This randomized clinical trial was conducted at infertility department of Royan Institute from January 2017 to June 2018. The trial protocol was approved by the Institutional Review Board and Ethics Committee of Royan Institute (Ethics code: IR.ACECR.ROYAN.REC.1394.121). The eligible patients signed the informed consent. Patients with poor ovarian response undergoing IVF/ICSI and fresh embryo transfer (ET) cycles were evaluated. Poor ovarian response was defined according to the Bologna criteria and existence of at least two of the following criteria: 1) a previous history of POR (retrieved oocytes ≤3) in a conventional stimulation protocol, 2) advanced maternal age (≥40 years) or any other risk factors for POR (e.g. a history of ovarian surgery) and 3) abnormal ovarian reserve test (i.e. antral follicle count (AFC) < 5 follicles or anti-Mȕllerian hormone (AMH) < 1.1 ng/ml). The exclusion criteria were premature ovarian failure (basal follicle stimulating hormone (FSH) above 20 IU/l or no antral follicle in ultrasound examination), donor/recipient treatments, metabolic or endocrine disorders including hyperprolactinoma and hypo/hyperthyroidism, endometriosis, body mass index > 30 kg/m2, and azoospermic male partner. A minimum of 2 or more month’s interval from the previous ovarian stimulation was considered to prevent any potential source of error.
Block randomization method was designed by a fellow epidemiologist using Stata software version 13 and the number of blocks were 4. The random allocation list for patients was only accessible to the epidemiologist. In order to random allocation concealment, only the methodologist was aware of the design of the code. When the doctor confirmed patient’s eligibility, the methodologist provided the doctor with the envelope. The group was selected based on the type of group mentioned in the envelope. The outcome evaluators were also blinded to the random allocation process and type of treatment. Data analysis was performed by a statistician who was also unaware of all processes performed.
On second day of menstrual cycle, the eligible patients were randomly allocated into either delayed start or routine GnRH-antagonist stimulation protocol in a 1:1 ratio. A flexible regimen of GnRH-antagonist was used for all study participants. The serum estradiol (E
2) concentrations < 60 pg/mL and absence of ovarian cysts < 10 mm diameter on vaginal ultrasound scans on cycle day 2 were used to define ovarian quiescence. The baseline serum FSH and luteinizing hormone (LH) levels were also measured at initial assessment before gonadotropin stimulation. All patients received estrogen priming (Estraval®, 2 mg
, Aburaihan Co., Tehran
, Iran) starting one week after LH surge and continued until mensturation and prior to ovarian stimulation. In the delayed-start protocol, baseline ultrasounds were performed on cycle day 2 and after the completion of GnRH antagonist pretreatment to identify the absence of ovarian cyst or lead follicle > 10 mm. In conventional antagonist protocol (control group), ovarian stimulation with gonadotropins was started on day 2 of menstrual cycle. In the delayed-start protocol (experimental group), ovarian stimulation was started after 7 days of GnRH antagonist pretreatment (Cetrotide®, 0.25 mg cetrorelix acetate
, Serono,
Inc). In both protocols, 300 IU FSH (Gonal - F®, Serono Laboratories Ltd., Geneva, Switzerland) and 150 IU human menopausal gonadotrophins (HMG) (Menopur; Ferring) were used for ovarian stimulation. The serial vaginal ultrasound (sonographic device: Phillips, affinity 70) and measurements of serum estradiol (E
2) level were used to assess follicular maturation. The dosage of gonadotropins was adjusted according to the ovarian response. In both groups, when follicle(s) ≥13 mm were observed, the GnRH antagonist, cetrorelix (Cetrotide ®, Serono International, Geneva, Switzerland), 0.25 mg/day was started subcutaneously and continued until the day of triggering of ovulation. Progesterone and E
2 levels were evaluated in serum on day of human chorionic gonadotropin (hCG) administration. When at least one follicles measuring ≥18 mm in diameter and serum E
2 concentration ≥ 500 pg/mL were observed, the final stage of oocyte maturation was induced by two pre-filled syringes of
recombinant human chorionic gonadotropin (rhCG) (Ovitrelle®, 250 μg/0.5 ml, Merck, Serono, Inc). If these criteria have not been achieved after 10–12 days stimulation, the cycle has been cancelled for inadequate response. Transvaginal ultrasound-guided oocyte retrieval was performed 34–36 h after oocyte triggering. After stripping the cumulus cells, intracytoplasmic sperm injection (ICSI) was done with ejaculated sperm to metaphase II (MII) oocytes in all cycles. ICSI was performed in all cases to prevent infrequent cases of fertilization failures with conventional IVF. Embryos were cultured in a commercially available culture medium until the day of transfer. The obtained embryos at cleavage stage were replaced by an embryo transfer catheter (Guardia™, Access ET Catheter, Cook Medical), 2 or 3 days after oocytes retrieval. Embryo quality was determined according to the number and regularity of blastomeres and the degree of embryonic fragmentation that has been explained previously [
11]. All patients received luteal phase support in the form of 400 mg vaginal progesterone suppository twice daily (Cyclogest® (400 mg), Actavis, Barnstaple, UK) starting on the evening of the oocyte retrieval and it was continued for 10 weeks in cases with positive pregnancy test. A serum ß-hCG analysis was done 14 days after ET, and the clinical pregnancy (presence of gestational sac with heartbeat) was determined by ultrasound scan 14 days later.
The main outcomes were the fertilization and cycle cancellation rates, the numbers of retrieved and MII oocytes, obtained and top quality embryos respectively. The secondary outcomes were the total gonadotropins dose, duration of ovarian stimulation, endometrial thickness, implantation and clinical pregnancy rates.
Statistical analysis
According to pilot study and by using G*Power software (version 3.1.9.2) with considering the effect size of 0.10, α = 0.05, and 80% power for fertilization rate as primary outcome; 60 subjects were needed in each study group. The statistical analysis was carried out by using Statistical Package for the Social Sciences, version 20, SPSS Inc., Chicago, Illinois, USA (SPSS). The differences between two groups were analyzed using the independent t-test and Mann-Whitney U test for the normal and non-normal continuous variables respectively. The Chi- square test was applied for comparison of the categorical variables between groups. Descriptive data are presented as mean ± standard deviation (SD) or median (interquartile range) as indicated. The Statistical significance level was set at p-value < 0.05.