0.05. The ratio between groups will be 1:1. The minimum sample size for each group will be 142; hence, 284 participants will achieve 90% of their efforts to detect a difference between the group proportions of 0.15. Assuming that the dropout rate of the study participants will be 10%, we expect to recruit 312 participants, 156 individuals in each group.

Statistical analysis

Analyses will be performed by a statistician at Lanzhou University via SAS statistical software (V.9.3). Data will be analysed according to the ‘intention-to-treat’ principle. A two-sided significance level of 5% will be used for all analyses.

Descriptive statistics will be used to summarise the demographic and clinical characteristics of the participants randomised to the intervention and control groups. The differences between the two groups in demographic characteristics, clinical characteristics, laboratory characteristics and follow-up retention rate will be compared using t-tests (or Wilcoxon rank sum test) and χ² test/Fisher’s exact test, as appropriate.

The primary outcome (on the third day after oocyte retrieval, embryos will be scored according to the Istanbul embryo scoring criteria (2011) and D3 high-quality embryos will be identified) analyses will be carried out using the χ² test considering the centre effect.

The differences in ovum and embryo parameters between the intervention and control groups will be determined using t-tests or Wilcoxon rank sum tests, as appropriate. Cox proportional hazards models will also be used to calculate HRs and 95% CIs, with other risk factors as covariates. For continuous variables, statistical significance will be assessed using the two-tailed Student’s t-test for unpaired data with Bonferroni correction for multiple comparisons. Fisher’s exact test and χ² will be used for discontinuous variables when appropriate. Statistical significance will be set at p<0.05. The differences in clinical and embryo indicators (number of MII ova and transferable embryos) between the intervention and control groups will be determined using repeated measures analysis of variance. Cox proportional hazards models will also be used to calculate HRs and 95% CIs with other risk factors (such as pregnancy outcome) as covariates.

The incidence of AEs between the two groups will be compared using the χ² test or Fisher’s exact test, and descriptive statistics will be used to summarise the incidence rates of AEs and serious AEs. Based on previous experience, we expect a data completion rate of at least 99.5% and we expect trivial influence on trial analysis or results due to missing data. In the unlikely event of unexpectedly high rates of missing data, the potential mechanisms for missing data (missing completely at random, missing at random or missing not at random) will be examined. We will compare the available characteristics of those with missing data to those with complete data. If necessary, imputation techniques may be used.

Safety and AEs monitoring

In previous studies, G-CSF was mainly used to reduce the incidence and severity of neutropenia caused by chemotherapy drugs, so most of the adverse reaction reports after drug use were from healthy people or these patients. In this study, G-CSF was used in patients with POR, and the possible adverse reaction treatment methods were referred to in the clear research report. If an unreported AE occurs, a joint consultation with an obstetrician-gynaecologist should be conducted immediately and recorded in detail.

The data and safety monitoring board (DSMB) consists of six experts other than the authors. Four obstetrics and gynaecology specialists and one pharmacy specialist are responsible for the monitoring and evaluation of adverse drug reactions, assisting researchers in regularly evaluating research progress and efficacy and then revising or early terminating research programmes to ensure the safety and health of subjects. A statistical expert assisted the researchers in monitoring patient safety data and indicators and analysing and calculating the study data.

The DSMB will receive unblinded data and advise on potential safety issues. If the subject’s white cell count is greater than 30×10⁹/L, appears intolerance or other more serious adverse reactions, the subject should be urgently blind, and early termination of the test. The choice of continuing therapy or trial participation based on AEs is at the discretion of the investigator and DSMB determinations. Taking safety data and efficacy into account, institutional data and safety monitoring board (IDSMB) can offer several different recommendations: (1) to continue the study without modifying the protocol; (2) to continue the study with modifying the protocol; (3) to temporarily stop the enrolment and (4) to early terminate the study.